Mary Helen Barcellos-Hoff, Ph.D.

Radiation and endoplasmic reticulum stress-inducer promote calreticulin translocation, contributing to immunogenic cell death of cancer cells
Golden E.; Demaria S.; Barcellos-Hoff M.H.
2012 ;35(1):100-100, Journal of immunotherapy (Hagerstown)
We hypothesize that cell damage and death from the effect of ionizing radiation (IR) and endoplasmic reticulum (ER) stressinducing agents could: (1) be monitored in vitro; and (2) contribute to an anti-tumor immune response via the induction of mediators of immunogenic cell death (ICD) of cancer cells. ICD promotes the cross-presentation of tumor-derived antigens by dendritic cells (DCs) to T cells (Semin Immunol. 2010;22:113-124). Calreticulin (CRT, an ER chaperone protein) redistribution to the surface of tumor cells acts as a potent 'eat me' signal for DCs involved in tumor associated antigen processing, thereby serving as a key step in ICD. In the clinical setting, IR or ER stress alone may not quantitatively and/or qualitatively achieve tumor cell death in a manner that specifically triggers immune-mediated tumor rejection. Thus, we hypothesized that clinically relevant doses of IR, when combined with thapsigargin (Tg, an ER stress-inducer via sarcoplasmic/ER calcium ATPase inhibition), may intensify CRT translocation to the cell surface. To test this, we employed the poorly immunogenic 4T1 mouse breast cancer cells. 4T1 cells were treated with IR (0, 6, or 20 Gy) followed by 24 hours. culture in the presence or absence of Tg (1 mM). Thereafter, the cells were assayed either via Western blot (WB) or immunofluorescence (IF). Cytotoxicity was determined via MTT assay at 12, 24, and 48 hours. Relative amounts of protein were determined via WB analysis with specific antibodies to phospho-EIF2-a, caspase-8, BAP-31, and PARP. Actin was used as a loading control. CRT redistribution was determined by IF analysis. When combined, IR (6 Gy) + Tg (1mM) triggered elevated phosphorylation of EIF2-a (a marker for ER stress and protein translation inhibition) in 4T1 cells. In addition, IR (6 and 20 Gy) + Tg (1 muM) increased the cleavage of the apoptotic markers caspase-8, BAP-31, and PARP. Finally, we observed that cell death by IR (6 and 10 Gy, single dose) in the presence of Tg (1 muM) was preceded by enhanced CRT translocation to the cell surface. In this in vitro model, IR (6Gy and 10 Gy) alone was unable to incite CRT redistribution. However, in the presence of Tg (1 muM), IR (6 Gy) CRT redistribution occurred and was superior to controls. Taken together, these findings suggest that IR combined with an ER stress-inducing agent is a novel application of radiotherapy that can potently trigger ICD and serve as a strategy to promote immune-mediated tumor rejection in cancer patients
— id: 149974, year: 2012, vol: 35, page: 100, stat: Journal Article,

Lack of radiation dose or quality dependence of epithelial-to-mesenchymal transition (EMT) mediated by transforming growth factor beta
Andarawewa, Kumari L; Costes, Sylvain V; Fernandez-Garcia, Ignacio; Chou, William S; Ravani, Shraddha A; Park, Howard; Barcellos-Hoff, Mary Helen
2011 Apr 1;79(5):1523-1531, International journal of radiation oncology biology physics
PURPOSE: Epithelial-to-mesenchymal transition (EMT) is a phenotype that alters cell morphology, disrupts morphogenesis, and increases motility. Our prior studies have shown that the progeny of human mammary epithelial cells (HMECs) irradiated with 2 Gy undergoes transforming growth factor beta (TGF-beta)-mediated EMT. In this study we determined whether radiation dose or quality affected TGF-beta-mediated EMT. METHODS AND MATERIALS: HMECs were cultured on tissue culture plastic or in Matrigel (BD Biosciences, San Jose, CA) and exposed to low or high linear energy transfer (LET) and TGF-beta (400 pg/mL). Image analysis was used to measure membrane-associated E-cadherin, a marker of functional epithelia, or fibronectin, a product of mesenchymal cells, as a function of radiation dose and quality. RESULTS: E-cadherin was reduced in TGF-beta-treated cells irradiated with low-LET radiation doses between 0.03 and 2 Gy compared with untreated, unirradiated cells or TGF-beta treatment alone. The radiation quality dependence of TGF-beta-mediated EMT was determined by use of 1 GeV/amu (gigaelectron volt/atomic mass unit) (56)Fe ion particles at the National Aeronautics and Space Administration's Space Radiation Laboratory. On the basis of the relative biological effectiveness of 2 for (56)Fe ion particles' clonogenic survival, TGF-beta-treated HMECs were irradiated with equitoxic 1-Gy (56)Fe ion or 2-Gy (137)Cs radiation in monolayer. Furthermore, TGF-beta-treated HMECs irradiated with either high- or low-LET radiation exhibited similar loss of E-cadherin and gain of fibronectin and resulted in similar large, poorly organized colonies when embedded in Matrigel. Moreover, the progeny of HMECs exposed to different fluences of (56)Fe ion underwent TGF-beta-mediated EMT even when only one-third of the cells were directly traversed by the particle. CONCLUSIONS: Thus TGF-beta-mediated EMT, like other non-targeted radiation effects, is neither radiation dose nor quality dependent at the doses examined
— id: 134227, year: 2011, vol: 79, page: 1523, stat: Journal Article,

Radiation-induced microenviornments and their role in carcinogenesis
Barcellos-Hoff MH; Nguyen DH
Tumor-Associated Fibroblasts and their Matrix Dordrecht : Springer, 2011,

— id: 5980, year: 2011, vol: , page: 267, stat: Chapter,

In honor of Mina J. Bissell
Barcellos-Hoff, Mary Helen
2011 Apr 1;3(4):253-254, Integrative Biology
— id: 130303, year: 2011, vol: 3, page: 253, stat: Journal Article,

TGFbeta Biology in Breast: 15 Years On
Barcellos-Hoff, Mary Helen
2011 Jun;16(2):65-66, Journal of mammary gland biology & neoplasia
— id: 132582, year: 2011, vol: 16, page: 65, stat: Journal Article,

What is the use of systems biology approaches in radiation biology?
Barcellos-Hoff, Mary Helen
2011 Mar;100(3):272-273, Health physics
— id: 150853, year: 2011, vol: 100, page: 272, stat: Journal Article,

Low-dose radiation knowledge worth the cost
Barcellos-Hoff, Mary Helen; Brenner, David J; Brooks, Antone L; Formenti, Silvia; Hlatky, Lyn; Locke, Paul A; Shore, Roy; Tenforde, Thomas; Travis, Elizabeth L; Williams, Jacqueline
2011 Apr 15;332(6027):305-306, Science
— id: 140490, year: 2011, vol: 332, page: 305, stat: Journal Article,

Increased radiosensitivity in vivo following inhibition of transforming growth factor beta in murine model of triple negative breast cancer
Bouquet S.F.; Pilones K.; Demaria S.; DeWyngaert K.J.; Lonning S.; Formenti S.; Barcellos-Hoff M.
2011 ;81(2 SUPPL 1):S714-S715, International journal of radiation oncology biology physics
Purpose/Objective(s): Ionizing radiation triggers activation of transforming growth factor beta1 (TGFbeta), a growth factor that promotes invasion and suppresses immune function. Either genetic or pharmaceutical TGFbeta inhibition prior to IR also inhibits the DNA damage response (DDR) in epithelial cells via blockade of ATM kinase activity (Cancer Res 62:5627, 2002; Cancer Res 66:10861, 2006). Moreover, transient TGFbeta inhibition increases the radiosensitivity of epithelial cancer cells (Bouquet et al. submitted). The current studies test the optimal combination of TGFbeta inhibition using neutralizing antibodies with radiotherapy (RT) in a model of triple negative breast cancer. Materials/Methods: The 4T1 murine mammary tumor model is highly metastatic when grown as subcutaneous tumors. 4T1 cells were injected s.c. in the flank of syngeneic Balb/c mice and about 13 days after implantation approximately 100 mm3 tumors were locally irradiated. An anti-TGFb murine monoclonal antibody, 1D11, was administered i.p. before irradiation. Mice were randomly assigned to four groups receiving control isotype monoclonal antibody, 1D11, RT (8 Gy) and isotype control antibody, or RT and 1D11. Tumor growth, DDR, lymphocyte infiltration, lung metastases and/or survival were evaluated. Results: TGFb TGFb inhibition in vivo reduced DDR as evidenced by gH2AX foci in irradiated tumors. A single injection of 1D11 (5 mg/kg) 24 hr before RT resulted in greater tumor growth delay compared to RT and control antibody (p<0.05). Furthermore, chronic treatment with a higher 1D11 dose (10 mg/kg or 50 mg/kg) in the context of fractionated RT significantly reduced tumor growth rate, decreased visible lung metastases and increased survival (p<0.05). Notably this RT+1D11 protocol increased tumor infiltration of CD8, but not CD4, T lymphocytes, which resulted in a significantly enhanced ratio of CD8+NKG2D+ effector cells to CD4+ T cells in the tumor, suggesting that TGFb inhibition also alters the immunological environment in irradiated tumors. Conclusions: Increased radiosensitivity of 4T1 tumor cells in vitro and in vivo supports the use of TGFb inhibitors as means to increase the response to RT. Moreover, an additional benefit may be that TGFb inhibition promotes an anti-tumor immune response to RT
— id: 150891, year: 2011, vol: 81, page: S714, stat: Journal Article,

TGFbeta1 Inhibition Increases the Radiosensitivity of Breast Cancer Cells In Vitro and Promotes Tumor Control by Radiation In Vivo
Bouquet, Fanny; Pal, Anupama; Pilones, Karsten A; Demaria, Sandra; Hann, Byron; Akhurst, Rosemary J; Babb, Jim S; Lonning, Scott M; Dewyngaert, J Keith; Formenti, Silvia C; Barcellos-Hoff, Mary Helen
2011 Nov 1;17(21):6754-6765, Clinical cancer research
PURPOSE: To determine whether inhibition of TGFbeta signaling prior to irradiation sensitizes human and murine cancer cells in vitro and in vivo. EXPERIMENTAL DESIGN: TGFbeta-mediated growth and Smad phosphorylation of MCF7, Hs578T, MDA-MB-231, and T47D human breast cancer cell lines were examined and correlated with clonogenic survival following graded radiation doses with and without pretreatment with LY364947, a small molecule inhibitor of the TGFbeta type I receptor kinase. The DNA damage response was assessed in irradiated MDA-MB-231 cells pretreated with LY364947 in vitro and LY2109761, a pharmacokinetically stable inhibitor of TGFbeta signaling, in vivo. The in vitro response of a syngeneic murine tumor, 4T1, was tested using a TGFbeta neutralizing antibody, 1D11, with single or fractionated radiation doses in vivo. RESULTS: Human breast cancer cell lines pretreated with TGFbeta small molecule inhibitor were radiosensitized, irrespective of sensitivity to TGFbeta growth inhibition. Consistent with increased clonogenic cell death, radiation-induced phosphorylation of H2AX and p53 was significantly reduced in MDA-MB-231 triple-negative breast cancer cells when pretreated in vitro or in vivo with a TGFbeta type I receptor kinase inhibitor. Moreover, TGFbeta neutralizing antibodies increased radiation sensitivity, blocked gammaH2AX foci formation, and significantly increased tumor growth delay in 4T1 murine mammary tumors in response to single and fractionated radiation exposures. CONCLUSION: These results show that TGFbeta inhibition prior to radiation attenuated DNA damage responses, increased clonogenic cell death, and promoted tumor growth delay, and thus may be an effective adjunct in cancer radiotherapy. Clin Cancer Res; 17(21); 6754-65. (c)2011 AACR
— id: 140531, year: 2011, vol: 17, page: 6754, stat: Journal Article,

Persistence of gamma-H2AX and 53BP1 foci in proliferating and non-proliferating human mammary epithelial cells after exposure to gamma-rays or iron ions
Groesser, Torsten; Chang, Hang; Fontenay, Gerald; Chen, James; Costes, Sylvain V; Helen Barcellos-Hoff, Mary; Parvin, Bahram; Rydberg, Bjorn
2011 Jul;87(7):696-710, International journal of radiation biology
PURPOSE: To investigate gamma-H2AX (phosphorylated histone H2AX) and 53BP1 (tumour protein 53 binding protein No. 1) foci formation and removal in proliferating and non-proliferating human mammary epithelial cells (HMEC) after exposure to sparsely and densely ionising radiation under different cell culture conditions. MATERIAL AND METHODS: HMEC cells were grown either as monolayers (2D) or in extracellular matrix to allow the formation of acinar structures in vitro (3D). Foci numbers were quantified by image analysis at various time points after exposure. RESULTS: Our results reveal that in non-proliferating cells under 2D and 3D cell culture conditions, iron-ion induced gamma-H2AX foci were still present at 72 h after exposure, although 53BP1 foci returned to control levels at 48 h. In contrast in proliferating HMEC, both gamma-H2AX and 53BP1 foci decreased to control levels during the 24-48 h time interval after irradiation under 2D conditions. Foci numbers decreased faster after gamma-ray irradiation and returned to control levels by 12 h regardless of marker, cell proliferation status, and cell culture condition. CONCLUSIONS: The disappearance of radiation-induced gamma-H2AX and 53BP1 foci in HMEC has different dynamics that depend on radiation quality and proliferation status. Notably, the general patterns do not depend on the cell culture condition (2D versus 3D). We speculate that the persistent gamma-H2AX foci in iron-ion irradiated non-proliferating cells could be due to limited availability of double-strand break (DSB) repair pathways in G0/G1-phase, or that repair of complex DSB requires replication or chromatin remodelling
— id: 150856, year: 2011, vol: 87, page: 696, stat: Journal Article,

Multiscale iterative voting for differential analysis of stress response for 2D and 3D cell culture models
Han, J; Chang, H; Yang, Q; Fontenay, G; Groesser, T; Barcellos-Hoff, M Helen; Parvin, B
2011 Mar;241(3):315-326, Journal of microscopy
Three-dimensional (2D) cell culture models have emerged as the basis for improved cell systems biology. However, there is a gap in robust computational techniques for segmentation of these model systems that are imaged through confocal or deconvolution microscopy. The main issues are the volume of data, overlapping subcellular compartments and variation in scale or size of subcompartments of interest, which lead to ambiguities for quantitative analysis on a cell-by-cell basis. We address these ambiguities through a series of geometric operations that constrain the problem through iterative voting and decomposition strategies. The main contributions of this paper are to (i) extend the previously developed 2D radial voting to an efficient 3D implementation, (ii) demonstrate application of iterative radial voting at multiple subcellular and molecular scales, and (iii) investigate application of the proposed technology to two endpoints between 2D and 3D cell culture models. These endpoints correspond to kinetics of DNA damage repair as measured by phosphorylation of gammaH2AX, and the loss of the membrane-bound E-cadherin protein as a result of ionizing radiation. Preliminary results indicate little difference in the kinetics of the DNA damage protein between 2D and 3D cell culture models; however, differences between membrane-bound E-cadherin are more pronounced
— id: 150854, year: 2011, vol: 241, page: 315, stat: Journal Article,

Development of a novel multiplexed-TGFbeta assay
Hardee M.E.; Marciscano A.E.; Rifkin D.B.; Barcellos-Hoff M.
2011 ;81(2 SUPPL 1):S755-S755, International journal of radiation oncology biology physics
Purpose/Objective(s): Transforming growth factor-beta (TGFbeta) levels are increased by radiation therapy. Pre-and post-radiotherapy systemic levels of TGFb measured by ELISA, mink lung epithelial cell luciferase assay (MLEC), and cell-based biological assays have been correlated with treatment outcomes and radiation-induced toxicity. However, aspects of these assays and TGFbeta biology can make TGFb measurements difficult to interpret. TGFbeta is secreted along with the latency associated peptide (LAP) that is activated extracellularly when needed; available assays measure active TGFbeta using artificial activation by acid or heat. Specimen contamination by platelets is another source of variability since latent TGFbeta is present in platelets in large quantities. Our goal is to develop an assay that eliminates artificial activation, includes an internal control for platelet contamination, and can reproducibly measure both active and latent TGFbeta. Materials/Methods: To measure active TGFbeta1, a commercially available TGFbeta1 ELISA using the Meso Scale Discovery (MSD) platform was used. MSD uses electrochemiluminescent detection that provides sensitivity over a concentration range of 5 logs and allows multiplexing of up to 10 assays. An ELISA for LAP-b1, a measure of latent TGFbeta1 was developed. Different preparations of plasma and serum were tested and contamination from platelet degranulation was measured using an ELISA of platelet factor-4 (PF4), which is released from a-granules of activated platelets. Results: LAP-s1 and PF4 assays using the MSD platform were successfully developed using commercially available antibodies. LAP-b1 ELISA was a highly reproducible measurement of total TGFbeta, as verified using MLEC assay of exogenously activated samples. Measurement of total TGFb in serum (19,085-156,058 pg/mL) was high and variable due to contamination by platelet activation as measured by PF4. By spiking plasma with increasing volumes of serum (to replicate platelet contamination), we determined a PF4 cutoff value of 1.5 mg/mL, below which total TGFbeta levels were constant. Collection of plasma in EDTA containing tubes (vs heparin tubes) and double centrifugation was most effective at reducing platelet contamination and lead to the most reproducible TGFbeta measurements that averaged 344 pg/ml.We determined that plasma contains undetectable levels of active TGFbeta but that as little as 62 pg/mL exogenous TGFbeta could be detected in plasma. Conclusions:We developed an improved TGFbeta assay for biological fluids that simultaneously measures active and latent TGFbeta. By avoiding the uncertainty introduced by exogenous activation in currently available assays and including an internal control for platelet contamination, this assay will be more robust for future correlative studies of systemic TGFbeta levels and radiation treatment outcomes and toxicity
— id: 150889, year: 2011, vol: 81, page: S755, stat: Journal Article,

TGFbeta inhibition radiosensitizes murine glioblastoma cells and decreases neurosphere-forming capacity
Hardee M.E.; Marciscano A.E.; Zagzag D.; Narayana A.; Barcellos-Hoff M.
2011 ;81(2 SUPPL 1):S714-S714, International journal of radiation oncology biology physics
S714 Purpose/Objective(s): Transforming growth factor-beta (TGFbeta) is a pleotropic cytokine in the tumor microenvironment that can promote malignant behaviors, including invasion and motility. Glioblastomas produce abundant TGFbeta, are routinely treated with radiation, and have a very poor prognosis. A role for TGFbeta in the DNA damage response has recently been discovered in which TGFb inhibition in vitro and in vivo compromises ATM-kinase activity induced by ionizing radiation (Cancer Research 66:10861-68; 62:5627-31). These data suggest that TGFbeta could protect cancer cells from the cytotoxic effects of radiation by promoting ATM dependent responses; if so, TGFbeta inhibitors, which are in clinical trials, might increase therapeutic response to radiation. Materials/Methods: We used the murine glioblastoma cell line, GL261, to test the effects of TGFbeta inhibition by LY364947 (a small molecule inhibitor of the TGFbeta type I receptor kinase) on proliferation, radiosensitivity, and neurosphere-forming capacity. Experiments were performed in triplicate and differences tested by ANOVA. Results: GL261 cells produce 0.9 ng/mL per 106 cells of total TGFbeta in media conditioned for 24 hr, the majority of which is latent TGFb2. They also respond to exogenous TGFbeta1 with an increase in Smad2 phosphorylation. Despite intact TGFb receptor kinase activity, GL261 cells displayed no growth modulation response to exogenous TGFbeta1 (0.5-2ng/mL) treatment or to inhibition by LY364947 (400nM). Nonetheless, inhibition of TGFbeta with LY364947 for 24 hours prior to radiation treatment significantly increased GL261 radiosensitivity in the clonogenic assay (p<0.001), with a 1.25 dose enhancement ratio at 10% surviving fraction. This correlated with a significant 55% decrease in H2AX foci following radiation treatment with 2Gy (p<0.0001). Irradiation of GL261 cells with 2Gy also decreased primary neurosphere-forming capacity by 28% (p<0.001, ANOVA), but had no effect on secondary neurosphere formation. Treatment with LY364947 alone had no effect on neurosphere formation. In contrast, treatment for 24 hours prior to irradiation decreased the primary neurosphere-forming capacity of irradiated GL261 cells by an additional 47% (p<0.001) and decreased secondary neurosphere formation by 68% (p<0.001). Conclusions: Given the radiosensitization and specifically the response of the neurosphere assay, which is thought to measure the glioma initiating cell population, our results suggest that inhibition of TGFb in combination with radiation represents a promising therapeutic strategy. By targeting the putative stem cells long term benefit of TGFb inhibition in glioblastoma may be achieved compared to poor response rates seen with the standard regimen of chemotherapy and radiotherapy
— id: 150892, year: 2011, vol: 81, page: S714, stat: Journal Article,

The pivotal role of insulin-like growth factor I in normal mammary development
Kleinberg, David L; Barcellos-Hoff, Mary Helen
2011 Sep;40(3):461-471, Endocrinology & metabolism clinics of North America
Mammary development begins in puberty in response to an estrogen (E(2)) surge. E(2) does not act alone. It relies on pituitary growth hormone (GH) to induce insulin-like growth factor I (IGF-I) production in the mammary stromal compartment. In turn, IGF-I permits E(2) (and progesterone) action. During puberty, E(2) and IGF-I synergize for ductal morphogenesis. During pregnancy, progesterone joins IGF-I and E(2) to stimulate secretory differentiation necessary to produce milk. Prolactin stimulates milk production, while transforming growth factor-beta inhibits proliferation. The orchestrated action of hormones, growth factors, and receptors necessary for mammary development and function are also critical in breast cancer
— id: 137076, year: 2011, vol: 40, page: 461, stat: Journal Article,

Interplay between BRCA1 and RHAMM regulates epithelial apicobasal polarization and may influence risk of breast cancer
Maxwell, Christopher A; Benitez, Javier; Gomez-Baldo, Laia; Osorio, Ana; Bonifaci, Nuria; Fernandez-Ramires, Ricardo; Costes, Sylvain V; Guino, Elisabet; Chen, Helen; Evans, Gareth J R; Mohan, Pooja; Catala, Isabel; Petit, Anna; Aguilar, Helena; Villanueva, Alberto; Aytes, Alvaro; Serra-Musach, Jordi; Rennert, Gad; Lejbkowicz, Flavio; Peterlongo, Paolo; Manoukian, Siranoush; Peissel, Bernard; Ripamonti, Carla B; Bonanni, Bernardo; Viel, Alessandra; Allavena, Anna; Bernard, Loris; Radice, Paolo; Friedman, Eitan; Kaufman, Bella; Laitman, Yael; Dubrovsky, Maya; Milgrom, Roni; Jakubowska, Anna; Cybulski, Cezary; Gorski, Bohdan; Jaworska, Katarzyna; Durda, Katarzyna; Sukiennicki, Grzegorz; Lubinski, Jan; Shugart, Yin Yao; Domchek, Susan M; Letrero, Richard; Weber, Barbara L; Hogervorst, Frans B L; Rookus, Matti A; Collee, J Margriet; Devilee, Peter; Ligtenberg, Marjolijn J; Luijt, Rob B van der; Aalfs, Cora M; Waisfisz, Quinten; Wijnen, Juul; Roozendaal, Cornelis E P van; Easton, Douglas F; Peock, Susan; Cook, Margaret; Oliver, Clare; Frost, Debra; Harrington, Patricia; Evans, D Gareth; Lalloo, Fiona; Eeles, Rosalind; Izatt, Louise; Chu, Carol; Eccles, Diana; Douglas, Fiona; Brewer, Carole; Nevanlinna, Heli; Heikkinen, Tuomas; Couch, Fergus J; Lindor, Noralane M; Wang, Xianshu; Godwin, Andrew K; Caligo, Maria A; Lombardi, Grazia; Loman, Niklas; Karlsson, Per; Ehrencrona, Hans; Wachenfeldt, Anna von; Barkardottir, Rosa Bjork; Hamann, Ute; Rashid, Muhammad U; Lasa, Adriana; Caldes, Trinidad; Andres, Raquel; Schmitt, Michael; Assmann, Volker; Stevens, Kristen; Offit, Kenneth; Curado, Joao; Tilgner, Hagen; Guigo, Roderic; Aiza, Gemma; Brunet, Joan; Castellsague, Joan; Martrat, Griselda; Urruticoechea, Ander; Blanco, Ignacio; Tihomirova, Laima; Goldgar, David E; Buys, Saundra; John, Esther M; Miron, Alexander; Southey, Melissa; Daly, Mary B; Schmutzler, Rita K; Wappenschmidt, Barbara; Meindl, Alfons; Arnold, Norbert; Deissler, Helmut; Varon-Mateeva, Raymonda; Sutter, Christian; Niederacher, Dieter; Imyamitov, Evgeny; Sinilnikova, Olga M; Stoppa-Lyonne, Dominique; Mazoyer, Sylvie; Verny-Pierre, Carole; Castera, Laurent; de Pauw, Antoine; Bignon, Yves-Jean; Uhrhammer, Nancy; Peyrat, Jean-Philippe; Vennin, Philippe; Fert Ferrer, Sandra; Collonge-Rame, Marie-Agnes; Mortemousque, Isabelle; Spurdle, Amanda B; Beesley, Jonathan; Chen, Xiaoqing; Healey, Sue; Barcellos-Hoff, Mary Helen; Vidal, Marc; Gruber, Stephen B; Lazaro, Conxi; Capella, Gabriel; McGuffog, Lesley; Nathanson, Katherine L; Antoniou, Antonis C; Chenevix-Trench, Georgia; Fleisch, Markus C; Moreno, Victor; Pujana, Miguel Angel
2011 Nov;9(11):e1001199-e1001199, PLoS biology
Differentiated mammary epithelium shows apicobasal polarity, and loss of tissue organization is an early hallmark of breast carcinogenesis. In BRCA1 mutation carriers, accumulation of stem and progenitor cells in normal breast tissue and increased risk of developing tumors of basal-like type suggest that BRCA1 regulates stem/progenitor cell proliferation and differentiation. However, the function of BRCA1 in this process and its link to carcinogenesis remain unknown. Here we depict a molecular mechanism involving BRCA1 and RHAMM that regulates apicobasal polarity and, when perturbed, may increase risk of breast cancer. Starting from complementary genetic analyses across families and populations, we identified common genetic variation at the low-penetrance susceptibility HMMR locus (encoding for RHAMM) that modifies breast cancer risk among BRCA1, but probably not BRCA2, mutation carriers: n = 7,584, weighted hazard ratio ((w)HR) = 1.09 (95% CI 1.02-1.16), p(trend) = 0.017; and n = 3,965, (w)HR = 1.04 (95% CI 0.94-1.16), p(trend) = 0.43; respectively. Subsequently, studies of MCF10A apicobasal polarization revealed a central role for BRCA1 and RHAMM, together with AURKA and TPX2, in essential reorganization of microtubules. Mechanistically, reorganization is facilitated by BRCA1 and impaired by AURKA, which is regulated by negative feedback involving RHAMM and TPX2. Taken together, our data provide fundamental insight into apicobasal polarization through BRCA1 function, which may explain the expanded cell subsets and characteristic tumor type accompanying BRCA1 mutation, while also linking this process to sporadic breast cancer through perturbation of HMMR/RHAMM
— id: 150852, year: 2011, vol: 9, page: e1001199, stat: Journal Article,

TGF-beta biology in mammary development and breast cancer
Moses, Harold; Barcellos-Hoff, Mary Helen
2011 Jan;3(1):a003277-a003277, Cold Spring Harbor perspectives in biology
Transforming growth factor-beta1 (TGF-beta) was first implicated in mammary epithelial development by Daniel and Silberstein in 1987 and in breast cancer cells and hormone resistance by Lippman and colleagues in 1988. TGF-beta is critically important for mammary morphogenesis and secretory function through specific regulation of epithelial proliferation, apoptosis, and extracellular matrix. Differential TGF-beta effects on distinct cell types are compounded by regulation at multiple levels and the influence of context on cellular responses. Studies using controlled expression and conditional-deletion mouse models underscore the complexity of TGF-beta biology across the cycle of mammary development and differentiation. Early loss of TGF-beta growth regulation in breast cancer evolves into fundamental deregulation that mediates cell interactions and phenotypes driving invasive disease. Two outstanding issues are to understand the mechanisms of biological control in situ and the circumstances by which TGF-beta regulation is subverted in neoplastic progression
— id: 138152, year: 2011, vol: 3, page: a003277, stat: Journal Article,

The biological impact of radiation exposure on breast cancer development
Nguyen, David H; Bochaca, Irineu Illa; Barcellos-Hoff, Mary Helen
Environment and breast cancer New York : Springer, c2011,
— id: 5981, year: 2011, vol: , page: 185, stat: Chapter,

Consequences of Epithelial or Stromal TGFbeta1 Depletion in the Mammary Gland
Nguyen, David H; Martinez-Ruiz, Haydeliz; Barcellos-Hoff, Mary Helen
2011 Jun;16(2):147-155, Journal of mammary gland biology & neoplasia
Transforming growth factor beta1 (TGFbeta) affects stroma and epithelial composition and interactions that mediate mammary development and determine the course of cancer. The reduction of TGFbeta in Tgfbeta1 heterozygote mice, which are healthy and long-lived, provides an important model to dissect the contribution of TGFbeta in mammary gland biology and cancer. We used both intact mice and mammary chimeras in conjunction with Tgfbeta1 genetic depletion and TGFbeta neutralizing antibodies to evaluate how stromal or epithelial TGFbeta depletion affect mammary development and response to physiological stimuli. Our studies of radiation carcinogenesis have revealed new aspects of TGFbeta biology and suggest that the paradoxical TGFbeta switch from tumor suppressor to tumor promoter can be resolved by assessing distinct stromal versus epithelial actions
— id: 134199, year: 2011, vol: 16, page: 147, stat: Journal Article,

Radiation Acts on the Microenvironment to Affect Breast Carcinogenesis by Distinct Mechanisms that Decrease Cancer Latency and Affect Tumor Type
Nguyen, David H; Oketch-Rabah, Hellen A; Illa-Bochaca, Irineu; Geyer, Felipe C; Reis-Filho, Jorge S; Mao, Jian-Hua; Ravani, Shraddha A; Zavadil, Jiri; Borowsky, Alexander D; Jerry, D Joseph; Dunphy, Karen A; Seo, Jae Hong; Haslam, Sandra; Medina, Daniel; Barcellos-Hoff, Mary Helen
2011 May 17;19(5):640-651, Cancer cell
Tissue microenvironment is an important determinant of carcinogenesis. We demonstrate that ionizing radiation, a known carcinogen, affects cancer frequency and characteristics by acting on the microenvironment. Using a mammary chimera model in which an irradiated host is transplanted with oncogenic Trp53 null epithelium, we show accelerated development of aggressive tumors whose molecular signatures were distinct from tumors arising in nonirradiated hosts. Molecular and genetic approaches show that TGFbeta mediated tumor acceleration. Tumor molecular signatures implicated TGFbeta, and genetically reducing TGFbeta abrogated the effect on latency. Surprisingly, tumors from irradiated hosts were predominantly estrogen receptor negative. This effect was TGFbeta independent and linked to mammary stem cell activity. Thus, the irradiated microenvironment affects latency and clinically relevant features of cancer through distinct and unexpected mechanisms
— id: 132592, year: 2011, vol: 19, page: 640, stat: Journal Article,

Multiplicity of Favorable Effects after Transforming Growth Factor-beta Inhibition in 4T1 Murine Mammary Tumors: Clinical Implications
Barcellos-Hoff, M. H.; Bouquet, S. F.; Pilones, K. A.; Demaria, S.; Formenti, S. C.
2010 OCT 13 ;78(3):S648-S648, International journal of radiation oncology biology physics
— id: 114022, year: 2010, vol: 78, page: S648, stat: Journal Article,

Stromal mediation of radiation carcinogenesis
Barcellos-Hoff, Mary Helen
2010 Dec;15(4):381-387, Journal of mammary gland biology & neoplasia
Ionizing radiation is a well-established carcinogen in human breast and rodent mammary gland. This review addresses evidence that radiation elicits the critical stromal context for cancer, affecting not only frequency but the type of cancer. Recent data from the breast tumors of women treated with radiation therapy and the cellular mechanisms evident in experimental models suggest that radiation effects on stromal-epithelial interactions and tissue composition are a major determinant of cancer development
— id: 116242, year: 2010, vol: 15, page: 381, stat: Journal Article,

Spatiotemporal characterization of ionizing radiation induced DNA damage foci and their relation to chromatin organization
Costes, S V; Chiolo, I; Pluth, J M; Barcellos-Hoff, M H; Jakob, B
2010 Apr-Jun;704(1-3):78-87, Mutation research
DNA damage sensing proteins have been shown to localize to the sites of DNA double strand breaks (DSB) within seconds to minutes following ionizing radiation (IR) exposure, resulting in the formation of microscopically visible nuclear domains referred to as radiation-induced foci (RIF). This review characterizes the spatiotemporal properties of RIF at physiological doses, minutes to hours following exposure to ionizing radiation, and it proposes a model describing RIF formation and resolution as a function of radiation quality and chromatin territories. Discussion is limited to RIF formed by three interrelated proteins ATM (Ataxia telangiectasia mutated), 53BP1 (p53 binding protein 1) and gammaH2AX (phosphorylated variant histone H2AX), with an emphasis on the later. This review discusses the importance of not equating RIF with DSB in all situations and shows how dose and time dependence of RIF frequency is inconsistent with a one to one equivalence. Instead, we propose that RIF mark regions of the chromatin that would serve as scaffolds rigid enough to keep broken DNA from diffusing away, but open enough to allow the repair machinery to access the damage site. We review data indicating clear kinetic and physical differences between RIF emerging from dense and uncondensed regions of the nucleus. We suggest that persistent RIF observed days following exposure to ionizing radiation are nuclear marks of permanent rearrangement of the chromatin architecture. Such chromatin alterations may not always lead to growth arrest as cells have been shown to replicate these in progeny. Thus, heritable persistent RIF spanning over tens of Mbp may reflect persistent changes in the transcriptome of a large progeny of cells. Such model opens the door to a 'non-DNA-centric view' of radiation-induced phenotypes
— id: 116247, year: 2010, vol: 704, page: 78, stat: Journal Article,

In situ analysis of cell populations: long-term label-retaining cells
Fernandez-Gonzalez, Rodrigo; Illa-Bochaca, Irineu; Shelton, Dawne N; Welm, Bryan E; Barcellos-Hoff, Mary Helen; Ortiz-de-Solorzano, Carlos
2010 ;621:1-28, Methods in molecular biology
The mammary gland consists of an epithelial ductal tree embedded in a fat pad. Adult mammary epithelium has been demonstrated to have outstanding regenerative potential, consistent with the presence of resident, adult stem cells. However, there are currently no bona fide markers to identify these cells within their tissue context. Here, we introduce long-term label retention as a method to investigate the location of quiescent cells (a property attributed to adult stem cells) in situ. Long-term label retaining cells divide actively during tissue development and remain quiescent at homeostasis. These two properties have been attributed to adult stem cells. Therefore, label-retaining cells can be used to identify populations that contain stem cells. We describe the materials and methods necessary to identify and image mammary label-retaining cells, to carry out morphometric analysis on these cells and to map their distribution of the mammary epithelium. The morphometric and spatial analyses described here are generally applicable to any mammary cell populations, and will therefore be useful to characterize mammary stem cells once bona fide mammary stem cell markers become available
— id: 116244, year: 2010, vol: 621, page: 1, stat: Journal Article,

Multidimensional profiling of cell surface proteins and nuclear markers
Han, Ju; Chang, Hang; Andarawewa, Kumari; Yaswen, Paul; Barcellos-Hoff, Mary Helen; Parvin, Bahram
2010 Jan-Mar;7(1):80-90, IEEE/ACM transactions on computational biology & bioinformatics
Cell membrane proteins play an important role in tissue architecture and cell-cell communication. We hypothesize that segmentation and multidimensional characterization of the distribution of cell membrane proteins, on a cell-by-cell basis, enable improved classification of treatment groups and identify important characteristics that can otherwise be hidden. We have developed a series of computational steps to 1) delineate cell membrane protein signals and associate them with a specific nucleus; 2) compute a coupled representation of the multiplexed DNA content with membrane proteins; 3) rank computed features associated with such a multidimensional representation; 4) visualize selected features for comparative evaluation through heatmaps; and 5) discriminate between treatment groups in an optimal fashion. The novelty of our method is in the segmentation of the membrane signal and the multidimensional representation of phenotypic signature on a cell-by-cell basis. To test the utility of this method, the proposed computational steps were applied to images of cells that have been irradiated with different radiation qualities in the presence and absence of other small molecules. These samples are labeled for their DNA content and E-cadherin membrane proteins. We demonstrate that multidimensional representations of cell-by-cell phenotypes improve predictive and visualization capabilities among different treatment groups, and identify hidden variables
— id: 116245, year: 2010, vol: 7, page: 80, stat: Journal Article,

Limiting-dilution transplantation assays in mammary stem cell studies
Illa-Bochaca, Irineu; Fernandez-Gonzalez, Rodrigo; Shelton, Dawne N; Welm, Bryan E; Ortiz-de-Solorzano, Carlos; Barcellos-Hoff, Mary Helen
2010 ;621:29-47, Methods in molecular biology
Mammary reconstitution assays can be used to measure the stem cell frequency within an epithelial population by transplanting increasingly diluted single-cell preparations of the population of interest. There are fundamental steps in the single-cell isolation protocol which are directly related to the number of single epithelial cells obtained. Once single-cell suspensions have been obtained, serial dilutions are prepared and transplanted into the cleared fat pads of the host mice. After 8-10 weeks, the transplanted fat pads are reevaluated for the presence of epithelial outgrowths. Based on the frequency of no outgrowth for each one of the transplanted dilutions, it is possible to estimate the frequency of mammary repopulating cells present in a given cell population. Here, we give details on how to carry out all these steps
— id: 109217, year: 2010, vol: 621, page: 29, stat: Journal Article,

Promotion of variant human mammary epithelial cell outgrowth by ionizing radiation: an agent-based model supported by in vitro studies
Mukhopadhyay, Rituparna; Costes, Sylvain V; Bazarov, Alexey V; Hines, William C; Barcellos-Hoff, Mary Helen; Yaswen, Paul
2010 ;12(1):R11-R11, Breast cancer research
INTRODUCTION: Most human mammary epithelial cells (HMEC) cultured from histologically normal breast tissues enter a senescent state termed stasis after 5 to 20 population doublings. These senescent cells display increased size, contain senescence associated beta-galactosidase activity, and express cyclin-dependent kinase inhibitor, p16INK4A (CDKN2A; p16). However, HMEC grown in a serum-free medium, spontaneously yield, at low frequency, variant (v) HMEC that are capable of long-term growth and are susceptible to genomic instability. We investigated whether ionizing radiation, which increases breast cancer risk in women, affects the rate of vHMEC outgrowth. METHODS: Pre-stasis HMEC cultures were exposed to 5 to 200 cGy of sparsely (X- or gamma-rays) or densely (1 GeV/amu 56Fe) ionizing radiation. Proliferation (bromodeoxyuridine incorporation), senescence (senescence-associated beta-galactosidase activity), and p16 expression were assayed in subcultured irradiated or unirradiated populations four to six weeks following radiation exposure, when patches of vHMEC became apparent. Long-term growth potential and p16 promoter methylation in subsequent passages were also monitored. Agent-based modeling, incorporating a simple set of rules and underlying assumptions, was used to simulate vHMEC outgrowth and evaluate mechanistic hypotheses. RESULTS: Cultures derived from irradiated cells contained significantly more vHMEC, lacking senescence associated beta-galactosidase or p16 expression, than cultures derived from unirradiated cells. As expected, post-stasis vHMEC cultures derived from both unirradiated and irradiated cells exhibited more extensive methylation of the p16 gene than pre-stasis HMEC cultures. However, the extent of methylation of individual CpG sites in vHMEC samples did not correlate with passage number or treatment. Exposure to sparsely or densely ionizing radiation elicited similar increases in the numbers of vHMEC compared to unirradiated controls. Agent-based modeling indicated that radiation-induced premature senescence of normal HMEC most likely accelerated vHMEC outgrowth through alleviation of spatial constraints. Subsequent experiments using defined co-cultures of vHMEC and senescent cells supported this mechanism. CONCLUSIONS: Our studies indicate that ionizing radiation can promote the outgrowth of epigenetically altered cells with pre-malignant potential
— id: 116246, year: 2010, vol: 12, page: R11, stat: Journal Article,

Use of stem cell markers in dissociated mammary populations
Shelton, Dawne N; Fernandez-Gonzalez, Rodrigo; Illa-Bochaca, Irineu; Ortiz-de-Solorzano, Carlos; Barcellos-Hoff, Mary Helen; Welm, Bryan E
2010 ;621:49-55, Methods in molecular biology
The regenerative potential of mammary epithelium facilitates assessment of the 'stemness' of any epithelial subpopulation in transplantation assays. Thus, mammary tissue can be dissociated into single cells, stained for cell surface markers of interest and classified using fluorescence-activated cell sorting. The selected cells can then be transplanted into epithelium-devoided fat pads from recipient hosts. Recent publications have described markers that enrich for mammary repopulating potential. Here, we describe the materials and methods necessary to sort cells according to these markers. This approach can be used interchangeably with other cell surface markers with slight variation to the protocol
— id: 116243, year: 2010, vol: 621, page: 49, stat: Journal Article,

Transforming growth factor-beta in breast cancer: too much, too late
Barcellos-Hoff, Mary Helen; Akhurst, Rosemary J
2009 ;11(1):202-202, Breast cancer research
The contribution of transforming growth factor (TGF)beta to breast cancer has been studied from a myriad perspectives since seminal studies more than two decades ago. Although the action of TGFbeta as a canonical tumor suppressor in breast is without a doubt, there is compelling evidence that TGFbeta is frequently subverted in a malignant plexus that drives breast cancer. New knowledge that TGFbeta regulates the DNA damage response, which underlies cancer therapy, reveals another facet of TGFbeta biology that impedes cancer control. Too much TGFbeta, too late in cancer progression is the fundamental motivation for pharmaceutical inhibition
— id: 99208, year: 2009, vol: 11, page: 202, stat: Journal Article,

Integrating biology and technology
Barcellos-Hoff, Mary Helen; Beebe, David
2009 Jan;1(1):14-14, Integrative Biology
— id: 116250, year: 2009, vol: 1, page: 14, stat: Journal Article,

Editorial: Resistance to radio- and chemotherapy and the tumour microenvironment
Barcellos-Hoff, Mary Helen; Cordes, Nils
2009 Nov;85(11):920-922, International journal of radiation biology
— id: 105535, year: 2009, vol: 85, page: 920, stat: Journal Article,

Therapeutic targets in malignant glioblastoma microenvironment
Barcellos-Hoff, Mary Helen; Newcomb, Elizabeth W; Zagzag, David; Narayana, Ashwatha
2009 Jul;19(3):163-170, Seminars in radiation oncology
There is considerable evidence that the tissue microenvironment can suppress cancer and that microenvironment disruption is required for cancer growth and progression. Distortion of the microenvironment by tumor cells can promote growth, recruit nonmalignant cells that provide physiological resources, and facilitate invasion. Compared with the variable routes taken by cells to become cancers, the response of normal tissue to cancer is relatively consistent such that controlling cancer may be more readily achieved indirectly via the microenvironment. Here, we discuss 3 ideas about how the microenvironment, consisting of a vasculature, inflammatory cells, immune cells, growth factors, and extracellular matrix, might provide therapeutic targets in glioblastoma (GBM) in the context of radiotherapy (RT): (1) viable therapeutic targets exist in the GBM microenvironment, (2) RT alters the microenvironment of tissues and tumors; and (3) a potential benefit may be achieved by targeting the microenvironments induced by RT
— id: 99219, year: 2009, vol: 19, page: 163, stat: Journal Article,

Radiation carcinogenesis in context: how do irradiated tissues become tumors?
Barcellos-Hoff, MH; Nguyen, DH
2009 ;97(5):446-457, Health physics
It is clear from experimental studies that genotype is an important determinant of cancer susceptibility in general, and for radiation carcinogenesis specifically. It has become increasingly clear that genotype influences not only the ability to cope with DNA damage but also influences the cooperation of other tissues, like the vasculature and immune system, necessary for the establishment of cancer. Our experimental data and that of others suggest that the carcinogenic action of ionizing radiation (IR) can also be considered a two-compartment problem: while IR can alter genomic sequence as a result of DNA damage, it can also induce signals that alter multicellular interactions and phenotypes that underpin carcinogenesis. Rather than being accessory or secondary to genetic damage, we propose that such non-targeted radiation effects create the critical context that promotes cancer development. This review focuses on experimental studies that clearly define molecular mechanisms by which cell interactions contribute to cancer in different organs, and addresses how non-targeted radiation effects may similarly act though the microenvironment. The definition of non-targeted radiation effects and their dose dependence could modify the current paradigms for radiation risk assessment since radiation non-targeted effects, unlike DNA damage, are amenable to intervention. The implications of this perspective in terms of reducing cancer risk after exposure are discussed. $$:
— id: 104662, year: 2009, vol: 97, page: 446, stat: Journal Article,

The development of integrative biology: bridging the gap--a view from the Scientific Editors. [An interview with David Beebe and Mary Helen Barcellos-Hoff by Kathleen Too]
Beebe, David; Barcellos-Hoff, Mary Helen
2009 Feb;1(2):145-147, Integrative Biology
— id: 116248, year: 2009, vol: 1, page: 145, stat: Journal Article,

Mapping mammary gland architecture using multi-scale in situ analysis
Fernandez-Gonzalez, Rodrigo; Illa-Bochaca, Irineu; Welm, Bryan E; Fleisch, Markus C; Werb, Zena; Ortiz-de-Solorzano, Carlos; Barcellos-Hoff, Mary Helen
2009 Jan;1(1):80-89, Integrative Biology
We have built a novel computational microscopy platform that integrates image acquisition, storage, processing and analysis to study cell populations in situ. This platform allows high-content studies where multiple features are measured and linked at multiple scales. We used this approach to study the cellular composition and architecture of the mouse mammary gland by quantitatively tracking the distribution and type, position, proliferative state, and hormone receptor status of epithelial cells that incorporated bromodeoxyuridine while undergoing DNA synthesis during puberty and retained this label in the adult gland as a function of tissue structure. Immunofluorescence was used to identify label-retaining cells, as well as epithelial cells expressing the proteins progesterone receptor and P63. Only 3.6% of luminal cells were label-retaining cells, the majority of which did not express the progesterone receptor. Multi-scale in situ analysis revealed that luminal label-retaining cells have a distinct nuclear morphology, are enriched 3.4-fold in large ducts, and are distributed asymmetrically across the tissue. We postulated that LRC enriched in the ventral mammary gland represent progenitor cells. Epithelial cells isolated from the ventral versus the dorsal portion of the gland were enriched for the putative stem cell markers CD24 and CD49f as measured by fluorescence activated cell sorting. Thus, quantitative analysis of the cellular composition of the mammary epithelium across spatial scales identified a previously unrecognized architecture in which the ventral-most, large ducts contain a reservoir of undifferentiated, putative stem cells
— id: 116249, year: 2009, vol: 1, page: 80, stat: Journal Article,

Host biology affects the frequency of ER plus breast cancer
Nguyen, David; Oketch-Rabah, Hellen; Barcellos-Hoff, Mary Helen
2009 ;50(Suppl. S):577-577, Proceedings (American Association for Cancer Research)
— id: 104661, year: 2009, vol: 50, page: 577, stat: Journal Article,

Estrogen and progesterone receptors have distinct roles in the establishment of the hyperplastic phenotype in PR-A transgenic mice
Simian, Marina; Bissell, Mina J; Barcellos-Hoff, Mary Helen; Shyamala, Gopalan
2009 Sep 29;11(5):R72-R72, Breast cancer research
ABSTRACT: INTRODUCTION: Expression of the A and B forms of progesterone receptor (PR) in an appropriate ratio is critical for mammary development. Mammary glands of PR-A transgenic mice, carrying an additional A form of PR as a transgene, exhibit morphological features associated with the development of mammary tumors. Our objective was to determine the roles of estrogen (E) and progesterone (P) in the genesis of mammary hyperplasias/preneoplasias in PR-A transgenics. METHODS: We subjected PR-A mice to hormonal treatments and analyzed mammary glands for the presence of hyperplasias and used BrdU incorporation to measure proliferation. Quantitative image analysis was carried out to compare levels of latency-associated peptide and transforming growth factor beta 1 (TGFbeta1) between PR-A and PR-B transgenics. Basement membrane disruption was examined by immunofluorescence and proteolytic activity by zymography. RESULTS: The hyperplastic phenotype of PR-A transgenics is inhibited by ovariectomy, and is reversed by treatment with E + P. Studies using the antiestrogen ICI 182,780 or antiprogestins RU486 or ZK 98,299 show that the increase in proliferation requires signaling through E/estrogen receptor alpha but is not sufficient to give rise to hyperplasias, whereas signaling through P/PR has little impact on proliferation but is essential for the manifestation of hyperplasias. Increased proliferation is correlated with decreased TGFbeta1 activation in the PR-A transgenics. Analysis of basement membrane integrity showed loss of laminin-5, collagen III and collagen IV in mammary glands of PR-A mice, which is restored by ovariectomy. Examination of matrix metalloproteases (MMPs) showed that total levels of MMP-2 correlate with the steady-state levels of PR, and that areas of laminin-5 loss coincide with those of activation of MMP-2 in PR-A transgenics. Activation of MMP-2 is dependent on treatment with E and P in ovariectomized wild-type mice, but is achieved only by treatment with P in PR-A mice. CONCLUSIONS: These data establish a link between hormonal response, proliferation, modulation of MMP activity and maintenance of basement membrane integrity that depend on a balance in the expression levels of PR-A and PR-B isoforms. Notably, concomitant increased proliferation, due to inhibition of TGFbeta1 activation, and loss of basement membrane integrity, via increased MMP-2 activity, appear to be prerequisites for the PR-A hyperplastic phenotype
— id: 105536, year: 2009, vol: 11, page: R72, stat: Journal Article,

Persistent phenotypic responses of human mammary epithelial cells induced by ionizing radiation
Andarawewa K.L.; Chou W.S.; Costes S.V.; Barcellos-Hoff M.H.
2008 ;53(SUPPL.):61-63, Acta Medica Nagasakiensia
Ionizing radiation (IR) is a known human breast carcinogen. Although the mutagenic capacity of IR is widely acknowledged as the basis for its action as a carcinogen, we and others have shown that IR can also induce growth factors and extracellular matrix remodeling. We have shown that irradiating human mammary epithelial cells (HMEC) cultured wilh transforming growth factor beta1 (TGFbeta) can generate a persistent phenotype in daughter cells characterized by spindle cell morphology, increased mesenchymal markers, decreased epithelial markers and increased cellular motility and invasion, which are hallmarks of epithelial to mesenchymal transition (EMT). Neither radiation nor TGFbeta alone elicited EMT, although IR increased chronic TGFbeta signaling and activity. Gene expression profiling revealed that double-treated cells exhibit a specific 10-gene signature associated with Erk/MAPK signaling. We hypothesized that IR-induced MAPK activation primes nonmalignant HMEC to undergo TGFbeta-mediated EMT. Consistent with this Erk phosphorylation was transiently induced by irradiation and persisted in irradiated cells treated with TGFbeta, and inhibition of Erk activation, blocked the EMT phenotype. Preliminary studies suggest that eqi-toxic doses of sparsely and densely ionizing radiation resulted incomparable EMT when cells were cultivated in the presence of TGFbeta. Furthermore radiation dose response studies show that this effect has a very low threshold in that a single exposure of 3-200 cGy radiation elicits the 'same' phenotypic switch, which is consistent with non-targeted effects. Together, these data show that the interactions between radiation-induced signaling pathways elicit heritable phenotypes that could contribute to radiation carcinogenesis
— id: 99261, year: 2008, vol: 53, page: 61, stat: Journal Article,

TGF beta: Roles in DNA damage responses
Andarawewa, Kumari L.; Kirshner, Julia; Mott, Joni D.; Barcellos-Hoff, Mary Helen
Transforming growth factor-[beta] in cancer therapy Totowa, N.J. : Humana Press, 2008,
This review focuses on an emerging role for TGF beta in DNA damage and repair and how this function may contribute to its role as an epithelial tumor suppressor. TGF beta is rapidly and widely activated in response to ionizing radiation (IR). IR is a carcinogen, a mode of cancer therapy, and a probe used by biologists to understand how cells and organisms deal with DNA damage. Thus understanding how TGF beta participates in DNA damage response has therapeutic implications. Our data suggest that TGF beta 1, in addition to its role in homeostatic growth control, plays a more complex role in regulating tissue response to damage, the failure of which would contribute to the development of cancer. Future studies are likely to uncover evidence of how TGF beta may act to integrate signaling across multiple scales of cell organization, underlining the broadening role of TGF beta as a fulcrum between physiology and pathology
— id: 5234, year: 2008, vol: , page: 321, stat: Chapter,

Bioavailability of TGF-beta in breast cancer
Barcellos-Hoff, Mary H; Bochaca, Irineu I
Ft. Belvoir VA : Defense Technical Information Center, 2008,
The Transforming Growth Factor beta (TGF-) superfamily includes three isoforms designated TGF-1, 2 and 3. All three isoforms are secreted as latent complex where the TGF- cytokine is non-covalently associated with an isoform specific latency-associated peptide (LAP). Mature cytokine binds cell surface receptors only after release from its LAP making extracellular activation a critical regulatory point for TGF- bioavailability. Proposed activation mechanisms include proteolysis and conformational changes. Previous work from our laboratory showed that latent TGF-1 (LTGF-1) is efficiently activated upon exposure to reactive oxygen species (ROS). ROS activation is restricted to the LTGF-1 isoform. Because of the amino acid sequence differences between the three LAPs, we postulate that the specificity of this activation mechanism lies within the LAP. Furthermore, we hypothesize that the presence of a metal in the latent complex could provide a redox active center for this process. Redox mediated activation provides a novel mechanism for TGF- participation in tissues undergoing oxidative stress. Moreover, this would allow TGF-1 to act both as a sensor of oxidative stress within tissues as well as a transducer of that signal by binding to its cellular receptors
— id: 2092, year: 2008, vol: , page: , stat: ,

Cancer as an emergent phenomenon in systems radiation biology
Barcellos-Hoff, Mary Helen
2008 Feb;47(1):33-38, Radiation & environmental biophysics
Radiation-induced DNA damage elicits dramatic cell signaling transitions, some of which are directed towards deciding the fate of that particular cell, while others lead to signaling to other cells. Each irradiated cell type and tissue has a characteristic pattern of radiation-induced gene expression, distinct from that of the unirradiated tissue and different from that of other irradiated tissues. It is the sum of such events, highly modulated by genotype that sometimes leads to cancer. The challenge is to determine as to which of these phenomena have persistent effect that should be incorporated into models of how radiation increases the risk of developing cancer. The application of systems biology to radiation effects may help to identify which biological responses are significant players in radiation carcinogenesis. In contrast to the radiation biology paradigm that focuses on genomic changes, systems biology seeks to integrate responses at multiple scales (e.g. molecular, cellular, organ, and organism). A key property of a system is that some phenomenon emerges as a property of the system rather than of the parts. Here, the idea that cancer in an organism can be considered as an emergent phenomenon of a perturbed system is discussed. Given the current research goal to determine the consequences of high and low radiation exposures, broadening the scope of radiation studies to include systems biology concepts should benefit risk modeling of radiation carcinogenesis
— id: 83249, year: 2008, vol: 47, page: 33, stat: Journal Article,

The genomic differences in radiated breast - Epithelial cells is centrosome given - Implication for the increased risk during continually radiated breast cancer
Fleisch, MC; Maxwell, CA; Costes, SV; Barcellos-Hoff, MH
2008 ;68(20):S104-S104, Geburtshilfe & Frauenheilkunde
— id: 104664, year: 2008, vol: 68, page: S104, stat: Journal Article,

Integrated profiling of cell surface protein and nuclear marker for discriminant analysis
Ju Han; Hang Chang; Andarawewa, K.; Yaswen, P.; Barcellos-Hoff, M.H.; Parvin, B.J.
2008 5th IEEE International Symposium on Biomedical Imaging from nano to macro : proceedings [Piscataway, N.J.] : IEEE, 2008,
Cell membrane proteins play an important role in tissue architecture and cell-cell communication. We hypothesize that segmentation and multivariate characterization of the distribution of cell membrane proteins, on a cell-cell basis, enable improved classification of treatment groups and identify important characteristics that can otherwise be hidden. We have developed a series of computational steps to (i) delineate cell membrane protein signals and associate them with specific nuclei, (ii) compute a coupled representation of the multiplexed DNA content with membrane proteins and other end points, (iii) evaluate computed features associated with such a multivariate representation, and (iv) discriminate between treatment groups in an optimal fashion. The novelty of our method is in the segmentation of the membrane signal and the multivariate representation of phenotypes on a cell-cell basis. To test the utility of the new method, the proposed computational steps were applied to images of cells that have been irradiated with different radiation qualities in the presence and absence of TGF beta . These samples are labeled for their DNA content and E-cadherin membrane protein. We demonstrate that multivariate representation of cell-cell phenotypes improves predictive and visualization capabilities among different treatment groups, and increases quantitative sensitivity of cellular responses
— id: 5225, year: 2008, vol: , page: 1343, stat: Chapter,

Targeted and nontargeted effects of ionizing radiation that impact genomic instability
Maxwell, Christopher A; Fleisch, Markus C; Costes, Sylvain V; Erickson, Anna C; Boissiere, Arnaud; Gupta, Rishi; Ravani, Shraddha A; Parvin, Bahram; Barcellos-Hoff, Mary Helen
2008 Oct 15;68(20):8304-8311, Cancer research
Radiation-induced genomic instability, in which the progeny of irradiated cells display a high frequency of nonclonal genomic damage, occurs at a frequency inconsistent with mutation. We investigated the mechanism of this nontargeted effect in human mammary epithelial cells (HMEC) exposed to low doses of radiation. We identified a centrosome-associated expression signature in irradiated HMEC and show here that centrosome deregulation occurs in the first cell cycle after irradiation, is dose dependent, and that viable daughters of these cells are genomically unstable as evidenced by spontaneous DNA damage, tetraploidy, and aneuploidy. Clonal analysis of genomic instability showed a threshold of >10 cGy. Treatment with transforming growth factor beta1 (TGFbeta), which is implicated in regulation of genomic stability and is activated by radiation, reduced both the centrosome expression signature and centrosome aberrations in irradiated HMEC. Furthermore, TGFbeta inhibition significantly increased centrosome aberration frequency, tetraploidy, and aneuploidy in nonirradiated HMEC. Rather than preventing radiation-induced or spontaneous centrosome aberrations, TGFbeta selectively deleted unstable cells via p53-dependent apoptosis. Together, these studies show that radiation deregulates centrosome stability, which underlies genomic instability in normal human epithelial cells, and that this can be opposed by radiation-induced TGFbeta signaling
— id: 93861, year: 2008, vol: 68, page: 8304, stat: Journal Article,

Inhibition of transforming growth factor betal (TGF beta 1) signaling increases radiosensitivity in breast cancer cell lines
Pal, Anupama; Gascard, Philippe D.; Ravani, Shraddha A.; Barcellos-Hoff, Mary Helen
2008 ;49(1):98-98, Proceedings (American Association for Cancer Research)
— id: 104663, year: 2008, vol: 49, page: 98, stat: Journal Article,

Karyotypic instability and centrosome aberrations in the progeny of finite life-span human mammary epithelial cells exposed to sparsely or densely ionizing radiation
Sudo, Hiroko; Garbe, James; Stampfer, Martha R; Barcellos-Hoff, Mary Helen; Kronenberg, Amy
2008 Jul;170(1):23-32, Radiation research
The human breast is sensitive to radiation carcinogenesis, and genomic instability occurs early in breast cancer development. This study tests the hypothesis that ionizing radiation elicits genomic instability in finite life-span human mammary epithelial cells (HMEC) and asks whether densely ionizing radiation is a more potent inducer of instability. HMEC in a non-proliferative state were exposed to X rays or 1 GeV/nucleon iron ions followed by delayed plating. Karyotypic instability and centrosome aberrations were monitored in expanded clonal isolates. Severe karyotypic instability was common in the progeny of cells that survived X-ray or iron-ion exposure. There was a lower dose threshold for severe karyotypic instability after iron-ion exposure. More than 90% of X-irradiated colonies and >60% of iron-ion-irradiated colonies showed supernumerary centrosomes at levels above the 95% upper confidence limit of the mean for unirradiated clones. A dose response was observed for centrosome aberrations for each radiation type. There was a statistically significant association between the incidence of karyotypic instability and supernumerary centrosomes for iron-ion-exposed colonies and a weaker association for X-irradiated colonies. Thus genomic instability occurs frequently in finite life-span HMEC exposed to sparsely or densely ionizing radiation and may contribute to radiation-induced breast cancer
— id: 83266, year: 2008, vol: 170, page: 23, stat: Journal Article,

Genomic approaches to breast cancer subset identification and treatment
Albertson, D; Chin, K; Devries, S; Feiler, H; Pinkel, D; Spellman, P; Waldman, F; Wang, N; Hennessy, B; Mills, G; Barcellos, HMH; Bissell, M; Guan, Y; Hu, Z; Kuo, WL; McCormick, F; Neve, R; Stampfer, M; Wooster, R; Yaswen, P; Das, D; Fridlyand, J; Correll, E; Jin, J; Nordmeyer, B; Sudar, D; Chew, K; Dairkee, S; Ljung, BM; Hwang, S; Esserman, L; Arbushites, M; Benz, C; Koehler, M; Marks, JD; Zhou, Y; Park, J; Weber, B; Gray, J
2007 ;106(11-12):S10-S10, Breast cancer research & treatment
— id: 104667, year: 2007, vol: 106, page: S10, stat: Journal Article,

Ionizing radiation induces TGF beta mediated epithelial to mesenchy mal transition through persistent activation of Erk;
Andarawewa, Kumari L.; Erickson, Anna C.; Chou, William S.; Mott, Joni D.; Gascard, Pilippe; Barcellos-Hoff, Mary Helen
2007 ;48(20):1013-1013, Proceedings (American Association for Cancer Research)
— id: 104665, year: 2007, vol: 48, page: 1013, stat: Journal Article,

Ionizing radiation predisposes nonmalignant human mammary epithelial cells to undergo transforming growth factor beta induced epithelial to mesenchymal transition
Andarawewa, Kumari L; Erickson, Anna C; Chou, William S; Costes, Sylvain V; Gascard, Philippe; Mott, Joni D; Bissell, Mina J; Barcellos-Hoff, Mary Helen
2007 Sep 15;67(18):8662-8670, Cancer research
Transforming growth factor beta1 (TGFbeta) is a tumor suppressor during the initial stage of tumorigenesis, but it can switch to a tumor promoter during neoplastic progression. Ionizing radiation (IR), both a carcinogen and a therapeutic agent, induces TGFbeta activation in vivo. We now show that IR sensitizes human mammary epithelial cells (HMEC) to undergo TGFbeta-mediated epithelial to mesenchymal transition (EMT). Nonmalignant HMEC (MCF10A, HMT3522 S1, and 184v) were irradiated with 2 Gy shortly after attachment in monolayer culture or treated with a low concentration of TGFbeta (0.4 ng/mL) or double treated. All double-treated (IR + TGFbeta) HMEC underwent a morphologic shift from cuboidal to spindle shaped. This phenotype was accompanied by a decreased expression of epithelial markers E-cadherin, beta-catenin, and ZO-1, remodeling of the actin cytoskeleton, and increased expression of mesenchymal markers N-cadherin, fibronectin, and vimentin. Furthermore, double treatment increased cell motility, promoted invasion, and disrupted acinar morphogenesis of cells subsequently plated in Matrigel. Neither radiation nor TGFbeta alone elicited EMT, although IR increased chronic TGFbeta signaling and activity. Gene expression profiling revealed that double-treated cells exhibit a specific 10-gene signature associated with Erk/mitogen-activated protein kinase (MAPK) signaling. We hypothesized that IR-induced MAPK activation primes nonmalignant HMEC to undergo TGFbeta-mediated EMT. Consistent with this, Erk phosphorylation was transiently induced by irradiation and persisted in irradiated cells treated with TGFbeta, and treatment with U0126, a MAP/Erk kinase (MEK) inhibitor, blocked the EMT phenotype. Together, these data show that the interactions between radiation-induced signaling pathways elicit heritable phenotypes that could contribute to neoplastic progression
— id: 83248, year: 2007, vol: 67, page: 8662, stat: Journal Article,

New rationales for using TGFbeta inhibitors in radiotherapy
Andarawewa, Kumari L; Paupert, Jenny; Pal, Anupama; Barcellos-Hoff, Mary Helen
2007 Nov-Dec;83(11-12):803-811, International journal of radiation biology
PURPOSE: The first reports that ionizing radiation (IR) induces rapid and persistent activation of transforming growth factor beta1 (TGFbeta) were nearly two decades ago. Subsequent studies have shown that TGFbeta is a major mediator of cellular and tissue responses to IR and have revealed novel facets of its complex biology. RESULTS: We and others have recently shown that inhibition of production or signaling of TGFbeta in epithelial cells modulates radiosensitivity and impedes activation of the DNA damage response program. The primary transducer of cellular response to DNA damage caused by ionizing radiation is the nuclear protein kinase ataxia telangiectasia mutated, whose activity is severely compromised when TGFbeta is inhibited. Thus, in conjunction, with its well-recognized contribution to normal tissue fibrosis, the role of TGFbeta in the genotoxic stress program provides a previously unsuspected avenue to modulate radiotherapy. CONCLUSIONS: We hypothesize that identification of the circumstances and tumors in which TGFbeta manipulation enhances tumor cell radiosensitivity, while protecting normal tissues, could significantly increase therapeutic index
— id: 83251, year: 2007, vol: 83, page: 803, stat: Journal Article,

Radiation therapy and the microenvironment
Barcellos-Hoff, Mary Helen; Cordes, Nils
2007 Nov-Dec;83(11-12):723-725, International journal of radiation biology
— id: 83250, year: 2007, vol: 83, page: 723, stat: Journal Article,

Perceptual grouping of membrane signals in cell-based assays
Chang, H.; Andarawewa, K.L.; Han, J.; Barcellos-Hoff, M.H.; Parvin, B.
Piscataway, N.J. : IEEE, 2007,
Membrane proteins organize themselves in a linear fashion where adjacent cells are attached together along the basal-lateral region. Their intensity distributions are often heterogeneous and may lack specificity. Grouping of these linear structures can aid in segmentation and quantitative representation of protein localization. However, quantitative analysis of these signals is often hindered by noise, variation in scale, and perceptual features. This paper introduces an iterative voting method for inferring the membrane signal as it relates to continuity. A unique aspect of this technique is in the topography of the voting kernel, which is refined and reoriented iteratively. The technique can cluster and group membrane signals along the tangential direction. It has an excellent noise immunity and is tolerant to perturbations in scale. Application of this technique to quantitative analysis of cell-cell adhesion mediated by integral cell membrane proteins is demonstr
— id: 5226, year: 2007, vol: , page: 532, stat: Chapter,

Image-based modeling reveals dynamic redistribution of DNA damage into nuclear sub-domains
Costes, Sylvain V; Ponomarev, Artem; Chen, James L; Nguyen, David; Cucinotta, Francis A; Barcellos-Hoff, Mary Helen
2007 Aug;3(8):e155-e155, PLoS Computational Biology
Several proteins involved in the response to DNA double strand breaks (DSB) form microscopically visible nuclear domains, or foci, after exposure to ionizing radiation. Radiation-induced foci (RIF) are believed to be located where DNA damage occurs. To test this assumption, we analyzed the spatial distribution of 53BP1, phosphorylated ATM, and gammaH2AX RIF in cells irradiated with high linear energy transfer (LET) radiation and low LET. Since energy is randomly deposited along high-LET particle paths, RIF along these paths should also be randomly distributed. The probability to induce DSB can be derived from DNA fragment data measured experimentally by pulsed-field gel electrophoresis. We used this probability in Monte Carlo simulations to predict DSB locations in synthetic nuclei geometrically described by a complete set of human chromosomes, taking into account microscope optics from real experiments. As expected, simulations produced DNA-weighted random (Poisson) distributions. In contrast, the distributions of RIF obtained as early as 5 min after exposure to high LET (1 GeV/amu Fe) were non-random. This deviation from the expected DNA-weighted random pattern can be further characterized by 'relative DNA image measurements.' This novel imaging approach shows that RIF were located preferentially at the interface between high and low DNA density regions, and were more frequent than predicted in regions with lower DNA density. The same preferential nuclear location was also measured for RIF induced by 1 Gy of low-LET radiation. This deviation from random behavior was evident only 5 min after irradiation for phosphorylated ATM RIF, while gammaH2AX and 53BP1 RIF showed pronounced deviations up to 30 min after exposure. These data suggest that DNA damage-induced foci are restricted to certain regions of the nucleus of human epithelial cells. It is possible that DNA lesions are collected in these nuclear sub-domains for more efficient repair
— id: 83246, year: 2007, vol: 3, page: e155, stat: Journal Article,

Segmentation of mammosphere structures from volumetric data
Ju Han; Hang Chang; Qing Yang; Barcellos-Hoff , M.H.; Parvin, B.
2007 4th IEEE International Symposium on Biomedical Imaging from nano to macro : proceedings Piscataway, N.J. : IEEE, 2007,
3D cell culture assays have emerged as the basis of an improved model system for evaluating therapeutic agents, molecular probes, and exogenous stimuli. However, there is a gap in robust computational techniques for segmentation of image data that are collected through confocal or deconvolution microscopy. The main issue is the volume of data, overlapping subcellular compartments, and variation in scale and size of subcompartments of interest. A geometric technique has been developed to bound the solution of the problem by first localizing centers of mass for each cell and then partitioning clumps of cells along minimal intersecting surfaces. An approximate solution to the center of mass is realized through iterative spatial voting, which is tolerant to variation in shape morphologies and overlapping compartments and is shown to have an excellent noise immunity. These approximate estimates to centers of mass are then used to partition a clump of cells along minimal intersecting surfaces that are estimated by Radon transform. Examples on real data and performance of the system over a large population of data are evaluated. Furthermore, it is shown that the proposed methodology is extensible in terms of its application to protein localization studies
— id: 5227, year: 2007, vol: , page: 524, stat: Chapter,

The morphologies of breast cancer cell lines in three-dimensional assays correlate with their profiles of gene expression
Kenny, Paraic A; Lee, Genee Y; Myers, Connie A; Neve, Richard M; Semeiks, Jeremy R; Spellman, Paul T; Lorenz, Katrin; Lee, Eva H; Barcellos-Hoff, Mary Helen; Petersen, Ole W; Gray, Joe W; Bissell, Mina J
2007 Jun;1(1):84-96, Molecular oncology
3D cell cultures are rapidly becoming the method of choice for the physiologically relevant modeling of many aspects of non-malignant and malignant cell behavior ex vivo. Nevertheless, only a limited number of distinct cell types have been evaluated in this assay to date. Here we report the first large scale comparison of the transcriptional profiles and 3D cell culture phenotypes of a substantial panel of human breast cancer cell lines. Each cell line adopts a colony morphology of one of four main classes in 3D culture. These morphologies reflect, at least in part, the underlying gene expression profile and protein expression patterns of the cell lines, and distinct morphologies were also associated with tumor cell invasiveness and with cell lines originating from metastases. We further demonstrate that consistent differences in genes encoding signal transduction proteins emerge when even tumor cells are cultured in 3D microenvironments
— id: 83265, year: 2007, vol: 1, page: 84, stat: Journal Article,

Response of a human breast cell line to transforming growth factor-beta (TGF-beta) is context specific and involves components of TGF-beta and the epidermal growth factor receptor signaling pathways
Mott, Joni D.; Barcellos-Hoff, Mary Helen
2007 ;48(18):896-896, Proceedings (American Association for Cancer Research)
— id: 104666, year: 2007, vol: 48, page: 896, stat: Journal Article,

Iterative voting for inference of structural saliency and characterization of subcellular events
Parvin, Bahram; Yang, Qing; Han, Ju; Chang, Hang; Rydberg, Bjorn; Barcellos-Hoff, Mary Helen
2007 Mar;16(3):615-623, IEEE transactions on image processing
Saliency is an important perceptual cue that occurs at different levels of resolution. Important attributes of saliency are symmetry, continuity, and closure. Detection of these attributes is often hindered by noise, variation in scale, and incomplete information. This paper introduces the iterative voting method, which uses oriented kernels for inferring saliency as it relates to symmetry. A unique aspect of the technique is the kernel topography, which is refined and reoriented iteratively. The technique can cluster and group nonconvex perceptual circular symmetries along the radial line of an object's shape. It has an excellent noise immunity and is shown to be tolerant to perturbation in scale. The application of this technique to images obtained through various modes of microscopy is demonstrated. Furthermore, as a case example, the method has been applied to quantify kinetics of nuclear foci formation that are formed by phosphorylation of histone gammaH2AX following ionizing radiation. Iterative voting has been implemented in both 2-D and 3-D for multi image analysis
— id: 83237, year: 2007, vol: 16, page: 615, stat: Journal Article,

Geometric approach to segmentation and protein localization in cell culture assays
Raman, S; Maxwell, C A; Barcellos-Hoff, M H; Parvin, B
2007 Jan;225(Pt 1):22-30, Journal of microscopy
Cell-based fluorescence imaging assays are heterogeneous and require the collection of a large number of images for detailed quantitative analysis. Complexities arise as a result of variation in spatial nonuniformity, shape, overlapping compartments and scale (size). A new technique and methodology has been developed and tested for delineating subcellular morphology and partitioning overlapping compartments at multiple scales. This system is packaged as an integrated software platform for quantifying images that are obtained through fluorescence microscopy. Proposed methods are model based, leveraging geometric shape properties of subcellular compartments and corresponding protein localization. From the morphological perspective, convexity constraint is imposed to delineate and partition nuclear compartments. From the protein localization perspective, radial symmetry is imposed to localize punctate protein events at submicron resolution. Convexity constraint is imposed against boundary information, which are extracted through a combination of zero-crossing and gradient operator. If the convexity constraint fails for the boundary then positive curvature maxima are localized along the contour and the entire blob is partitioned into disjointed convex objects representing individual nuclear compartment, by enforcing geometric constraints. Nuclear compartments provide the context for protein localization, which may be diffuse or punctate. Punctate signal are localized through iterative voting and radial symmetries for improved reliability and robustness. The technique has been tested against 196 images that were generated to study centrosome abnormalities. Corresponding computed representations are compared against manual counts for validation
— id: 83232, year: 2007, vol: 225, page: 22, stat: Journal Article,

Persistent activation of Erk in the progeny of irradiated human mammary epithelial cells during TGF beta-induced epithelial to mesenchymal transition
Andarawewa, Kumari L.; Erickson, Anna C.; Chou, William S.; Barcellos-Hoff, Mary Helen
2006 ;47(1):651-651, Proceedings (American Association for Cancer Research)
— id: 104668, year: 2006, vol: 47, page: 651, stat: Journal Article,

A systems biology approach to multicellular and multi-generational radiation responses
Barcellos-Hoff, Mary Helen; Costes, Sylvain V
2006 May 11;597(1-2):32-38, Mutation research
Recent studies have highlighted crosstalk between irradiated cells and non-irradiated bystander cells and have uncovered high-frequency phenotypes of genomic instability in the progeny of irradiated cells that cannot be solely explained by radiation-induced mutation. It is difficult to explain these multicellular and multi-generational phenomena using the current paradigm of radiation biology. Radiation-induced bystander effect is a type of multicellular response to radiation that illustrates that the unit of function in multicellular organisms is neither the genome nor the cell. Cell function in complex three-dimensional tissues is coordinated by soluble signaling peptides and by small molecules within the context of insoluble scaffolding provided by the extracellular matrix. Adaptive response and radiation-induced genomic instability could thus result from persistent signaling perturbations following radiation exposures. A model of radiation response based on the systems biology principles of network interconnectivity and spatial organization should reconcile the apparent contradiction of these cellular phenotypes within the higher order structure of tissues and organisms
— id: 83213, year: 2006, vol: 597, page: 32, stat: Journal Article,

Multicellular and multigenerational responses to TGF beta
Barcellos-Hoff, MH
2006 ;27(1-2):32-32, Tumour biology
— id: 104669, year: 2006, vol: 27, page: 32, stat: Journal Article,

Imaging features that discriminate between foci induced by high- and low-LET radiation in human fibroblasts
Costes, Sylvain V; Boissiere, Arnaud; Ravani, Shraddha; Romano, Raquel; Parvin, Bahram; Barcellos-Hoff, Mary Helen
2006 May;165(5):505-515, Radiation research
In this study, we investigated the formation of radiation-induced foci in normal human fibroblasts exposed to X rays or 130 keV/mum nitrogen ions using antibodies to phosphorylated protein kinase ataxia telangiectasia mutated (ATMp) and histone H2AX (gamma-H2AX). High-content automatic image analysis was used to quantify the immunofluorescence of radiation-induced foci. The size of radiation-induced foci increased for both proteins over a 2-h period after nitrogen-ion irradiation, while the size of radiation-induced foci did not change after exposure to low-LET radiation. The number of radiation-induced ATMp foci showed a more rapid rise and greater frequency after X-ray exposure and was resolved more rapidly such that the frequency of radiation-induced foci decreased by 90% compared to 60% after exposure to high-LET radiation 2 h after 30 cGy. In contrast, the kinetics of radiation-induced gamma-H2AX focus formation was similar for high- and low-LET radiation in that it reached a plateau early and remained constant for up to 2 h. High-resolution 3D images of radiation-induced gamma-H2AX foci and dosimetry computation suggest that multiple double-strand breaks from nitrogen ions are encompassed within large nuclear domains of 4.4 Mbp. Our work shows that the size and frequency of radiation-induced foci vary as a function of radiation quality, dose, time and protein target. Thus, even though double-strand breaks and radiation-induced foci are correlated, the dynamic nature of both contradicts their accepted equivalence for low doses of different radiation qualities
— id: 83217, year: 2006, vol: 165, page: 505, stat: Journal Article,

Quantitative in vivo microscopy: the return from the 'omics'
Fernandez-Gonzalez, Rodrigo; Munoz-Barrutia, Arrate; Barcellos-Hoff, Mary Helen; Ortiz-de-Solorzano, Carlos
2006 Oct;17(5):501-510, Current opinion in biotechnology
The confluence of recent advances in microscopy instrumentation and image analysis, coupled with the widespread use of GFP-like proteins as reporters of gene expression, has opened the door to high-throughput in vivo studies that can provide the morphological and temporal context to the biochemical pathways regulating cell function. We are now able to quantify the concentration and three-dimensional distribution of multiple spectrally resolved GFP-tagged proteins. Using automatic segmentation and tracking we can then measure the dynamics of the processes in which these elements are involved. In this way, parallel studies are feasible where multiple cell colonies treated with drugs or gene expression repressors can be monitored and analyzed to study the dynamics of relevant biological processes
— id: 83223, year: 2006, vol: 17, page: 501, stat: Journal Article,

The pleiotropic roles of transforming growth factor beta in homeostasis and carcinogenesis of endocrine organs
Fleisch, Markus C; Maxwell, Christopher A; Barcellos-Hoff, Mary-Helen
2006 Jun;13(2):379-400, Endocrine-related cancer
Transforming growth factor beta (TGF-beta) is a ubiquitous cytokine that plays a critical role in numerous pathways regulating cellular and tissue homeostasis. TGF-beta is regulated by hormones and is a primary mediator of hormone response in uterus, prostate and mammary glands. This review will address the role of TGF-beta in regulating hormone-dependent proliferation and morphogenesis. The subversion of TGF-beta regulation during the processes of carcinogenesis, with particular emphasis on its effects on genetic stability and epithelial to mesenchymal transition, will also be examined. An understanding of the multiple and complex mechanisms of TGF-beta regulation of epithelial function, and the ultimate loss of TGF-beta function during carcinogenesis, will be critical in the design of novel therapeutic interventions for endocrine-related cancers
— id: 83219, year: 2006, vol: 13, page: 379, stat: Journal Article,

Intensity-based signal separation algorithm for accurate quantification of clustered centrosomes in tissue sections
Fleisch, Markus C; Maxwell, Christopher A; Kuper, Claudia K; Brown, Erika T; Barcellos-Hoff, Mary Helen; Costes, Sylvain V
2006 Dec;69(12):964-972, Microscopy research & technique
Centrosomes are small organelles that organize the mitotic spindle during cell division and are also involved in cell shape and polarity. Within epithelial tumors, such as breast cancer, and some hematological tumors, centrosome abnormalities (CAs) are common, occur early in disease etiology, and correlate with chromosomal instability and disease stage. In situ quantification of CA by optical microscopy is hampered by overlap and clustering of these organelles, which appear as focal structures. CA has been frequently associated with Tp53 status in premalignant lesions and tumors. Here the authors described an approach to accurately quantify centrosome frequencies in tissue sections and tumors, independently of background or noise levels. Applying simple optical rules in nondeconvolved conventional 3D images of stained tissue sections, the authors showed that they could evaluate more accurately and rapidly centrosome frequencies than with traditional investigator-based visual analysis or threshold-based techniques. The resulting population-based frequency of centrosomes per nucleus could then be used to approximate the proportion of cells with CA in that same population. This was done by taking into account baseline centrosome amplification and proliferation rates measured in the tissue. Using this technique, the authors showed that 20-30% of cells have amplified centrosomes in Tp53 null mammary tumors
— id: 83224, year: 2006, vol: 69, page: 964, stat: Journal Article,

Loss of TGF-beta promotes accumulation of centrosome amplification and karyotypic abnormalities in p53 competent mammary cells and tissues
Fleisch, MC; Maxwell, CA; Kuper, CK; Costes, SV; Barcellos-Hoff, MH
2006 ;100(12):S290-S291, Breast cancer research & treatment
— id: 104670, year: 2006, vol: 100, page: S290, stat: Journal Article,

Isoform-specific activation of latent transforming growth factor beta (LTGF-beta) by reactive oxygen species
Jobling, Michael F; Mott, Joni D; Finnegan, Monica T; Jurukovski, Vladimir; Erickson, Anna C; Walian, Peter J; Taylor, Scott E; Ledbetter, Steven; Lawrence, Catherine M; Rifkin, Daniel B; Barcellos-Hoff, Mary Helen
2006 Dec;166(6):839-848, Radiation research
The three mammalian transforming growth factor beta (TGF-beta) isoforms are each secreted in a latent complex in which TGF-beta homodimers are non-covalently associated with homodimers of their respective pro-peptide called the latency-associated peptide (LAP). Release of TGF-beta from its LAP, called activation, is required for binding of TGF-beta to cellular receptors, making extracellular activation a critical regulatory point for TGF-beta bioavailability. Our previous work demonstrated that latent TGF-beta1 (LTGF-beta1) is efficiently activated by ionizing radiation in vivo and by reactive oxygen species (ROS) generated by Fenton chemistry in vitro. In the current study, we determined the specific ROS and protein target that render LTGF-beta1 redox sensitive. First, we compared LTGF-beta1, LTGF-beta2 and LTGF-beta3 to determine the generality of this mechanism of activation and found that redox-mediated activation is restricted to the LTGF-beta1 isoform. Next, we used scavengers to determine that ROS activation was a function of OH(.) availability, confirming oxidation as the primary mechanism. To identify which partner of the LTGF-beta1 complex was functionally modified, each was exposed to ROS and tested for the ability to form a latent complex. Exposure of TGF-beta1 did not alter its ability to associate with LAP, but exposing LAP-beta1 to ROS prohibited this phenomenon, while treatment of ROS-exposed LAP-beta1 with a mild reducing agent restored its ability to neutralize TGF-beta1 activity. Taken together, these results suggest that ROS-induced oxidation in LAP-beta1 triggers a conformational change that releases TGF-beta1. Using site-specific mutation, we identified a methionine residue at amino acid position 253 unique to LAP-beta1 as critical to ROS-mediated activation. We propose that LTGF-beta1 contains a redox switch centered at methionine 253, which allows LTGF-beta1 to act uniquely as an extracellular sensor of oxidative stress in tissues
— id: 83229, year: 2006, vol: 166, page: 839, stat: Journal Article,

3D segmentation of mammospheres for localization studies
Ju Han; Hang Chang; Qing Yang; Barcellos-Hoff, M.H.; Parvin, B.
Advances in visual computing : second international symposium, ISVC 2006, Lake Tahoe, NV, USA, November 6-8, 2006 : proceedings New York : Springer, 2006,
Three dimensional cell culture assays have emerged as the basis of an improved model system for evaluating therapeutic agents, molecular probes, and exogenous stimuli. However, there is a gap in robust computational techniques for segmentation of image data that are collected through confocal or deconvolution microscopy. The main issue is the volume of data, overlapping subcellular compartments, and variation in scale and size of subcompartments of interest. A geometric technique has been developed to bound the solution of the problem by first localizing centers of mass for each cell and then partitioning clump of cells along minimal intersecting surfaces. An approximate solution to the center of mass is realized through iterative spatial voting, which is tolerant to variation in shape morphologies and overlapping compartments and is shown to have an excellent noise immunity. These centers of mass are then used to partition a clump of cells along minimal intersecting surfaces that are estimated by Radon transform. Examples on real data and performance of the system over a large population of data are evaluated. Although proposed strategies have been developed and tested on data collected through fluorescence microscopy, they are applicable to other problems in low level vision and medical imaging
— id: 5228, year: 2006, vol: , page: 518, stat: Chapter,

Inhibition of transforming growth factor-beta1 signaling attenuates ataxia telangiectasia mutated activity in response to genotoxic stress
Kirshner, Julia; Jobling, Michael F; Pajares, Maria Jose; Ravani, Shraddha A; Glick, Adam B; Lavin, Martin J; Koslov, Sergei; Shiloh, Yosef; Barcellos-Hoff, Mary Helen
2006 Nov 15;66(22):10861-10869, Cancer research
Ionizing radiation causes DNA damage that elicits a cellular program of damage control coordinated by the kinase activity of ataxia telangiectasia mutated protein (ATM). Transforming growth factor beta (TGFbeta)-1, which is activated by radiation, is a potent and pleiotropic mediator of physiologic and pathologic processes. Here we show that TGFbeta inhibition impedes the canonical cellular DNA damage stress response. Irradiated Tgfbeta1 null murine epithelial cells or human epithelial cells treated with a small-molecule inhibitor of TGFbeta type I receptor kinase exhibit decreased phosphorylation of Chk2, Rad17, and p53; reduced gammaH2AX radiation-induced foci; and increased radiosensitivity compared with TGFbeta competent cells. We determined that loss of TGFbeta signaling in epithelial cells truncated ATM autophosphorylation and significantly reduced its kinase activity, without affecting protein abundance. Addition of TGFbeta restored functional ATM and downstream DNA damage responses. These data reveal a heretofore undetected critical link between the microenvironment and ATM, which directs epithelial cell stress responses, cell fate, and tissue integrity. Thus, Tgfbeta1, in addition to its role in homoeostatic growth control, plays a complex role in regulating responses to genotoxic stress, the failure of which would contribute to the development of cancer; conversely, inhibiting TGFbeta may be used to advantage in cancer therapy
— id: 83228, year: 2006, vol: 66, page: 10861, stat: Journal Article,

Activated type I TGFbeta receptor kinase enhances the survival of mammary epithelial cells and accelerates tumor progression
Muraoka-Cook, R S; Shin, I; Yi, J Y; Easterly, E; Barcellos-Hoff, M H; Yingling, J M; Zent, R; Arteaga, C L
2006 Jun 8;25(24):3408-3423, Oncogene
We have examined the effects of transforming growth factor-beta (TGFbeta) signaling on mammary epithelial cell survival. Transgenic mice expressing an active mutant of Alk5 in the mammary gland (MMTV-Alk5(T204D)) exhibited reduced apoptosis in terminal endbuds and during postlactational involution. Transgene-expressing mammary cells contained lower Smad2/3 and higher c-myc levels than controls, high ligand-independent phosphatidylinositol-3 kinase (PI3K) and Akt activities, and were insensitive to TGFbeta-mediated growth arrest. Treatment with a proteasome inhibitor increased Smad2/3 levels and ligand-independent Smad transcriptional reporter activity, as well as reduced both c-myc protein and basal cell proliferation. Treatment with an Alk5 kinase small-molecule inhibitor upregulated Smad2/3 levels, reduced PI3K activity, P-Akt, and c-myc, and inhibited cell survival. Although Alk5(T204D)-expressing mice did not develop mammary tumors, bigenic MMTV-Alk(T204D) x Neu mice developed cancers that were more metastatic than those occurring in MMTV-Neu transgenics. These data suggest that (1) TGFbeta can signal to PI3K/Akt and enhance mammary epithelial cell survival in vivo before cytological or histological evidence of transformation, and (2) TGFbeta signaling can provide epithelial cells with a 'gain-of-function' effect that synergizes with oncogene-induced transformation
— id: 83206, year: 2006, vol: 25, page: 3408, stat: Journal Article,

How tissues respond to damage at the cellular level: orchestration by transforming growth factor-{beta} (TGF-{beta})
Barcellos-Hoff, M H
2005 ;27:123-127, BJR supplement / BIR
When the human body is exposed to high doses of radiation, a complex, rapidly evolving, deleterious biological response is initiated that may culminate in multi-organ failure (MOF). Although this process begins with energy deposits in cellular targets, it is propagated and amplified by the tissue response to cell damage. I will argue that if the biology of wound healing is at the root of MOF following surgical trauma, and inflammation is the basis for MOF in sepsis, then the biology of the irradiated tissue uniquely initiates radiogenic MOF. The present review summarises data suggesting that tissue response to radiation damage is initiated and co-ordinated by extracellular signalling. In particular, research from the author's laboratory demonstrates that transforming growth factor-beta1 orchestrates the biology of irradiated tissue via a novel function as a tissue level sensor of oxidative stress, and is integral to the cellular DNA damage response. Thus, the means to therapeutically control radiogenic MOF lies in the mechanisms by which tissues respond to global cellular damage
— id: 83200, year: 2005, vol: 27, page: 123, stat: Journal Article,

Integrative radiation carcinogenesis: interactions between cell and tissue responses to DNA damage
Barcellos-Hoff, Mary Helen
2005 Apr;15(2):138-148, Seminars in cancer biology
Tissue function requires coordinated multicellular behavior as a consequence of diverse signals integrated through the tissue microenvironment; importantly, these cell-cell and cell-microenvironment interactions also actively suppress cancer. Ionizing radiation (IR) elicits a well-defined cellular response to DNA damage that mediates the fate of the individual cell, concomitantly with a less well-characterized overarching tissue stress response that coordinates the response of multiple cell types via microenvironment signaling. We have now shown that these programs to reestablish homeostasis intersect via mutual regulation by transforming growth factor beta1 (TGF beta 1), which acts as an extracellular sensor and signal of stress. In this review, the concept that this type of functional integration of cell and tissue stress response programs is essential to cancer suppression will be discussed. Our experiments using IR, and several recent studies that experimentally manipulate stromal TGF beta, show that disruption of microenvironment signaling actively promotes malignant progression. Understanding the dynamic interactions between tissue and cell stress responses will be necessary for an accurate assessment of cancer risk and may also provide targets for prevention
— id: 83196, year: 2005, vol: 15, page: 138, stat: Journal Article,

New highlights on stroma-epithelial interactions in breast cancer
Barcellos-Hoff, Mary Helen; Medina, Daniel
2005 ;7(1):33-36, Breast cancer research
Although the stroma in which carcinomas arise has been previously regarded as a bystander to the clonal expansion and acquisition of malignant characteristics of tumor cells, it is now generally acknowledged that stromal changes are required for the establishment of cancer. In the present article, we discuss three recent publications that highlight the complex role the stroma has during the development of cancer and the potential for targeting the stroma by therapeutic approaches
— id: 83195, year: 2005, vol: 7, page: 33, stat: Journal Article,

Radiation and the microenvironment - tumorigenesis and therapy
Barcellos-Hoff, Mary Helen; Park, Catherine; Wright, Eric G
2005 Nov;5(11):867-875, Nature reviews. Cancer
Radiation rapidly and persistently alters the soluble and insoluble components of the tissue microenvironment. This affects the cell phenotype, tissue composition and the physical interactions and signalling between cells. These alterations in the microenvironment can contribute to carcinogenesis and alter the tissue response to anticancer therapy. Examples of these responses and their implications are discussed with a view to therapeutic intervention
— id: 83210, year: 2005, vol: 5, page: 867, stat: Journal Article,

Proliferation of estrogen receptor-alpha-positive mammary epithelial cells is restrained by transforming growth factor-beta1 in adult mice
Ewan, Kenneth B R; Oketch-Rabah, Hellen A; Ravani, Shraddha A; Shyamala, G; Moses, Harold L; Barcellos-Hoff, Mary Helen
2005 Aug;167(2):409-417, American journal of pathology
Transforming growth factor (TGF)-beta1 is a potent inhibitor of mammary epithelial proliferation. In human breast, estrogen receptor (ER)-alpha cells rarely co-localize with markers of proliferation, but their increased frequency correlates with breast cancer risk. To determine whether TGF-beta1 is necessary for the quiescence of ER-alpha-positive populations, we examined mouse mammary epithelial glands at estrus. Approximately 35% of epithelial cells showed TGF-beta1 activation, which co-localized with nuclear receptor-phosphorylated Smad 2/3, indicating that TGF-beta signaling is autocrine. Nuclear Smad co-localized with nuclear ER-alpha. To test whether TGF-beta inhibits proliferation, we examined genetically engineered mice with different levels of TGF-beta1. ER-alpha co-localization with markers of proliferation (ie, Ki-67 or bromodeoxyuridine) at estrus was significantly increased in the mammary glands of Tgf beta1 C57/bl/129SV heterozygote mice. This relationship was maintained after pregnancy but was absent at puberty. Conversely, mammary epithelial expression of constitutively active TGF-beta1 via the MMTV promoter suppressed proliferation of ER-alpha-positive cells. Thus, TGF-beta1 activation functionally restrains ER-alpha-positive cells from proliferating in adult mammary gland. Accordingly, we propose that TGF-beta1 dysregulation may promote proliferation of ER-alpha-positive cells associated with breast cancer risk in humans
— id: 83202, year: 2005, vol: 167, page: 409, stat: Journal Article,

A tool for the quantitative spatial analysis of complex cellular systems
Fernandez-Gonzalez, Rodrigo; Barcellos-Hoff, Mary Helen; Ortiz-de-Solorzano, Carlos
2005 Sep;14(9):1300-1313, IEEE transactions on image processing
Spatial events largely determine the biology of cells, tissues, and organs. In this paper, we present a tool for the quantitative spatial analysis of heterogeneous cell populations, and we show experimental validation of this tool using both artificial and real (mammary gland tissue) data, in two and three dimensions. We present the refined relative neighborhood graph as a means to establish neighborhood between cells in an image while modeling the topology of the tissue. Then, we introduce the M function as a method to quantitatively evaluate the existence of spatial patterns within one cell population or the relationship between the spatial distributions of multiple cell populations. Finally, we show a number of examples that demonstrate the feasibility of our approach
— id: 83207, year: 2005, vol: 14, page: 1300, stat: Journal Article,

A tool for the quantitative spatial analysis of heterogeneous cell populations
Fernandez-Gonzalez, Rodrigo; Ortiz de Solorzano, Carlos; Barcellos-Hoff, Mary Helen
2005 ;46(2):384-385, Proceedings (American Association for Cancer Research)
— id: 104671, year: 2005, vol: 46, page: 384, stat: Journal Article,

Cell cycle defects contribute to a block in hormone-induced mammary gland proliferation in CCAAT/enhancer-binding protein (C/EBPbeta)-null mice
Grimm, Sandra L; Contreras, Alejandro; Barcellos-Hoff, Mary-Helen; Rosen, Jeffrey M
2005 Oct 28;280(43):36301-36309, Journal of biological chemistry
In contrast to hormone-dependent breast cancer, steroid hormone-induced proliferation in the normal mammary gland does not occur in the steroid-receptor positive cells but rather in adjacent cells via paracrine signaling involving several local growth factors. To help elucidate the mechanisms involved in the block in proliferation in hormone-receptor positive cells, we have utilized a CCAAT/enhancer binding protein (C/EBPbeta)-null mouse model. Loss of this transcription factor results in increased steroid and prolactin receptor expression concomitant with a 10-fold decrease in proliferation in response to pregnancy hormones. To determine the basis for this decrease, several markers of cell cycle progression were analyzed in wild type and C/EBPbeta-null mammary epithelial cells (MECs). These studies indicated that cell cycle progression in C/EBPbeta-null MECs is blocked at the G1/S transition. C/EBPbeta-null mammary glands display substantially increased levels of the activated form of transforming growth factor beta, a potent inhibitor of epithelial cell proliferation, as well as increased downstream Smad2 expression and signaling. While cyclin D1 levels were equivalent, cyclin E expression was markedly reduced in C/EBPbeta-null as compared with wildtype MECs. In addition, increased p27 stability and retention in the nucleus and decreased levels of the cdc25a phosphatase contributed to a significant loss of cdk2 kinase activity. Collectively, these changes prevent C/EBPbeta-null mammary epithelial cells from responding to hormone-induced proliferative signals
— id: 83204, year: 2005, vol: 280, page: 36301, stat: Journal Article,

Three-dimensional histology of the mammary gland: An application to the study of hormone receptor expression during normal mammary gland development
Laribi, Ouahiba; Hartland, Abbey; Fernandez-Gonzalez, Rodrigo; Arganda-Carreras, Ignacio; Idica, Adam; Barcellos-Hoff, Mary Helen; Ortiz de Solorzano, Carlos
2005 ;46(43):1241-1241, Proceedings (American Association for Cancer Research)
— id: 104672, year: 2005, vol: 46, page: 1241, stat: Journal Article,

Targeting cell-extracellular matrix (ECM) interactions to enhance radiation efficacy for breast cancer treatment
Park, Catherine C.; Zhang, Hui J.; Yao, Evelyn; Barcellos-Hoff, Mary Helen; Bissell, Mina J.
2005 ;46(43):390-391, Proceedings (American Association for Cancer Research)
— id: 104673, year: 2005, vol: 46, page: 390, stat: Journal Article,

Geometric approach to segmentation and protein localization in cell cultured assays
Raman, S; Parvin, B; Maxwell, C; Barcellos-Hoff, MH
2005 ;3804(43):427-436, Lecture notes in computer science
Cell-based fluorescence imaging assays are heterogeneous requiring collection of a large number of images for detailed quantitative analysis. Complexities arise as a result of variation in spatial nonuniformity, shape, overlapping compartments, and scale. A new technique and methodology has been developed and tested for delineating subcellular morphology and partitioning overlapping compartments at multiple scales. This system is packaged as an integrated software platform for quantifying images that are obtained through fluorescence microscopy. Proposed methods are model-based, leveraging geometric shape properties of subcellular compartments and corresponding protein localization. From the morphological perspective, convexity constraint is imposed to delineate, partition, and group nuclear compartments. From the protein localization perspective, radial symmetry is imposed to localize punctate protein events at sub-micron resolution. The technique has been tested against 196 images that were generated to study centrosome abnormalities. Computed representations are evaluated against the ground truth annotation for comparative analysis. $$:
— id: 104674, year: 2005, vol: 3804, page: 427, stat: Journal Article,

Quantitative image analysis in mammary gland biology
Fernandez-Gonzalez, Rodrigo; Barcellos-Hoff, Mary Helen; Ortiz-de-Solorzano, Carlos
2004 Oct;9(4):343-359, Journal of mammary gland biology & neoplasia
In this paper we present a summary of recent quantitative approaches used for the analysis of macro and microscopic images in mammary gland biology. The advantages and disadvantages of whole mount analysis, reconstruction of serial tissue sections and nucleus/cell segmentation of either conventional and confocal images are discussed, as are applications of quantitative image analysis, such as quantification of protein levels or vasculature measurements in normal tissue and cancer. Integration of quantitative imaging into the further study of the mammary gland holds the promise of better understanding its tissue complexity that evolves during development, differentiation and disease
— id: 83198, year: 2004, vol: 9, page: 343, stat: Journal Article,

Conditional overexpression of active transforming growth factor beta1 in vivo accelerates metastases of transgenic mammary tumors
Muraoka-Cook, Rebecca S; Kurokawa, Hirokazu; Koh, Yasuhiro; Forbes, James T; Roebuck, L Renee; Barcellos-Hoff, Mary Helen; Moody, Susan E; Chodosh, Lewis A; Arteaga, Carlos L
2004 Dec 15;64(24):9002-9011, Cancer research
To address the role of transforming growth factor (TGF) beta in the progression of established tumors while avoiding the confounding inhibitory effects of TGF-beta on early transformation, we generated doxycycline (DOX)-inducible triple transgenic mice in which active TGF-beta1 expression could be conditionally regulated in mouse mammary tumor cells transformed by the polyomavirus middle T antigen. DOX-mediated induction of TGF-beta1 for as little as 2 weeks increased lung metastases >10-fold without a detectable effect on primary tumor cell proliferation or tumor size. DOX-induced active TGF-beta1 protein and nuclear Smad2 were restricted to cancer cells, suggesting a causal association between autocrine TGF-beta and increased metastases. Antisense-mediated inhibition of TGF-beta1 in polyomavirus middle T antigen-expressing tumor cells also reduced basal cell motility, survival, anchorage-independent growth, tumorigenicity, and metastases. Therefore, induction and/or activation of TGF-beta in hosts with established TGF-beta-responsive cancers can rapidly accelerate metastatic progression
— id: 83193, year: 2004, vol: 64, page: 9002, stat: Journal Article,

Localization of saliency through iterative voting
Qing Yang; Parvin, B.; Barcellos-Hoff, M.H.
Proceedings of the 17th International Conference on Pattern Recognition : ICPR 2004 Los Alamitos, Calif. : IEEE Computer Society Press, 2004,
Saliency is an important perceptual cue that occurs at different scales of resolution. Important attributes of saliency are symmetry, continuity, and closure. Detection of these attributes is often hindered by noise, variation in scale, and incomplete information. An iterative voting method using oriented kernels is introduced for inferring saliency as it relates to symmetry or continuity. A unique aspect of the technique is in the kernel topography, which is refined and reoriented iteratively. The technique can cluster and group nonconvex perceptual circular symmetries along the radial line or sparse features along the tangential direction. It has an excellent noise immunity, and is shown to be tolerant to perturbation in scale. Applications of this approach to blobs with incomplete and noisy boundaries and to scientific images are demonstrated
— id: 5229, year: 2004, vol: , page: 63, stat: Chapter,

Models for evaluating agents intended for the prophylaxis, mitigation and treatment of radiation injuries. Report of an NCI Workshop, December 3-4, 2003
Stone, Helen B; Moulder, John E; Coleman, C Norman; Ang, K Kian; Anscher, Mitchell S; Barcellos-Hoff, Mary Helen; Dynan, William S; Fike, John R; Grdina, David J; Greenberger, Joel S; Hauer-Jensen, Martin; Hill, Richard P; Kolesnick, Richard N; Macvittie, Thomas J; Marks, Cheryl; McBride, William H; Metting, Noelle; Pellmar, Terry; Purucker, Mary; Robbins, Mike E; Schiestl, Robert H; Seed, Thomas M; Tomaszewski, Joseph E; Travis, Elizabeth L; Wallner, Paul E; Wolpert, Mary; Zaharevitz, Daniel
2004 Dec;162(6):711-728, Radiation research
To develop approaches to prophylaxis/protection, mitigation and treatment of radiation injuries, appropriate models are needed that integrate the complex events that occur in the radiation-exposed organism. While the spectrum of agents in clinical use or preclinical development is limited, new research findings promise improvements in survival after whole-body irradiation and reductions in the risk of adverse effects of radiotherapy. Approaches include agents that act on the initial radiochemical events, agents that prevent or reduce progression of radiation damage, and agents that facilitate recovery from radiation injuries. While the mechanisms of action for most of the agents with known efficacy are yet to be fully determined, many seem to be operating at the tissue, organ or whole animal level as well as the cellular level. Thus research on prophylaxis/protection, mitigation and treatment of radiation injuries will require studies in whole animal models. Discovery, development and delivery of effective radiation modulators will also require collaboration among researchers in diverse fields such as radiation biology, inflammation, physiology, toxicology, immunology, tissue injury, drug development and radiation oncology. Additional investment in training more scientists in radiation biology and in the research portfolio addressing radiological and nuclear terrorism would benefit the general population in case of a radiological terrorism event or a large-scale accidental event as well as benefit patients treated with radiation
— id: 83189, year: 2004, vol: 162, page: 711, stat: Journal Article,

The role of TGF-beta in radiation responses
Barcellos-Hoff, M. H.
2003 ;1(5):S227-S227, EJC Supplements
— id: 104677, year: 2003, vol: 1, page: S227, stat: Journal Article,

How radiation-induced phenotypes contribute to neoplastic progression
Barcellos-Hoff, MH
2003 ;85(1):84-84, Health physics
— id: 104675, year: 2003, vol: 85, page: 84, stat: Journal Article,

Radiation-induced versus endogenous DNA damage: commentary on Pollycove and Feinendegen
Barcellos-Hoff, MH
2003 ;22(6):307-307, Human & Experimental Toxicology
— id: 104676, year: 2003, vol: 22, page: 307, stat: Journal Article,

Education and training for radiation scientists: radiation research program and American Society of Therapeutic Radiology and Oncology Workshop, Bethesda, Maryland, May 12-14, 2003
Coleman, C Norman; Stone, Helen B; Alexander, George A; Barcellos-Hoff, Mary Helen; Bedford, Joel S; Bristow, Robert G; Dynlacht, Joseph R; Fuks, Zvi; Gorelic, Lester S; Hill, Richard P; Joiner, Michael C; Liu, Fei-Fei; McBride, William H; McKenna, W Gillies; Powell, Simon N; Robbins, Michael E C; Rockwell, Sara; Schiff, Peter B; Shaw, Edward G; Siemann, Dietmar W; Travis, Elizabeth L; Wallner, Paul E; Wong, Rosemary S L; Zeman, Elaine M
2003 Dec;160(6):729-737, Radiation research
Current and potential shortfalls in the number of radiation scientists stand in sharp contrast to the emerging scientific opportunities and the need for new knowledge to address issues of cancer survivorship and radiological and nuclear terrorism. In response to these challenges, workshops organized by the Radiation Research Program (RRP), National Cancer Institute (NCI) (Radiat. Res. 157, 204-223, 2002; Radiat. Res. 159, 812-834, 2003), and National Institute of Allergy and Infectious Diseases (NIAID) (Nature, 421, 787, 2003) have engaged experts from a range of federal agencies, academia and industry. This workshop, Education and Training for Radiation Scientists, addressed the need to establish a sustainable pool of expertise and talent for a wide range of activities and careers related to radiation biology, oncology and epidemiology. Although fundamental radiation chemistry and physics are also critical to radiation sciences, this workshop did not address workforce needs in these areas. The recommendations include: (1) Establish a National Council of Radiation Sciences to develop a strategy for increasing the number of radiation scientists. The strategy includes NIH training grants, interagency cooperation, interinstitutional collaboration among universities, and active involvement of all stakeholders. (2) Create new and expanded training programs with sustained funding. These may take the form of regional Centers of Excellence for Radiation Sciences. (3) Continue and broaden educational efforts of the American Society for Therapeutic Radiology and Oncology (ASTRO), the American Association for Cancer Research (AACR), the Radiological Society of North America (RSNA), and the Radiation Research Society (RRS). (4) Foster education and training in the radiation sciences for the range of career opportunities including radiation oncology, radiation biology, radiation epidemiology, radiation safety, health/government policy, and industrial research. (5) Educate other scientists and the general public on the quantitative, basic, molecular, translational and applied aspects of radiation sciences
— id: 83174, year: 2003, vol: 160, page: 729, stat: Journal Article,

The not-so innocent bystander: the microenvironment as a therapeutic target in cancer
Erickson, Anna C; Barcellos-Hoff, Mary Helen
2003 Feb;7(1):71-88, Expert opinion on therapeutic targets
The microenvironment in which cancer arises is often regarded as a bystander to the clonal expansion and acquisition of malignant characteristics of the tumour. However, a major function of the microenvironment is to suppress cancer, and its disruption is required for the establishment of cancer. In addition, tumour cells can further distort the microenvironment to promote growth, recruit non-malignant cells that provide physiological resources, and facilitate invasion. In this review, the authors discuss the contribution of the microenvironment, i.e., the stroma and its resident vasculature, inflammatory cells, growth factors and the extracellular matrix (ECM), in the development of cancer, and focus on two components as potential therapeutic targets in breast cancer. First, the ECM, which imparts crucial signalling via integrins and other receptors, is a first-line barrier to invasion, modulates aggressive behaviour and may be manipulated to provide novel impediments to tumour growth. Second, the authors discuss the involvement of TGF-beta1 as an example of one of many growth factors that can regulate ECM composition and degradation and that play complex roles in cancer. Compared to the variable routes taken by cells to become cancers, the response of tissues to cancer is relatively consistent. Therefore, controlling and eliminating cancer may be more readily achieved indirectly via the tissue microenvironment
— id: 83157, year: 2003, vol: 7, page: 71, stat: Journal Article,

Isoform specificity of redox-mediated TGF-beta activation
Jobling, M; Erickson, A; Taylor, S; Finnigan, M; Ledbetter, S; Lawrence, C; Barcellos-Hoff, MH
2003 ;35(1):25-25, Free radical biology & medicine
— id: 104678, year: 2003, vol: 35, page: 25, stat: Journal Article,

"Radiation-induced p53 Ser-18 phosphorylation is TGF-beta1 dependent in primary epithelial, but not fibroblast, cells"
Pajares, Maria J.; Glick, Adam; Jobling, Michael F.; Barcellos-Hoff, Mary Helen
2003 ;44(1):465-465, Proceedings (American Association for Cancer Research)
— id: 104679, year: 2003, vol: 44, page: 465, stat: Journal Article,

Ionizing radiation induces heritable disruption of epithelial cell interactions
Park, Catherine C; Henshall-Powell, Rhonda L; Erickson, Anna C; Talhouk, Rabih; Parvin, Bahram; Bissell, Mina J; Barcellos-Hoff, Mary Helen
2003 Sep 16;100(19):10728-10733, Proceedings of the National Academy of Sciences of the United States of America
Ionizing radiation (IR) is a known human breast carcinogen. Although the mutagenic capacity of IR is widely acknowledged as the basis for its action as a carcinogen, we and others have shown that IR can also induce growth factors and extracellular matrix remodeling. As a consequence, we have proposed that an additional factor contributing to IR carcinogenesis is the potential disruption of critical constraints that are imposed by normal cell interactions. To test this hypothesis, we asked whether IR affected the ability of nonmalignant human mammary epithelial cells (HMEC) to undergo tissue-specific morphogenesis in culture by using confocal microscopy and imaging bioinformatics. We found that irradiated single HMEC gave rise to colonies exhibiting decreased localization of E-cadherin, beta-catenin, and connexin-43, proteins necessary for the establishment of polarity and communication. Severely compromised acinar organization was manifested by the majority of irradiated HMEC progeny as quantified by image analysis. Disrupted cell-cell communication, aberrant cell-extracellular matrix interactions, and loss of tissue-specific architecture observed in the daughters of irradiated HMEC are characteristic of neoplastic progression. These data point to a heritable, nonmutational mechanism whereby IR compromises cell polarity and multicellular organization
— id: 83169, year: 2003, vol: 100, page: 10728, stat: Journal Article,

BioSig: an imaging bioinformatics system for phenotypic analysis
Parvin, B; Yang, Qing; Fontenay, G; Barcellos-Hoff, M H
2003 ;33(5):814-824, IEEE transactions on systems, man, & cybernetics. Part B, Cybernetics
Organisms express their genomes in a cell-specific manner, resulting in a variety of cellular phenotypes or phenomes. Mapping cell phenomes under a variety of experimental conditions is necessary in order to understand the responses of organisms to stimuli. Representing such data requires an integrated view of experimental and informatic protocols. The proposed system, named BioSig, provides the foundation for cataloging cellular responses as a function of specific conditioning, treatment, staining, etc., for either fixed tissue or living cell studies. A data model has been developed to capture experimental variables and map them to image collections and their computed representation. This representation is hierarchical and spans across sample tissues, cells, and organelles, which are imaged with light microscopy. At each layer, content is represented with an attributed graph, which contains information about cellular morphology, protein localization, and cellular organization in tissue or cell culture. The Web-based multilayer informatics architecture uses the data model to provide guided workflow access for content exploration
— id: 83255, year: 2003, vol: 33, page: 814, stat: Journal Article,

Transforming growth factor beta1 mediates radiation-induced apoptosis and p53 response in situ
Barcellos-Hoff, Mary H.; Henshall-Powell, Rhonda; Ewan, Kenneth B.; Ravani, Shraddha A.; Arteaga, Carlos; Akhurst, Rosemary; Warters, Ray; Parvin, Bahram
2002 ;43(4):147-147, Proceedings (American Association for Cancer Research)
— id: 104680, year: 2002, vol: 43, page: 147, stat: Journal Article,

The contribution of radiation-induced microenvironments to neoplastic progression
Barcellos-Hoff, MH
2002 ;158(6):792-793, Radiation research
— id: 104681, year: 2002, vol: 158, page: 792, stat: Journal Article,

Radiation quality and tissue-specific microenvironments following exposure to 1 GeV/amu Fe
Costes, S; Barcellos-Hoff, M H
2002 ;30(4):865-870, Advances in space research : the official journal of the Committee on Space Research (COSPAR)
This paper summarizes quantitative in vivo laminin immunofluorescence analysis of mammary glands and skin epithelial structures from mice exposed to 1 GeV/amu Fe ions. Digital confocal microscopic images were quantified and linked to the rough 'core-penumbra' Fe track physical description. Comparison to gamma-ray sparsely ionizing radiation suggested the core of the Fe track being responsible for a biological response only seen with energetic Fe particles. Conclusions for modeling in vivo responses to radiation were then implied
— id: 83154, year: 2002, vol: 30, page: 865, stat: Journal Article,

Radiation alters cytoskeletal association of E-cadherin in TGF-beta 1 treated human mammary epithelial cells
Erickson, AC; Chou, WS; Henshall-Powell, R; Bissell, MJ; Barcellos-Hoff, MH
2002 ;13(2):409-409, Molecular biology of the cell
— id: 104682, year: 2002, vol: 13, page: 409, stat: Journal Article,

Transforming growth factor-beta1 mediates cellular response to DNA damage in situ
Ewan, Kenneth B; Henshall-Powell, Rhonda L; Ravani, Shraddha A; Pajares, Maria Jose; Arteaga, Carlos; Warters, Ray; Akhurst, Rosemary J; Barcellos-Hoff, Mary Helen
2002 Oct 15;62(20):5627-5631, Cancer research
Transforming growth factor (TGF)-beta1 is rapidly activated after ionizing radiation, but its specific role in cellular responses to DNA damage is not known. Here we use Tgfbeta1 knockout mice to show that radiation-induced apoptotic response is TGF-beta1 dependent in the mammary epithelium, and that both apoptosis and inhibition of proliferation in response to DNA damage decrease as a function of TGF-beta1 gene dose in embryonic epithelial tissues. Because apoptosis in these tissues has been shown previously to be p53 dependent, we then examined p53 protein activation. TGF-beta1 depletion, by either gene knockout or by using TGF-beta neutralizing antibodies, resulted in decreased p53 Ser-18 phosphorylation in irradiated mammary gland. These data indicate that TGF-beta1 is essential for rapid p53-mediated cellular responses that mediate cell fate decisions in situ
— id: 83149, year: 2002, vol: 62, page: 5627, stat: Journal Article,

Latent transforming growth factor-beta activation in mammary gland: regulation by ovarian hormones affects ductal and alveolar proliferation
Ewan, Kenneth B; Shyamala, Gopalan; Ravani, Shraddha A; Tang, Yang; Akhurst, Rosemary; Wakefield, Lalage; Barcellos-Hoff, Mary Helen
2002 Jun;160(6):2081-2093, American journal of pathology
Transforming growth factor-beta1 (TGF-beta 1) is a pluripotent cytokine that can inhibit epithelial proliferation and induce apoptosis, but is also widely implicated in breast cancer progression. Understanding its biological action in mammary development is critical for understanding its role in cancer. TGF-beta 1 is produced as a latent complex that requires extracellular activation before receptor binding. To better understand the spatial and temporal regulation of its action during mammary gland development, we examined the pattern of activation in situ using antibodies selected to distinguish between latent and active TGF-beta. Activation was highly restricted. TGF-beta 1 activation was localized primarily to the epithelium, and within the epithelium it was restricted to luminal epithelial cells but absent from either cap or myoepithelial cells. Within the luminal epithelium, we noted a further restriction. During periods of proliferation (ie, puberty, estrus and pregnancy), which are stimulated by ovarian hormones, TGF-beta 1 activation decreased in some cells, consistent with preparation for proliferation. Paradoxically, other cells simultaneously increase TGF-beta 1 immunoreactivity, which suggests that TGF-beta 1 differentially restrains epithelial subpopulations from responding to hormonal signals to proliferate. These data suggest that endogenous TGF-beta 1 activation and thus activity are regulated by ovarian hormones. To determine the specific consequences of TGF-beta 1 activity, we manipulated TGF-beta 1 levels in vivo using Tgfbeta 1 knockout mice and undertook tissue recombination experiments with heterozygous tissue. In Tgfbeta 1 heterozygous mice, which have <10% wild-type levels of TGF-beta1, ductal development during puberty and alveolar development during pregnancy were accelerated, consistent with its role as a growth inhibitor. The proliferative index of Tgfbeta 1+/- epithelium was increased approximately twofold in quiescent tissue and fourfold in proliferating tissue but both ducts and alveoli were grossly and histologically normal. To test whether epithelial TGF-beta1 was critical to the proliferative phenotype, Tgfbeta 1+/+ and +/- epithelium were transplanted into +/+ mammary stroma. The outgrowth of Tgfbeta 1+/- epithelium was accelerated in wild-type hosts, indicating that the phenotype was intrinsic to the epithelium. Moreover, proliferation was 15-fold greater in Tgfbeta 1+/- than wild-type mice after ovariectomy and treatment with estrogen and progesterone, suggesting that TGF-beta 1 acts in an autocrine or juxtacrine manner to regulate epithelial proliferation. Together these data indicate that ovarian hormones regulate TGF-beta 1 activation, which in turn restricts proliferative response to hormone signaling
— id: 83144, year: 2002, vol: 160, page: 2081, stat: Journal Article,

System for combined three-dimensional morphological and molecular analysis of thick tissue specimens
Fernandez-Gonzalez, Rodrigo; Jones, Arthur; Garcia-Rodriguez, Enrique; Chen, Ping Yuan; Idica, Adam; Lockett, Stephen J; Barcellos-Hoff, Mary Helen; Ortiz-De-Solorzano, Carlos
2002 Dec 15;59(6):522-530, Microscopy research & technique
We present a new system for simultaneous morphological and molecular analysis of thick tissue samples. The system is composed of a computer-assisted microscope and a JAVA-based image display, analysis, and visualization program that allows acquisition, annotation, meaningful storage, three-dimensional reconstruction, and analysis of structures of interest in thick sectioned tissue specimens. We describe the system in detail and illustrate its use by imaging, reconstructing, and analyzing two complete tissue blocks that were differently processed and stained. One block was obtained from a ductal carcinoma in situ (DCIS) lumpectomy specimen and stained alternatively with Hematoxilyn and Eosin (H&E), and with a counterstain and fluorescence in situ hybridization (FISH) to the ERB-B2 gene. The second block contained a fully sectioned mammary gland of a mouse, stained for histology with H&E. We show how the system greatly reduces the amount of interaction required for the acquisition and analysis and is, therefore, suitable for studies that require morphologically driven, wide-scale (e.g., whole gland) analysis of complex tissue samples or cultures
— id: 83151, year: 2002, vol: 59, page: 522, stat: Journal Article,

Redox-mediated TGFbeta activation: Site specific oxidation of TGFbeta1
Jobling, Michael; Ledbetter, Steven; Finnigan, Monica; Lawrence, Cathy; Barcellos-Hoff, Mary Helen
2002 ;43(6):385-385, Proceedings (American Association for Cancer Research)
— id: 104683, year: 2002, vol: 43, page: 385, stat: Journal Article,

Applications of quantitative digital image analysis to breast cancer research
Ortiz De Solorzano, C; Costes, S; Callahan, D E; Parvin, B; Barcellos-Hoff, M H
2002 Oct 15;59(2):119-127, Microscopy research & technique
Our studies of radiogenic carcinogenesis in mouse and human models of breast cancer are based on the view that cell phenotype, microenvironment composition, communication between cells and within the microenvironment are important factors in the development of breast cancer. This is complicated in the mammary gland by its postnatal development, cyclic evolution via pregnancy and involution, and dynamic remodeling of epithelial-stromal interactions, all of which contribute to breast cancer susceptibility. Microscopy is the tool of choice to examine cells in context. Specific features can be defined using probes, antibodies, immunofluorescence, and image analysis to measure protein distribution, cell composition, and genomic instability in human and mouse models of breast cancer. We discuss the integration of image acquisition, analysis, and annotation to efficiently analyze large amounts of image data. In the future, cell and tissue image-based studies will be facilitated by a bioinformatics strategy that generates multidimensional databases of quantitative information derived from molecular, immunological, and morphological probes at multiple resolutions. This approach will facilitate the construction of an in vivo phenotype database necessary for understanding when, where, and how normal cells become cancer
— id: 83148, year: 2002, vol: 59, page: 119, stat: Journal Article,

BioSig: An imaging bioinformatic system for studying phenomics
Parvin, B; Yang, Q; Fontenay, G; Barcellos-Hoff, MH
2002 ;35(7):65-+, Computer (IEEE Computer Society)
Using genomic information to understand complex organisms requires comprehensive knowledge of the dynamics of phenotype generation and maintenance. A phenotype results from selective expression of the genome, creating a history of the cell and its response to the extracellular environment. Defining cell phenomes requires tracking the kinetics and quantities of multiple constituent proteins, their cellular context, and their morphological features in large populations. The BioSig imaging bioinformatic system for characterizing phenomics answers these challenges. The BioSig approach to microscopy and quantitative image analysis helps to build a more detailed picture of the signaling that occurs between cells as a response to exogenous stimulus such as radiation or as a consequence of endogenous programs leading to biological functions. The system provides a data model for capturing experimental annotations and variables, computational techniques for summarizing large numbers of images, and a distributed architecture that facilitates distant collaboration. $$:
— id: 104684, year: 2002, vol: 35, page: 65, stat: Journal Article,

Microscopy environment for quantitative spatial and temporal analysis of multicellular interactions
Sudar, D; Parvin, B; Callahan, DE; Schwarz, RI; Knowles, DW; de Solorzano, CO; Barcellos-Hoff, MH
Three-dimensional and multidimensional microscopy : image acquisition and processing IX Bellingham, WA : SPIE, 2002,
Quantitative analysis of spatial and temporal concurrent responses of multiple markers in 3-dimensional cell cultures is hampered by the routine mode of sequential image acquisition, measurement and analysis of specific targets. A system was developed for detailed analysis of multi-dimensional, time-sequence responses and in order to relate features in novel and meaningful ways that will further our understanding of basic biology. Optical sectioning of the 3D structures is achieved with structured light illumination using the Wilson grating as described by Lanni. The automated microscopy system can image multicellular structures and track dynamic events, and is equipped for simultaneous/ sequential imaging of multiple fluorescent markers. Computer-controlled perfusion of external stimuli into the culture system allows (i) real-time observations of multiple cellular responses and (ii) automatic and intelligent adjustment of experimental parameters. This creates a feedback loop in real-time that directs desired responses in a given experiment. On-line image analysis routines provide cell-by-cell measurement results through segmentation and feature extraction (i.e. intensity, localization, etc.), and quantitation of meta-features such as dynamic responses of cells or correlations between different cells. Off-line image and data analysis is used to derive models of the processes involved, which will deepen the understanding of the basic biology
— id: 5230, year: 2002, vol: , page: 47, stat: Chapter,

Phenotypic reversion or death of cancer cells by altering signaling pathways in three-dimensional contexts
Wang, Fei; Hansen, Rhonda K; Radisky, Derek; Yoneda, Toshiyuki; Barcellos-Hoff, Mary Helen; Petersen, Ole W; Turley, Eva A; Bissell, Mina J
2002 Oct 2;94(19):1494-1503, Journal of the National Cancer Institute
BACKGROUND: We previously used a three-dimensional (3D) reconstituted basement membrane (rBM) assay to demonstrate that tumorigenic HMT-3522 T4-2 human breast cells can be induced to form morphologically normal structures ('reversion') by treatment with inhibitors of beta1 integrin, the epidermal growth factor receptor (EGFR), or mitogen-activated protein kinase (MAPK). We have now used this assay to identify reversion and/or death requirements of several more aggressive human breast cancer cell lines. METHODS: Breast tumor cell lines MCF7, Hs578T, and MDA-MB-231 were cultured in 3D rBM and treated with inhibitors of beta1 integrin, MAPK, or phosphatidylinositol 3-kinase (PI3K). MDA-MB-231 cells, which lack E-cadherin, were transfected with an E-cadherin cDNA. The extent of reversion was assessed by changes in morphology and polarity, growth in 3D rBM or soft agar, level of invasiveness, and tumor formation in nude mice. RESULTS: All three cell lines showed partial reversion (MCF7 the greatest and Hs578T the least) of tumorigenic properties treated with a single beta1 integrin, MAPK, or PI3K inhibitor. Combined inhibition of beta1 integrin and either PI3K or MAPK resulted in nearly complete phenotypic reversion (MDA-MB-231, MCF7) or in cell death (Hs578T). E-cadherin-transfected MDA-MB-231 cells showed partial reversion, but exposure of the transfectants to an inhibitor of beta1 integrin, PI3K, or MAPK led to nearly complete reversion. CONCLUSION: The 3D rBM assay can be used to identify signaling pathways that, when manipulated in concert, can lead to the restoration of morphologically normal breast structures or to death of the tumor cells, even highly metastatic cells. This approach may be useful to design therapeutic intervention strategies for aggressive breast cancers
— id: 83147, year: 2002, vol: 94, page: 1494, stat: Journal Article,

Epigenetics and breast cancer
Asch, B B; Barcellos-Hoff, M H
2001 Apr;6(2):151-152, Journal of mammary gland biology & neoplasia
— id: 83134, year: 2001, vol: 6, page: 151, stat: Journal Article,

Epigenetic mechanisms controlling gene expression in breast cancer
Asch, Bonnie B; Barcellos-Hoff, Mary Helen
New York NY : Kluwer Academic/Plenum, 2001,
— id: 2094, year: 2001, vol: , page: , stat: ,

It takes a tissue to make a tumor: epigenetics, cancer and the microenvironment
Barcellos-Hoff, M H
2001 Apr;6(2):213-221, Journal of mammary gland biology & neoplasia
How do normal tissues limit the development of cancer? This review discusses the evidence that normal cells effectively restrict malignant behavior, and that such tissue forces must be subjugated to establish a tumor. The action of ionizing radiation will be specifically discussed regarding the disruption of the microenvironment that promotes the transition from preneoplastic to neoplastic growth. Unlike the highly unpredictable nature of genetic mutations, the response of normal cells to radiation damage follows an epigenetic program similar to wound healing and other damage responses. Our hypothesis is that the persistent disruption of the microenvironment in irradiated tissue compromises its ability to suppress carcinogenesis
— id: 83135, year: 2001, vol: 6, page: 213, stat: Journal Article,

Three down and counting: the transformation of human mammary cells from normal to malignant in three steps
Barcellos-Hoff, M H
2001 Apr;7(4):142-143, Trends in molecular medicine
An array of genetic mutations associated with human breast cancers has been identified. However, which specific combination of mutations permit normal cells to form breast cancer remains unknown. Elenbaas et al. recently described an experimental system for studying the genetic requirements for the development of breast cancer
— id: 83131, year: 2001, vol: 7, page: 142, stat: Journal Article,

Extracellular signaling through the microenvironment: a hypothesis relating carcinogenesis, bystander effects, and genomic instability
Barcellos-Hoff, M H; Brooks, A L
2001 Nov;156(5 Pt 2):618-627, Radiation research
Cell growth, differentiation and death are directed in large part by extracellular signaling through the interactions of cells with other cells and with the extracellular matrix; these interactions are in turn modulated by cytokines and growth factors, i.e. the microenvironment. Here we discuss the idea that extracellular signaling integrates multicellular damage responses that are important deterrents to the development of cancer through mechanisms that eliminate abnormal cells and inhibit neoplastic behavior. As an example, we discuss the action of transforming growth factor beta (TGFB1) as an extracellular sensor of damage. We propose that radiation-induced bystander effects and genomic instability are, respectively, positive and negative manifestations of this homeostatic process. Bystander effects exhibited predominantly after a low-dose or a nonhomogeneous radiation exposure are extracellular signaling pathways that modulate cellular repair and death programs. Persistent disruption of extracellular signaling after exposure to relatively high doses of ionizing radiation may lead to the accumulation of aberrant cells that are genomically unstable. Understanding radiation effects in terms of coordinated multicellular responses that affect decisions regarding the fate of a cell may necessitate re-evaluation of radiation dose and risk concepts and provide avenues for intervention
— id: 83136, year: 2001, vol: 156, page: 618, stat: Journal Article,

Immunodetection of 3-nitrotyrosine in the liver of zymosan-treated rats with a new monoclonal antibody: comparison to analysis by HPLC
Girault, I; Karu, A E; Schaper, M; Barcellos-Hoff, M H; Hagen, T; Vogel, D S; Ames, B N; Christen, S; Shigenaga, M K
2001 Dec 1;31(11):1375-1387, Free radical biology & medicine
Zymosan-induced peritonitis is associated with an increased production of reactive nitrogen oxides that may contribute to the often-observed failure of multiple organ systems in this model of acute inflammation. Quantitative biochemical evidence is provided for a marked 13-fold increase in protein-bound 3-nitrotyrosine (NTyr), a biomarker of reactive nitrogen oxides, in liver tissue of zymosan-treated rats. In order to investigate the localization of NTyr in this affected tissue, a monoclonal antibody, designated 39B6, was raised against 3-(4-hydroxy-3-nitrophenylacetamido) propionic acid-bovine serum albumin conjugate and its performance characterized. 39B6 was judged by competition ELISA to be approximately 2 orders of magnitude more sensitive than a commercial anti-NTyr monoclonal antibody. Binding characteristics of 39B6 were similar, but not identical, to that of a commercial affinity-purified polyclonal antibody in ELISA and immunohistochemical analyses. Western blot experiments revealed high specificity of 39B6 against NTyr and increased immunoreactivity of specific proteins from liver tissue homogenates of zymosan-treated rats. Immunohistochemical analysis of liver sections indicated a marked zymosan-induced increase in immunofluorescent staining, which was particularly intense in or adjacent to nonparenchymal cells, but not in the parenchymal cells of this tissue. Quantitative analysis of fractions enriched in these cell populations corroborated the immunofluorescent data, although the relative amounts detected in response to zymosan treatment was greatly reduced compared to whole liver tissue. These results demonstrate the high specificity of the newly developed antibody and its usefulness in Western blot and immunohistochemical analysis for NTyr, confirm the presence of NTyr by complementary methods, and suggest the possible involvement of reactive nitrogen oxides in hepatic vascular dysfunction
— id: 83139, year: 2001, vol: 31, page: 1375, stat: Journal Article,

Loss of heterozygosity at the mannose 6-phosphate insulin-like growth factor 2 receptor (M6P/IGF2R) locus predisposes patients to radiation-induced lung injury
Kong, F M; Anscher, M S; Sporn, T A; Washington, M K; Clough, R; Barcellos-Hoff, M H; Jirtle, R L
2001 Jan 1;49(1):35-41, International journal of radiation oncology biology physics
PURPOSE: To investigate the relationship between loss of heterozygosity (LOH) at the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) gene locus and the development of radiation-induced lung injury. MATERIAL AND METHODS: Thirty-five lung cancer patients with both stored plasma for Transforming Growth Factor beta1 (TGFbeta1) analysis and sufficient quantities of archival pathology tissue to screen for LOH were studied. All patients had been treated with thoracic radiotherapy for their malignancy and had radiographically detectable tumor present before beginning radiotherapy. Tumor and normal cells were microdissected from archival lung cancer pathology specimens. Two polymorphisms in the 3' untranslated region of the M6P/IGF2R were used to screen for LOH. Plasma TGFbeta1 levels were measured using acid-ethanol extraction and an ELISA. TGFbeta1 and M6P/IGF2R protein expression was estimated by immunofluorescence and immunohistochemical staining. Symptomatic radiation pneumonitis was scored according to National Cancer Institute Common Toxicity Criteria without knowledge of the results of TGFbeta or LOH analyses. RESULTS: Of the 35 patients, 10 were homozygous for this polymorphism (noninformative) and were excluded. Of the 25 informative patients, 13 had LOH. Twelve of 13 patients with LOH had increased pretreatment plasma TGFbeta1 levels, vs. 3/12 patients without LOH (p < 0.01). A decrease or loss of M6P/IGF2R protein in the malignant cell accompanied by increased latent TGFbeta1 protein in extracellular matrix and tumor stroma was found in tumors with LOH, suggesting that this mutation resulted in loss of function of the receptor. Seven of 13 (54%) LOH patients developed symptomatic radiation-induced lung injury vs. 1/12 (8%) of patients without LOH (p = 0.05). CONCLUSION: Loss of the M6P/IGF2R gene strongly correlates with the development of radiation pneumonitis after thoracic radiotherapy (RT). Furthermore, patients with LOH (in the setting of measurable tumor) are much more likely to have elevated plasma TGFbeta, suggesting an inability to normally process this cytokine. Thus, loss of the M6P/IGF2R gene may predispose patients to the development of radiation-induced lung injury
— id: 83129, year: 2001, vol: 49, page: 35, stat: Journal Article,

Progesterone receptor signaling controls MMP activities in mouse mammary glands
Simian, M; Barcellos-Hoff, MH; Bissell, MJ; Harris, GS
2001 ;12(1):1090-1090, Molecular biology of the cell
— id: 104685, year: 2001, vol: 12, page: 1090, stat: Journal Article,

Transforming growth factor-beta and breast cancer: Mammary gland development
Barcellos-Hoff, M H; Ewan, K B
2000 ;2(2):92-99, Breast cancer research
Transforming growth factor (TGF)-beta1 is a pluripotent cytokine that profoundly inhibits epithelial proliferation, induces apoptosis, and influences morphogenesis by mediating extracellular matrix deposition and remodeling. The physiologic roles of the action of TGF-beta in mammary gland, indeed in most tissues, are poorly understood. In order to understand the actions of TGF-beta, we need to take into account the complexity of its effects on different cell types and the influence of context on cellular responses. This task is further compounded by multiple mechanisms for regulating TGF-beta transcription, translation, and activity. One of the most significant factors that obscures the action of TGF-beta is that it is secreted as a stable latent complex, which consists of the 24-kDa cytokine and the 80-kDa dimer of its prepro region, called latency-associated peptide. Latency imposes a critical restraint on TGF-beta activity that is often overlooked.The extracellular process known as activation, in which TGF-beta is released from the latent complex, is emphasized in the present discussion of the role of TGF-beta in mammary gland development. Definition of the spatial and temporal patterns of latent TGF-beta activation in situ is essential for understanding the specific roles that TGF-beta plays during mammary gland development, proliferation, and morphogenesis
— id: 83130, year: 2000, vol: 2, page: 92, stat: Journal Article,

Irradiated mammary gland stroma promotes the expression of tumorigenic potential by unirradiated epithelial cells
Barcellos-Hoff, M H; Ravani, S A
2000 Mar 1;60(5):1254-1260, Cancer research
We have shown that ionizing radiation, a known carcinogen of human breast, elicits rapid, persistent, and global changes in the mammary microenvironment as evidenced by altered extracellular matrix composition and growth factor activities. To address whether these events contribute to radiogenic carcinogenesis, we evaluated the effect of irradiated mammary stroma on the neoplastic potential of COMMA-D mammary epithelial cells. Although COMMA-D cells harbor mutations in both alleles of p53, they are nontumorigenic when injected s.c. into syngeneic hosts. Unirradiated COMMA-D cells transplanted to mammary fat pads cleared previously of epithelia preferentially formed tumors in irradiated hosts. Tumor incidence at 6 weeks was 81% +/- 12 SE when animals were irradiated with 4 Gy, 3 days prior to transplantation, compared with 19% +/- 2 SE (P < 0.005) in sham-irradiated hosts. This effect was evident when cells were transplanted 1 to 14 days after irradiation. Furthermore, tumors were significantly larger (243.1 +/- 61.3 mm3 versus 30.8 +/- 8.7 mm3) and arose more quickly (100% by 6 weeks versus 39% over 10 weeks in sham hosts) in fat pads in irradiated hosts. The contribution of local versus systemic effects was evaluated using hemibody (left versus right) irradiation; tumors formed only in fat pads on the irradiated side. These data indicate that radiation-induced changes in the stromal microenvironment can contribute to neoplastic progression in vivo. Disruption of solid tissue interactions is a heretofore unrecognized activity of ionizing radiation as a carcinogen
— id: 83119, year: 2000, vol: 60, page: 1254, stat: Journal Article,

Particle irradiation induces FGF2 expression in normal human lens cells
Chang, P Y; Bjornstad K, A; Chang, E; McNamara, M; Barcellos-Hoff, M H; Lin, S P; Aragon, G; Polansky, J R; Lui, G M; Blakely, E A
2000 Nov;154(5):477-484, Radiation research
Particle Irradiation Induces FGF2 Expression in Normal Human Lens Cells. Particle radiations, including both proton and helium-ion beams, have been used to successfully treat choroidal melanoma, but with the complication of radiation-induced cataract. We have investigated a role for radiation-induced changes in the expression of basic fibroblast growth factor (FGF2) gene expression as part of the mechanism(s) underlying lens cell injury associated with cataract. Normal human lens epithelial (HLE) cells were cultured in vitro on extracellular matrix (ECM) originated from bovine corneal endothelial cells. This study reports evidence for rapid but transient induction of FGF2 transcripts, an increase of between 5- and 8-fold, within 0.5 h after exposure to particle radiation, followed by another wave of increased transcription at 2-3 h postirradiation. Immunofluorescence results confirm the enhanced levels of FGF2 protein rapidly after exposure to protons or helium ions, followed by another wave of increased activity unique to helium at 6 h postirradiation. This second wave of increased immunoreactivity was not observed in the proton-irradiated samples. Total FGF2 protein analysis after helium-ion exposures shows induced expression of three FGF2 isoforms, with an increase of up to 2-fold in the 18-kDa low-molecular-weight species. Studies of the effects of protons on individual FGF2 protein isoforms are in progress. Several mechanisms involving a role for FGF2 in radiation-induced cataract are discussed
— id: 83127, year: 2000, vol: 154, page: 477, stat: Journal Article,

Quantitative image analysis of laminin immunoreactivity in skin basement membrane irradiated with 1 GeV/nucleon iron particles
Costes, S; Streuli, C H; Barcellos-Hoff, M H
2000 Oct;154(4):389-397, Radiation research
We previously reported that laminin immunoreactivity in mouse mammary epithelium is altered shortly after whole-body irradiation with 0.8 Gy from 600 MeV/nucleon iron ions but is unaffected after exposure to sparsely ionizing radiation. This observation led us to propose that the effect could be due to protein damage from the high ionization density of the ion tracks. If so, we predicted that it would be evident soon after radiation exposure in basement membranes of other tissues and would depend on ion fluence. To test this hypothesis, we used immunofluorescence, confocal laser scanning microscopy, and image segmentation techniques to quantify changes in the basement membrane of mouse skin epidermis. At 1 h after exposure to 1 GeV/nucleon iron ions with doses from 0.03 to 1.6 Gy, neither the visual appearance nor the mean pixel intensity of laminin in the basement membrane of mouse dorsal skin epidermis was altered compared to sham-irradiated tissue. This result does not support the hypothesis that particle traversal directly affects laminin protein integrity. However, the mean pixel intensity of laminin immunoreactivity was significantly decreased in epidermal basement membrane at 48 and 96 h after exposure to 0.8 Gy 1 GeV/nucleon iron ions. We confirmed this effect with two additional antibodies raised against affinity-purified laminin 1 and the E3 fragment of the long-arm of laminin 1. In contrast, collagen type IV, another component of the basement membrane, was unaffected. Our studies demonstrate quantitatively that densely ionizing radiation elicits changes in skin microenvironments distinct from those induced by sparsely ionizing radiation. Such effects may might contribute to the carcinogenic potential of densely ionizing radiation by altering cellular signaling cascades mediated by cell-extracellular matrix interactions
— id: 83126, year: 2000, vol: 154, page: 389, stat: Journal Article,

The mammary phenotype of TGF-beta 1 null heterozygotes indicates that TGF-beta actively restrains ovarian steroid hormone-induced proliferation
Ewan, KB; Wakefield, L; Shyamala, WG; Barcellos-Hoff, MH
2000 ;11(4):140-140, Molecular biology of the cell
— id: 104686, year: 2000, vol: 11, page: 140, stat: Journal Article,

Three-dimensional (3D) cultures of human mammary epithelial cells treated with low-dose radiation and transforming growth factor-beta (TGF beta) exhibit altered cell-cell and cell-extracellular matrix interactions
Henshall-Powell, RL; Park, CC; Bissell, MJ; Barcellos-Hoff, MH
2000 ;11(4):2485-2485, Molecular biology of the cell
— id: 104687, year: 2000, vol: 11, page: 2485, stat: Journal Article,

The influence of the microenvironment on the malignant phenotype
Park, C C; Bissell, M J; Barcellos-Hoff, M H
2000 Aug;6(8):324-329, Molecular medicine today
Normal tissue homeostasis is maintained by dynamic interactions between epithelial cells and their microenvironment. As tissue becomes cancerous, there are reciprocal interactions between neoplastic cells, adjacent normal cells such as stroma and endothelium, and their microenvironments. The current dominant paradigm wherein multiple genetic lesions provide both the impetus for, and the Achilles heel of, cancer might be inadequate to understand cancer as a disease process. In the following brief review, we will use selected examples to illustrate the influence of the microenvironment in the evolution of the malignant phenotype. We will also discuss recent studies that suggest novel therapeutic interventions might be derived from focusing on microenvironment and tumor cells interactions
— id: 83124, year: 2000, vol: 6, page: 324, stat: Journal Article,

Low-dose ionizing radiation and transforming growth factor-beta (TGF-beta) are synergistic in altering interactions of human mammary epithelial cells with basement membrane (BM) cultured in a 3-dimensional (3D) matrix
Park, Catherine C.; Wang, Fei; Bissell, Mina J.; Barcellos-Hoff, Mary Helen
2000 ;6(41):376-376, Proceedings (American Association for Cancer Research)
— id: 104688, year: 2000, vol: 6, page: 376, stat: Journal Article,

BioSig: a bioinformatic system for studying the mechanism of inter-cell signaling
Parvin, B.; Cong, G.; Fontenay, G.; Taylor, J.; Henshall, R.; Barcellos-Hoff, M.H.
IEEE International Symposium on Bio-Informatics and Biomedical Engineering Los Alamitos, Ca. : IEEE Computer Society, 2000,
Mapping inter-cell signaling pathways requires an integrated view of experimental and informatic protocols. BioSig provides the foundation of cataloging inter-cell responses as a function of particular conditioning, treatment, staining, etc. for either in vivo or in vitro experiments. This paper outlines the system architecture, a functional data model for representing experimental protocols, algorithms for image analysis, and the required statistical analysis. The architecture provides remote shared operation of an inverted optical microscope, and couples instrument operation with images acquisition and annotation. The information is stored in an object-oriented database. The algorithms extract structural information such as morphology and organization, and map it to functional information such as inter-cellular responses. An example of usage of this system is included
— id: 5231, year: 2000, vol: , page: ?, stat: Chapter,

Modification of multiple pathways revert the malignant phenotype of metastatic mammary carcinoma cells MDA-MB-231
Wang, Fei; Boudreau, Rosanne M.; Yoneda, Toshiyuki; Barcellos-Hoff, Mary Helen; Bissell, Mina J.
2000 ;6(41):473-473, Proceedings (American Association for Cancer Research)
— id: 104689, year: 2000, vol: 6, page: 473, stat: Journal Article,

Characterization of nitrosated latency-associated peptide (LAP), the endogenous neutralizer of the iNOS-inhibitor cytokine transforming growth factor-beta1 (TGF-beta1)
Zamora, Ruben; Wink, David; Barcellos-Hoff, Mary Helen; Billiar, Timothy R.; Vodovotz, Yoram
2000 ;4(3):313-313, Nitric oxide : biology & chemistry
— id: 104690, year: 2000, vol: 4, page: 313, stat: Journal Article,

Immunocytochemical localization of latent transforming growth factor-beta1 activation by stimulated macrophages
Chong, H; Vodovotz, Y; Cox, G W; Barcellos-Hoff, M H
1999 Mar;178(3):275-283, Journal of cellular physiology
Transforming growth factor-beta1 (TGF-beta) is secreted in a latent form consisting of mature TGF-beta noncovalently associated with its amino-terminal propeptide, which is called latency associated peptide (LAP). Biological activity depends upon the release of TGF-beta from the latent complex following extracellular activation, which appears to be the key regulatory mechanism controlling TGF-beta action. We have identified two events associated with latent TGF-beta (LTGF-beta) activation in vivo: increased immunoreactivity of certain antibodies that specifically detect TGF-beta concomitant with decreased immunoreactivity of antibodies to LAP. Macrophages stimulated in vitro with interferon-gamma and lipopolysaccharide reportedly activate LTGF-beta via cell membrane-bound protease activity. We show through dual immunostaining of paraformaldehyde-fixed macrophages that such physiological TGF-beta activation is accompanied by a loss of LAP immunoreactivity with concomitant revelation of TGF-beta epitopes. The induction of TGF-beta immunoreactivity colocalized with immunoreactive betaglycan/RIII in activated macrophages, suggesting that LTGF-beta activation occurs on the cell surface. Confocal microscopy of metabolically active macrophages incubated with antibodies to TGF-beta and betaglycan/RIII prior to fixation supported the localization of activation to the cell surface. The ability to specifically detect and localize LTGF-beta activation provides an important tool for studies of its regulation
— id: 83328, year: 1999, vol: 178, page: 275, stat: Journal Article,

Radiation-induced alterations in rat mesangial cell Tgfb1 and Tgfb3 gene expression are not associated with altered secretion of active Tgfb isoforms
O'Malley, Y; Zhao, W; Barcellos-Hoff, M H; Robbins, M E
1999 Dec;152(6):622-628, Radiation research
Despite evidence of selective radiation-induced modulation of expression of rat mesangial cell Tgfb gene isoforms, it is unclear whether these changes in gene expression are accompanied by changes in protein secretion. To address this issue, primary cultures of rat mesangial cells (passage number 6- 11) were placed in serum-free medium 24 h prior to irradiation with single doses of 0.5-20 Gy of (137)Cs gamma rays. After irradiation, cells were maintained in serum-free medium for a further 24 h. Irradiation of quiescent mesangial cells resulted in a significant (P </= 0.05) dose-independent increase in steady-state levels of Tgfb1 mRNA 24 h postirradiation. In contrast, steady-state levels of Tgfb3 mRNA exhibited a dose-dependent reduction after irradiation; this reduction was statistically significant after doses of 5 and 10 Gy compared to control cells (P </= 0.05). These radiation-induced changes in Tgfb gene expression were associated with modest increases in Tgfb protein as determined using mink lung epithelial cells transfected with the Pai1 promoter-luciferase construct. Twenty-four hours after a single dose of 5 Gy, the total Tgfb protein secreted by the mesangial cells was 181 +/- 2.0% of that secreted by unirradiated control cells (P </= 0.01). However, this increase was seen in terms of latent Tgfb protein; radiation failed to increase significantly the amount of active Tgfb protein secreted by mesangial cells. Both quiescent and irradiated rat mesangial cells secreted active Tgfb as primarily the Tgfb3 isoform. These data reinforce the need to interpret changes in Tgfb gene expression with caution
— id: 83118, year: 1999, vol: 152, page: 622, stat: Journal Article,

DeepView: a channel for distributed microscopy and informatics
Parvin, B.; Taylor, J.; Cong, G.; O'Keefe, M.A.; Barcellos-Hoff, M.H.
SC 99 : proceedings of the ACM/IEEE SC99 Conference on high performance networking and computing [New York, NY] : ACM, 1999,
This paper outlines the requirements, architecture, and design of a 'Microscopy Channel' over the wide area network. A microscopy channel advertises a listing of available online microscopes, where users can seamlessly participate in an experiment, acquire expert opinions, collect and process data, and store this information in their electronic notebook. The proposed channel is a collaborative problem solving environment (CPSE) that allows for both synchronous and asynchronous collaboration. Our testbed includes several unique electron and optical microscopes with applications ranging from material science to cell biology. We have studied current commercial CORBA services and concluded that three basic services are needed to meet the extensibility and functionality constraints. These include: Instrument Services (IS), Exchange Services (ES), and Computational Services (CS). These services sit on top of CORBA and its enabling services (naming, trading, security, and notification). IS provide a layer of abstraction for controlling any type of microscope. ES provide a common set of utilities for information management and transaction. CS provide the analytical capabilities needed for online microscopy and PSE
— id: 5232, year: 1999, vol: , page: ?, stat: Chapter,

Ionizing radiation accelerates aortic lesion formation in fat-fed mice via SOD-inhibitable processes
Tribble, D L; Barcellos-Hoff, M H; Chu, B M; Gong, E L
1999 Jun;19(6):1387-1392, Arteriosclerosis, thrombosis, & vascular biology
Ionizing radiation promotes formation of reactive oxygen species, including the superoxide anion (O2-). To evaluate whether O2- or O2--mediated perturbations may contribute to the known atherogenic effects of radiation, we examined aortic lesion formation in irradiated C57BL/6 mice and evaluated the effects of CuZn-superoxide dismutase (CuZn-SOD) overexpression. Ten-week-old mice were exposed to a 2-, 4-, or 8-Gy dose of 250-keV x-rays to the upper thorax and then placed on a high-fat diet for 18 weeks. Based on quantitative lipid staining of serial sections of the proximal aorta, mean lesion area was increased with increasing radiation dose and was 3-fold greater in 8-Gy-irradiated than sham-irradiated mice (7800+/-2140 versus 2635+/-709 micrometer(2), P<0.05). These effects were absolutely dependent on a high-fat diet, which had to be introduced within 1 to 2 weeks of the radiation exposure, suggesting the early involvement of atherogenic lipoproteins that were elevated in response to the diet. The importance of radiation-induced oxidative stress was supported by the observation of a 2-fold lower mean lesion area in irradiated CuZn-SOD transgenic mice than in their irradiated, nontransgenic littermates (3026+/-1590 versus 6102+/-1834 micrometer(2), P<0.05). Lucigenin-enhanced chemiluminescence, used as an index of aortic O2- concentrations, was significantly elevated in the postradiation period, and this response was reduced in CuZn-SOD transgenics. On the basis of these results, we propose that radiation may be a useful tool for initiating oxidative or redox-regulated events that promote atherogenesis and for testing the antiatherogenic properties of antioxidants
— id: 83114, year: 1999, vol: 19, page: 1387, stat: Journal Article,

Regulation of transforming growth factor beta1 by nitric oxide
Vodovotz, Y; Chesler, L; Chong, H; Kim, S J; Simpson, J T; DeGraff, W; Cox, G W; Roberts, A B; Wink, D A; Barcellos-Hoff, M H
1999 May 1;59(9):2142-2149, Cancer research
Many tumor cells or their secreted products suppress the function of tumor-infiltrating macrophages. Tumor cells often produce abundant transforming growth factor beta1 (TGF-beta1), which in addition to other immunosuppressive actions suppresses the inducible isoform of NO synthase. TGF-beta1 is secreted in a latent form, which consists of TGF-beta1 noncovalently associated with latency-associated peptide (LAP) and which can be activated efficiently by exposure to reactive oxygen species. Coculture of the human lung adenocarcinoma cell line A549 and ANA-1 macrophages activated with IFN-gamma plus lipopolysaccharide resulted in increased synthesis and activation of latent TGF-beta1 protein by both A549 and ANA-1 cells, whereas unstimulated cultures of either cell type alone expressed only latent TGF-beta1. We investigated whether exposure of tumor cells to NO influences the production, activation, or activity of TGF-beta1.A549 human lung adenocarcinoma cells exposed to the chemical NO donor diethylamine-NONOate showed increased immunoreactivity of cell-associated latent and active TGF-beta1 in a time- and dose-dependent fashion at 24-48 h after treatment. Exposure of latent TGF-beta1 to solution sources of NO neither led to recombinant latent TGF-beta1 activation nor modified recombinant TGF-beta1 activity. A novel mechanism was observed, however: treatment of recombinant LAP with NO resulted in its nitrosylation and interfered with its ability to neutralize active TGF-beta1. These results provide the first evidence that nitrosative stress influences the regulation of TGF-beta1 and raise the possibility that NO production may augment TGF-beta1 activity by modifying a naturally occurring neutralizing peptide
— id: 83112, year: 1999, vol: 59, page: 2142, stat: Journal Article,

Modulation of protein expression and activity by radiation: relevance to intracoronary radiation for the prevention of restenosis
Vodovotz, Y; Mitchell, J B; Lucia, M S; McKinney, L; Kollum, M; Cottin, Y; Chan, R C; Barcellos-Hoff, M H; Waksman, R
1999 Oct-Dec;1(4):336-343, Cardiovascular radiation medicine
Restenosis is a common complication of percutaneous transluminal coronary angioplasty. Recent studies have demonstrated a striking reduction in the neointimal hyperplasia characteristic of restenosis following intracoronary radiation (IR), but the mechanisms by which radiation reduces neointima formation following balloon overstretch injury are not elucidated fully. In addition to direct antimitotic effects mediated via oxygen free radicals, ionizing radiation can induce the expression of numerous genes and thereby mediate indirect effects. Additionally, IR prevents restenosis at the cost of decreased healing and increased thrombosis, and we suggest that these adverse reactions can be modulated by adjunct pharmacology or gene-based strategies. This review discusses several genes and proteins modulated by radiation in the context of arterial injury, and their possible therapeutic relevance
— id: 83122, year: 1999, vol: 1, page: 336, stat: Journal Article,

Combinatorial modifications of multiple pathways reverts the malignant phenotype or mammary carcinoma cells MDA-MB-231
Wang, F; Yoneda, T; Barcellos-Hoff, MH; Bissell, MJ
1999 ;10(9):2024-2024, Molecular biology of the cell
— id: 104691, year: 1999, vol: 10, page: 2024, stat: Journal Article,

How do tissues respond to damage at the cellular level? The role of cytokines in irradiated tissues
Barcellos-Hoff, M H
1998 Nov;150(5 Suppl):S109-S120, Radiation research
The capacity of ionizing radiation to affect tissue function, control tumor growth and elicit pathological sequelae has been attributed in great part to its effects on cellular DNA, which, as the transmitter of genetic information, can both register damage and perpetuate it. Nonetheless, multicellular organisms function as the result of the cooperation of many cell types. What then occurs when individual cells are damaged by ionizing radiation? Is tissue response a sum of cellular effects such as cell death and DNA damage? Or does the tissue respond as a coherent unit to the damage of its parts? In this paper, data in support of the latter model that indicate a role for cytokines, in particular transforming growth factor beta1, as critical components of extracellular signaling pathways that mediate tissue response to radiation will be reviewed. The key to manipulating the consequences of radiation exposure lies in understanding the complex interplay of events initiated at the cellular level, but acting on the tissue
— id: 83325, year: 1998, vol: 150, page: S109, stat: Journal Article,

The potential influence of radiation-induced microenvironments in neoplastic progression
Barcellos-Hoff, M H
1998 Apr;3(2):165-175, Journal of mammary gland biology & neoplasia
Ionizing radiation is a complete carcinogen, able both to initiate and promote neoplastic progression and is a known carcinogen of human and murine mammary gland. Tissue response to radiation is a composite of genetic damage, cell death and induction of new gene expression patterns. Although DNA damage is believed to initiate carcinogenesis, the contribution of these other aspects of radiation response are beginning to be explored. Our studies demonstrate that radiation elicits rapid and persistent global alterations in the mammary gland microenvironment. We postulate that radiation-induced microenvironments may affect epithelial cells neoplastic transformation by altering their number or susceptibility. Alternatively, radiation induced microenvironments may exert a selective force on initiated cells and/or be conducive to progression. A key impetus for these studies is the possibility that blocking these events could be a strategy to interrupt neoplastic progression
— id: 83121, year: 1998, vol: 3, page: 165, stat: Journal Article,

Carcinogen-induced microenvironment in breast cancer
Barcellos-Hoff, Mary
[S.l. : University of California at Berkeley, 1998,
These studies address the question of how abnormal stromal-epithelial interactions affect the progression of cancer cells. Our studies in mouse mammary gland reveal that ionizing radiation, a known human breast carcinogen, elicits rapid and persistent global changes in the tissue microenvironment. If the microenvironments induced by carcinogens can shape the features and frequency of neoplastic phenotypes, then the carcinogen
— id: 2091, year: 1998, vol: , page: , stat: ,

A monoclonal antibody for 3-nitrotyrosine: Protein nitration is elevated in liver nonparenchymal cells
Shigenaga, MK; Girault, I; Karu, AE; Barcellos-Hoff, MH; Ames, BN
1998 ;25(5):187-187, Free radical biology & medicine
— id: 104692, year: 1998, vol: 25, page: 187, stat: Journal Article,

Transgenic mice carrying an imbalance in the native ratio of A to B forms of progesterone receptor exhibit developmental abnormalities in mammary glands
Shyamala, G; Yang, X; Silberstein, G; Barcellos-Hoff, M H; Dale, E
1998 Jan 20;95(2):696-701, Proceedings of the National Academy of Sciences of the United States of America
In this report we document the creation of transgenic mice in which the native ratio of A and B forms of progesterone receptor (PR) has been altered by the introduction of additional A form as transgene. We also show that in these mice there is an aberration in mammary development. In ovariectomized prepubertal PR-A transgenic mice, end buds with unusual morphology persist after ovariectomy, and in young adult nonovariectomized mice, mammary glands have extensive lateral branching. The glands of adult mice also exhibit ductal hyperplasia with a disorganized basement membrane and decreased cell-cell adhesion, features commonly associated with neoplasia. Because progesterone is a mitogenic hormone in mammary glands and PR is required for mammary development, these data provide direct evidence that in vivo a regulated expression of the two isoforms of PR is critical for appropriate cellular response to progesterone and that for mammary glands this may have major implications to carcinogenesis
— id: 83320, year: 1998, vol: 95, page: 696, stat: Journal Article,

Activation of latent transforming growth factor-beta1 by oxidative and nirosative stress: Role of heme, nitroxyl anion, and nitric oxide
Vodovotz, Y.; Chesler, L.; Simpson, J. L.; Chong, H.; Kim, S.-J.; Cox, G.; Degraff, W.; Roberts, A. B.; Barcellos-Hoff, M. H.; Wink, D. A.
1998 ;2(2):114-114, Nitric oxide : biology & chemistry
— id: 104693, year: 1998, vol: 2, page: 114, stat: Journal Article,

Latent transforming growth factor beta1 activation in situ: quantitative and functional evidence after low-dose gamma-irradiation
Ehrhart, E J; Segarini, P; Tsang, M L; Carroll, A G; Barcellos-Hoff, M H
1997 Oct;11(12):991-1002, FASEB journal
The biological activity of transforming growth factor beta1 (TGF-beta) is controlled by its secretion as a latent complex in which it is noncovalently associated with latency-associated peptide (LAP). Activation is the extracellular process in which TGF-beta is released from LAP, and is considered to be a primary regulatory control. We recently reported rapid and persistent changes in TGF-beta immunoreactivity in conjunction with extracellular matrix remodeling in gamma-irradiated mouse mammary gland. Our hypothesis is that these specific changes in immunoreactivity are indicative of latent TGF-beta activation. In the present study, we determined the radiation dose response and tested whether a functional relationship exists between radiation-induced TGF-beta and collagen type III remodeling. After radiation exposures as low as 0.1 Gy, we detected increased TGF-beta immunoreactivity in the mammary epithelium concomitant with decreased LAP immunostaining, which are events consistent with activation. Quantitative image analysis demonstrated a significant (P=0.0005) response at 0.1 Gy without an apparent threshold and a linear dose response to 5 Gy. However, in the adipose stroma, loss of LAP demonstrated a qualitative threshold at 0.5 Gy. Loss of LAP paralleled induction of collagen III immunoreactivity in this tissue compartment. We tested whether TGF-beta mediates collagen III expression by treating animals with TGF-beta panspecific monoclonal antibody, 1D11.16, administered i.p. shortly before irradiation. Radiation-induced collagen III staining in the adipose stroma was blocked in an antibody dose-dependent manner, which persisted through 7 days postirradiation. RNase protection assay revealed that radiation-induced elevation of total gland collagen III mRNA was also blocked by neutralizing antibody treatment. These data provide functional confirmation of the hypothesis that radiation exposure leads to latent TGF-beta activation, support our interpretation of the reciprocal shift in immunoreactivity as evidence of activation, and implicate TGF-beta as a mediator of tissue response to ionizing radiation. The sensitivity of activation to low radiation doses points to a potential role for TGF-beta in orchestrating tissue response to oxidative stress. As such, radiation may be useful as a probe to delineate the consequences of latent TGF-beta activation in situ
— id: 83319, year: 1997, vol: 11, page: 991, stat: Journal Article,

Immunohistochemical localization of transforming growth factor beta and tumor necrosis factor alpha in the lungs of fibrosis-prone and "non-fibrosing" mice during the latent period and early phase after irradiation
Franko, A J; Sharplin, J; Ghahary, A; Barcellos-Hoff, M H
1997 Feb;147(2):245-256, Radiation research
To evaluate the possibility that TGF-beta and TNF-alpha are involved in fibrosis induced in mouse lung by irradiation, the proportion of cells immunoreactive for each was compared in two strains of mice. C3HeB/FeJ mice develop only classical pneumonitis during the early phase, whereas C57L/J mice develop small, tightly packed areas of inflammation which undergo fibrosis during the latent period, and exhibit progressive fibrosis of large regions of intense inflammation during the early phase. Very few cells were immunoreactive for an antibody to the latency-associated peptide (LAP) of TGF-beta during the latent period in C3HeB/FeJ mice, and no cells were positive during the early phase. In contrast, between 0.7 and 10% of cells were positive in C57L/J mice in lesions without fibrosis and in lesions in the early stages of fibrosis. Fibroblasts positive for LAP were seen only in lesions containing fibrosis. A similar pattern of immunoreactivity was seen in C57L/J mice using an antibody which recognizes active TGF-beta, with the exception that positive fibroblasts were observed within areas of inflammation without fibrosis. Thus the association of active TGF-beta with fibroblasts might be a characteristic of the initiation of fibrosis in this model. TNF-alpha was detected in macrophages in all classes of lesions, and minor differences between the strains did not appear to be biologically meaningful
— id: 83312, year: 1997, vol: 147, page: 245, stat: Journal Article,

Transforming growth factor-beta in breast cancer: a working hypothesis
Reiss, M; Barcellos-Hoff, M H
1997 Aug;45(1):81-95, Breast cancer research & treatment
Transforming Growth Factor-beta (TGF beta) is the most potent known inhibitor of the progression of normal mammary epithelial cells through the cell cycle. During the early stages of breast cancer development, the transformed epithelial cells appear to still be sensitive to TGF beta-mediated growth arrest, and TGF beta can act as an anti-tumor promoter. In contrast, advanced breast cancers are mostly refractory to TGF beta-mediated growth inhibition and produce large amounts of TGF beta, which may enhance tumor cell invasion and metastasis by its effects on extracellular matrix. We postulate that this seemingly paradoxical switch in the responsiveness of tumor cells to TGF beta during progression is the consequence of the activation of the latent TGF beta that is produced and deposited into the tumor microenvironment, thereby driving the clonal expansion of TGF beta-resistant tumor cells. While tumor cells themselves may activate TGF beta, recent observations suggest that environmental tumor promoters or carcinogens, such as ionizing radiation, can cause stromal fibroblasts to activate TGF beta by epigenetic mechanisms. As the biological effects of the anti-estrogen tamoxifen may well be mediated by TGF beta, this model has a number of important implications for the clinical uses of tamoxifen in the prevention and treatment of breast cancer. In addition, it suggests a number of novel approaches to the treatment of advanced breast cancer
— id: 83317, year: 1997, vol: 45, page: 81, stat: Journal Article,

Zymosan peritonitis and NO-X-induced nitration
Shigenaga, M. K.; Girault, I.; Christen, S.; Barcellos-Hoff, M. E.; Shigeno, E.; Chang, H.; Ames, B. N.
1997 ;75(SUPPL. 1):74P-74P, Japanese Journal of Pharmacology
— id: 104694, year: 1997, vol: 75, page: 74P, stat: Journal Article,

In situ localization of progesterone receptors in normal mouse mammary glands: absence of receptors in the connective and adipose stroma and a heterogeneous distribution in the epithelium
Shyamala, G; Barcellos-Hoff, M H; Toft, D; Yang, X
1997 Nov-Dec;63(4-6):251-259, Journal of steroid biochemistry & molecular biology
In normal mammary glands of both rodents and humans, progesterone promotes the proliferation of epithelial cells and several lines of evidence suggest that this action of progesterone may be mediated by progesterone receptor (PR). It is well established that normal mammary development involves a complex interplay between the epithelial cells and the surrounding fatty stroma. Furthermore, during mammary development, there is a change in both the relative proportion of epithelial cells and the steady-state levels of PR. Therefore, towards understanding the precise role of PR in mammary development, we have generated a highly sensitive antibody against mouse PR and examined its pattern of localization. Immunoreactive PR was detected only in the epithelial cells of the ducts while both the adipose and fibrous stroma surrounding these ducts were receptor negative. Similarly, PR mRNA was also associated only with the ductal epithelial cells. Approximately only 45-50% of the ductal cells were receptor positive and this distribution remained unchanged whether or not the tissues had been exposed to estrogen, suggesting that they may represent a distinct subpopulation. The potential significance of these findings to mammary development is discussed
— id: 83321, year: 1997, vol: 63, page: 251, stat: Journal Article,

Ionizing radiation accelerates aortic lesion formation in fat-fed mice via a mechanism that is inhibited by overexpression of Cu/Zn superoxide dismutase
Tribble, Diane L.; Barcellos-Hoff, Mary H.; Rubin, Edward M.; Leeuwenburgh, Christian; Heinecke, Jay W.; Gong, Elaine L.
1997 ;96(8 SUPPL.):I416-I416, Circulation
— id: 104695, year: 1997, vol: 96, page: I416, stat: Journal Article,

Latency and activation in the control of TGF-beta
Barcellos-Hoff, M H
1996 ;1(4):353-363, Journal of mammary gland biology & neoplasia
The biological activity of the transforming growth factor-beta's (TGF-beta)3 is tightly controlled by their persistence in the extracellular compartment as latent complexes. Each of the three mammalian isoform genes encodes a product that is cleaved intracellularly to form two polypeptides, each of which dimerizes. Mature TGF-beta, a 24 kD homodimer, is noncovalently associated with the 80 kD latency-associated peptide (LAP). LAP is a fundamental component of TGF-beta that is required for its efficient secretion, prevents it from binding to ubiquitous cell surface receptors, and maintains its availability in a large extracellular reservoir that is readily accessed by activation. This latent TGF-beta complex (LTGF-beta) is secreted by all cells and is abundant both in circulating forms and bound to the extracellular matrix. Activation describes the collective events leading to the release of TGF-beta. Despite the importance of TGF-beta regulation of growth and differentiation in physiological and malignant tissue processes, remarkably little is known about the mechanisms of activation in situ. Recent studies of irradiated mammary gland reveal certain features of TGF-beta 1 activation that may shed light on its regulation and potential roles in the normal and neoplastic mammary gland
— id: 116251, year: 1996, vol: 1, page: 353, stat: Journal Article,

Latency and activation in the control of TGF-beta
Barcellos-Hoff, M H
1996 Oct;1(4):353-363, Journal of mammary gland biology & neoplasia
The biological activity of the transforming growth factor-beta's (TGF-beta) is tightly controlled by their persistance in the extracellular compartment as latent complexes. Each of the three mammalian isoform genes encodes a product that is cleaved intracellularly to form two polypeptides, each of which dimerizes. Mature TGF-beta, a 24 kD homodimer, is noncovalently associated with the 80 kD latency-associated peptide (LAP). LAP is a fundamental component of TGF-beta that is required for its efficient secretion, prevents it from binding to ubiquitous cell surface receptors, and maintains its availability in a large extracellular reservoir that is readily accessed by activation. This latent TGF-beta complex (LTGF-beta) is secreted by all cells and is abundant both in circulating forms and bound to the extracellular matrix. Activation describes the collective events leading to the release of TGF-beta. Despite the importance of TGF-beta regulation of growth and differentiation in physiological and malignant tissue processes, remarkably little is known about the mechanisms of activation in situ. Recent studies of irradiated mammary gland reveal certain features of TGF-beta 1 activation that may shed light on its regulation and potential roles in the normal and neoplastic mammary gland
— id: 150855, year: 1996, vol: 1, page: 353, stat: Journal Article,

Redox-mediated activation of latent transforming growth factor-beta 1
Barcellos-Hoff, M H; Dix, T A
1996 Sep;10(9):1077-1083, Molecular endocrinology
Transforming growth factor beta 1 (TGF beta) is a multifunctional cytokine that orchestrates response to injury via ubiquitous cell surface receptors. The biological activity of TGF beta is restrained by its secretion as a latent complex (LTGF beta) such that activation determines the extent of TGF beta activity during physiological and pathological events. TGF beta action has been implicated in a variety of reactive oxygen-mediated tissue processes, particularly inflammation, and in pathologies such as reperfusion injury, rheumatoid arthritis, and atherosclerosis. It was recently shown to be rapidly activated after in vivo radiation exposure, which also generates reactive oxygen species (ROS). In the present studies, the potential for redox-mediated LTGF beta activation was investigated using a cell-free system in which ROS were generated in solution by ionizing radiation or metal ion-catalyzed ascorbate reaction. Irradiation (100 Gray) of recombinant human LTGF beta in solution induced 26% activation compared with that elicited by standard thermal activation. Metal-catalyzed ascorbate oxidation elicited extremely efficient recombinant LTGF beta activation that matched or exceeded thermal activation. The efficiency of ascorbate activation depended on ascorbate concentrations and the presence of transition metal ions. We postulate that oxidation of specific amino acids in the latency-conferring peptide leads to a conformation change in the latent complex that allows release of TGF beta. Oxidative activation offers a novel route for the involvement of TGF beta in tissue processes in which ROS are implicated and endows LTGF beta with the ability to act as a sensor of oxidative stress and, by releasing TGF beta, to function as a signal for orchestrating the response of multiple cell types. LTGF beta redox sensitivity is presumably directed toward recovery of homeostasis; however, oxidation may also be a mechanism of LTGF beta activation that can be deleterious during disease mechanisms involving chronic ROS production
— id: 83310, year: 1996, vol: 10, page: 1077, stat: Journal Article,

Effects of nitric oxide on the production of transforming growth factor beta-1 by tumor cells
Chesler, Louis; Cox, George; Barcellos-Hoff, Mary Helen; Wolf, Jeffrey; Takeguchi, Heather; Roberts, Anita; Kim, Seong-Jin; Vodovotz, Yoram
1996 ;37(0):42-43, Proceedings (American Association for Cancer Research)
— id: 104696, year: 1996, vol: 37, page: 42, stat: Journal Article,

Immunohistochemical evidence of rapid extracellular matrix remodeling after iron-particle irradiation of mouse mammary gland
Ehrhart, E J; Gillette, E L; Barcellos-Hoff, M H
1996 Feb;145(2):157-162, Radiation research
High-LET radiation has unique physical and biological properties compared to sparsely ionizing radiation. Recent studies demonstrate that sparsely ionizing radiation rapidly alters the pattern of extracellular matrix expression in several tissues, but little is known about the effect of heavy-ion radiation. This study investigates densely ionizing radiation-induced changes in extracellular matrix localization in the mammary glands of adult female BALB/c mice after whole-body irradiation with 0.8 Gy 600 MeV iron particles. The basement membrane and interstitial extracellular matrix proteins of the mammary gland stroma were mapped with respect to time postirradiation using immunofluorescence. Collagen III was induced in the adipose stroma within 1 day, continued to increase through day 9 and was resolved by day 14. Immunoreactive tenascin was induced in the epithelium by day 1, was evident at the epithelial-stromal interface by day 5-9 and persisted as a condensed layer beneath the basement membrane through day 14. These findings parallel similar changes induced by gamma irradiation but demonstrate different onset and chronicity. In contrast, the integrity of epithelial basement membrane, which was unaffected by sparsely ionizing radiation, was disrupted by iron-particle irradiation. Laminin immunoreactivity was mildly irregular at 1 h postirradiation and showed discontinuities and thickening from days 1 to 9. Continuity was restored by day 14. Thus high-LET radiation, like sparsely ionizing radiation, induces rapid-remodeling of the stromal extracellular matrix but also appears to alter the integrity of the epithelial basement membrane, which is an important regulator of epithelial cell proliferation and differentiation
— id: 83308, year: 1996, vol: 145, page: 157, stat: Journal Article,

TGF-beta activation in situ: Role in extracellular matrix remodeling
Ehrhart, E. J.; Segarini, P.; Tsang, M. L.-S.; Barcellos-Hoff, M. H.
1996 ;7(SUPPL.):410A-410A, Molecular biology of the cell
— id: 104697, year: 1996, vol: 7, page: 410A, stat: Journal Article,

Immunohistochemical detection of active transforming growth factor-beta in situ using engineered tissue
Barcellos-Hoff, M H; Ehrhart, E J; Kalia, M; Jirtle, R; Flanders, K; Tsang, M L
1995 Nov;147(5):1228-1237, American journal of pathology
The biological activity of transforming growth factor-beta 1 (TGF-beta) is governed by dissociation from its latent complex. Immunohistochemical discrimination of active and latent TGF-beta could provide insight into TGF-beta activation in physiological and pathological processes. However, evaluation of immunoreactivity specificity in situ has been hindered by the lack of tissue in which TGF-beta status is known. To provide in situ analysis of antibodies to differentiate between these functional forms, we used xenografts of human tumor cells modified by transfection to overexpress latent TGF-beta or constitutively active TGF-beta. This comparison revealed that, whereas most antibodies did not differentiate between TGF-beta activation status, the immunoreactivity of some antibodies was activation dependent. Two widely used peptide antibodies to the amino-terminus of TGF-beta, LC(1-30) and CC(1-30) showed marked preferential immunoreactivity with active TGF-beta versus latent TGF-beta in cryosections. However, in formalin-fixed, paraffin-embedded tissue, discrimination of active TGF-beta by CC(1-30) was lost and immunoreactivity was distinctly extracellular, as previously reported for this antibody. Similar processing-dependent extracellular localization was found with a neutralizing antibody raised to recombinant TGF-beta. Antigen retrieval recovered cell-associated immunoreactivity of both antibodies. Two antibodies to peptides 78-109 showed mild to moderate preferential immunoreactivity with active TGF-beta only in paraffin sections. LC(1-30) was the only antibody tested that discriminated active from latent TGF-beta in both frozen and paraffin-embedded tissue. Thus, in situ discrimination of active versus latent TGF-beta depends on both the antibody and tissue preparation. We propose that tissues engineered to express a specific form of a given protein provide a physiological setting in which to evaluate antibody reactivity with specific functional forms of a protein
— id: 83290, year: 1995, vol: 147, page: 1228, stat: Journal Article,

REDOX MEDIATED LATENT TGF-BETA ACTIVATION
BARCELLOSHOFF, MH
1995 ;9(3):A125-A125, FASEB journal
— id: 104698, year: 1995, vol: 9, page: A125, stat: Journal Article,

Transforming growth factor-beta activation in irradiated murine mammary gland
Barcellos-Hoff, M H; Derynck, R; Tsang, M L; Weatherbee, J A
1994 Feb;93(2):892-899, Journal of clinical investigation
The biological activity of TGF-beta, an important modulator of cell proliferation and extracellular matrix formation, is governed by dissociation of mature TGF-beta from an inactive, latent TGF-beta complex in a process that is critical to its role in vivo. So far, it has not been possible to monitor activation in vivo since conventional immunohistochemical detection does not accurately discriminate latent versus active TGF-beta, nor have events associated with activation been defined well enough to serve as in situ markers of this process. We describe here a modified immunodetection method using differential antibody staining that allows the specific detection of active versus latent TGF-beta. Under these conditions, we report that an antibody raised to latency-associated peptide detects latent TGF-beta, and we demonstrate that LC(1-30) antibodies specifically recognize active TGF-beta 1 in tumor xenografts overproducing active TGF-beta 1, without cross-reactivity in tumors expressing similar levels of latent TGF-beta 1. We previously reported that TGF-beta immunoreactivity increases in murine mammary gland after whole-body 60Co-gamma radiation exposure. Using differential antibody staining we now show that radiation exposure specifically generates active TGF-beta 1. While latent TGF-beta 1 was widely distributed in unirradiated tissue, active TGF-beta 1 distribution was restricted. Active TGF-beta 1 increased significantly within 1 h of irradiation concomitant with decreased latent TGF-beta immunoreactivity. This rapid shift in immunoreactivity provides the first evidence for activation of TGF-beta in situ. This reciprocal pattern of expression persisted for 3 d and was accompanied by decreased recovery of latent TGF-beta 1 from irradiated tissue. Radiation-induced activation of TGF-beta may have profound implications for understanding tissue effects caused by radiation therapy
— id: 83299, year: 1994, vol: 93, page: 892, stat: Journal Article,

Multicellular reorganization by single cells on extracellular matrix
Barcellos-Hoff, M. H.
1994 ;30A(3 PART 2):48-48, In vitro cellular & developmental biology. Animal
— id: 104699, year: 1994, vol: 30A, page: 48, stat: Journal Article,

Redox mechanism for activation of latent TGF-beta
Barcellos-Hoff, M. H.
1994 ;5(SUPPL.):139A-139A, Molecular biology of the cell
— id: 104700, year: 1994, vol: 5, page: 139A, stat: Journal Article,

Radiation-induced transforming growth factor beta and subsequent extracellular matrix reorganization in murine mammary gland
Barcellos-Hoff, M H
1993 Sep 1;53(17):3880-3886, Cancer research
Little is known about radiation-induced protein expression in vivo nor has the relationship between early molecular events and subsequent tissue repair, fibrosis, or carcinogenesis been fully appraised. In this study, expression of proteins involved in tissue remodeling was examined in mammary gland immediately and shortly after ionizing radiation exposure. Using indirect immunofluorescence, selected antigens were followed as a function of time after 0, 5, or 10 Gy of whole body gamma-radiation in the mammary gland of adult female BALB/c mice. Rapid induction of transforming growth factor beta (TGF beta) immunoreactivity was observed at 1 h post radiation. Extracellular and intracellular TGF beta increased in the periepithelial stromal sheath as evidenced by immunoreactivity with antibodies CC(1-30) and LC(1-30), respectively. Furthermore, both extracellular and intracellular TGF beta were unexpectedly expressed in the previously negative adipose stroma. Elevated expression persisted for 7 days after irradiation. Thus an early response to radiation exposure is the induction of TGF beta, which mediates myriad events during tissue repair, growth, and extracellular matrix production. The distribution of extracellular matrix proteins was examined as a function of time post radiation exposure. Collagen III immunoreactivity decreased in the periepithelial stroma at day 1. In contrast, at day 3 collagen III was newly evident in the adipose stroma, and periepithelial collagen III had increased in both abundance and intensity. By day 7 collagen III expression in the adipose stroma had resolved but was enhanced in the periepithelial stroma. Over this same period stromal collagen I immunoreactivity surrounding the epithelium became diffuse and possibly diminished. Fibronectin, laminin, and collagen IV localization were unchanged over the time course. I postulate that radiation-induced TGF beta may mediate the remodeling of the stromal extracellular matrix in the irradiated mammary gland
— id: 83305, year: 1993, vol: 53, page: 3880, stat: Journal Article,

Immunohistochemical discrimination of active versus latent TGF beta in situ
Barcellos-Hoff, M. H.
1993 ;4(SUPPL.):294A-294A, Molecular biology of the cell
— id: 104701, year: 1993, vol: 4, page: 294A, stat: Journal Article,

Development and functional differentiation of cultured mammary epithelial cells
Aggeler, Judith; Barcellos-Hoff, Mary Helen; Bissell, Mina J.
1992 ;4(SUPPL.):21-33, Contemporary Issues in Clinical Nutrition
— id: 104702, year: 1992, vol: 4, page: 21, stat: Journal Article,

IONIZING RADIATION INDUCED WOUNDING LEADS TO ALTERED STROMAL EXPRESSION OF TRANSFORMING GROWTH FACTOR-BETA IN-VIVO
BARCELLOS-HOFF M H
1992 ;4(16 PART F):78-78, Journal of cellular biochemistry. Supplement
— id: 104703, year: 1992, vol: 4, page: 78, stat: Journal Article,

Mammary epithelial reorganization on extracellular matrix is mediated by cell surface galactosyltransferase
Barcellos-Hoff, M H
1992 Jul;201(1):225-234, Experimental cell research
When plated at appropriate densities in serum-free media, the COMMA-D mammary epithelial cell line rapidly reorganizes into multicellular spheres on the basement membrane matrix derived from Engelbreth-Holm-Swarm murine tumor. Using time-lapse video-microscopy, four stages of reorganization were discerned during the first 24 h of culture. In the first few hours, cells attached to the matrix, elongated, migrated, and formed chains. In the next 6 h, chains of cells linked together in anastomosing networks. In the period between 8 and 18 h postplating, the networks contracted, resulting in dense cords radiating from central aggregates. During the final 6 h, the cords were drawn into the aggregates, which condensed further into spheres. The events occurring during mammary epithelial cell reorganization on the matrix were shown to be mediated by cell surface beta-1,4-galactosyltransferase (GalTase), a receptor that binds N-acetylglucosamine residues on glycosylated proteins. GalTase activity was evident at the surface of cells cultured on reconstituted matrix for 3 h but was absent from cells on glass. The protein alpha-lactalbumin (alpha-LA) inhibits the association of GalTase with N-acetylglucosamine. alpha-LA present from the beginning of culture on reconstituted matrix had no effect on cell attachment but caused concentration-dependent inhibition of the first two steps of reorganization, i.e., cell elongation and network formation, which then interfered with subsequent events. These observations were replicated using polyclonal antibodies to GalTase. Reorganization was impaired when alpha-LA was added during the first two stages but no effect was observed when it was added during the last two stages. Cells cultured on plastic, which lack surface GalTase activity, were unperturbed by incubation with alpha-LA. Thus certain events (cell elongation and network elaboration) during mammary epithelial cell reorganization on reconstituted matrix are GalTase dependent, while others (attachment, network contraction, and compaction) are not. The functional and temporal specificity of GalTase involvement indicates that GalTase mediates cell-matrix, but not cell-cell, interactions during epithelial morphogenetic events in culture
— id: 83205, year: 1992, vol: 201, page: 225, stat: Journal Article,

Production of stable phenotypes from 9L rat brain tumor multicellular spheroids treated with 1,3-bis(2-chloroethyl)-1-nitrosourea
Barcellos-Hoff, M H; Linfoot, P A; Marton, L J; Deen, D F
1992 Sep 30;52(3):409-413, International journal of cancer
During chemotherapy and regrowth of brain tumors, tumor-cell heterogeneity, and possibly tumor progression, may change as a result of both the selective forces and mutagenic effects of treatment. We have isolated and characterized drug-response variants of multicellular rat 9L brain-tumor spheroids exposed to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Ten colonies were isolated from spheroids disaggregated immediately after treatment, and 10 colonies were isolated from treated spheroids disaggregated after 1 week in suspension culture. The sensitivity to BCNU was determined by assays of sister chromatid exchange and colony-forming efficiency in monolayer cultures of each subline after a 1-hr exposure to graded doses of BCNU. Three classes of response were found: BCNU sensitivity increased, decreased, or was comparable to that of uncloned, parent 9L cells. Resistant phenotypes were predominant (8/10) in sublines from spheroids disaggregated immediately after treatment, whereas hypersensitive phenotypes (4/8) were isolated only from spheroids disaggregated after 1 week of regrowth. Since subpopulations isolated immediately after treatment do not have the same biological characteristics as those isolated after a period of regrowth, these data suggest that tumor-cell heterogeneity may be generated by distinct processes at various times during therapy. The predominance of hypersensitive sublines obtained by the regrowth protocol may have resulted from the recovery of cells that would have died if isolated but were instead able to repair the drug-induced damage when left in contact with neighboring, possibly resistant cells. Two resistant and two hypersensitive sublines were studied further
— id: 83171, year: 1992, vol: 52, page: 409, stat: Journal Article,

ACTIVATION OF TRANSFORMING GROWTH FACTOR-BETA-1 (TGF-BETA) AND STROMAL EXTRACELLULAR-MATRIX REMODELING ARE INDUCED BY RADIATION
BARCELLOSHOFF, MH
1992 ;3(3):A29-A29, Molecular biology of the cell
— id: 104704, year: 1992, vol: 3, page: A29, stat: Journal Article,

Cytodifferentiation of mouse mammary epithelial cells cultured on a reconstituted basement membrane reveals striking similarities to development in vivo
Aggeler, J; Ward, J; Blackie, L M; Barcellos-Hoff, M H; Streuli, C H; Bissell, M J
1991 Jun;99 ( Pt 2):407-417, Journal of cell science
In the present study we provide evidence that the cytodifferentiation of primary mouse mammary epithelial cells within the alveolar-like structures formed after culture on a reconstituted basement membrane resembles development in vivo during late pregnancy and early lactation. During the first two days in culture on a basement membrane gel in the presence of lactogenic hormones, epithelial cells isolated from mid-pregnant mice are disorganized and central lumina are largely absent. Levels of mRNA for the milk proteins, beta-casein and transferrin, are dramatically reduced. By the second or third day in culture, cytoplasmic polarization becomes evident and prominent apical junctional complexes are formed. Synthesis of both mRNA and milk protein is reinitiated at this time. By day 4, well-defined lumina appear, and abundant synthesis and secretion of casein and lipid is observed. A striking feature of this differentiation in culture is the specific localization of milk protein gene expression (beta-casein mRNA) to luminal epithelial cells in the alveolar-like structures. At the ultrastructural level, increased milk protein synthesis and secretion are paralleled by a fourfold increase in rough ER that resembles the dramatic increase in the ER observed in vivo following parturition. One indication of tissue-specific differentiation observed in later cultures (days 4-11) is the synthesis and secretion of abundant casein micelles. A second characteristic of lactating mammary epithelial cells in vivo that has not previously been observed in culture is the secretion of milk fat globules. Taken together, these observations indicate that mammary epithelial cells plated onto a reconstituted basement membrane differentiate to the lactating phenotype in culture
— id: 83268, year: 1991, vol: 99 ( Pt 2), page: 407, stat: Journal Article,

The extracellular matrix
Talhouk, Rabih S.; Streuli, Charles H.; Barcellos-Hoff, Mary Helen; Bissell, Mina J.
Structural biology Greenwich, CT : JAI Press, 1991,
— id: 5233, year: 1991, vol: , page: 137, stat: Chapter,

MAMMARY ALVEOLAR-LIKE REORGANIZATION ON RECONSTITUTED BASEMENT MEMBRANE MATRIX IS GALACTOSYLTRANSFERASE DEPENDENT
BARCELLOS-HOFF M H
1990 ;111(5 PART 2):12A-12A, Journal of cell biology
— id: 104706, year: 1990, vol: 111, page: 12A, stat: Journal Article,

DEVELOPMENT OF THIN GELS CONTAINING VIRAL DNA FOR DETERMINATIONS OF STRAND-BREAK INDUCTION BY ULTRASOFT X-RAYS
BARCELLOS-HOFF M H; SHIRLEY G V; CHATTERJEE A
1990 ;99(14 PART A):71-71, Journal of cellular biochemistry. Supplement
— id: 104705, year: 1990, vol: 99, page: 71, stat: Journal Article,

Differential drug sensitivity conferred by growth status detected in a mixed population of cycling and noncycling cells
Barcellos-Hoff, M H; Marton, L J; Deen, D F
1990 Jun 15;50(12):3551-3555, Cancer research
The noncycling cell compartment of tumors is considered to be an important target for chemotherapeutic agents; yet, it has been difficult to accurately quantitate its contribution to tumor response because of a lack of methods that can readily discern the relative sensitivities of cycling and noncycling cells. We have used antibodies against bromodeoxyuridine-substituted DNA in a unique experimental protocol that provides a basis for distinguishing the cycling and noncycling cell compartments and detecting their respective levels of drug-induced chromosome damage. A mixed population of cycling and noncycling cells was obtained by culturing 9L rat brain tumor cells as multicellular spheroids. Cell cycle compartments and phase distributions were monitored with flow cytometry using bivariate analysis of DNA content and bromodeoxyuridine incorporation. Bromodeoxyuridine labeling was manipulated to differentially label metaphases from cycling and noncycling cells for sister chromatid exchange. This is based on the differential staining patterns of chromatids and metaphases that are obtained from cells that have replicated in the presence of bromodeoxyuridine. The chromosome damage in each cell cycle compartment following exposure to the chemotherapeutic agent 1,3-bis(2-chloroethyl)-1-nitrosourea was assessed by the number of sister chromatid exchanges induced by treatment. Noncycling cells were shown to be more sensitive to 1,3-bis(2-chloroethyl)-1-nitrosourea-induced damage than were cycling cells. However, when allowed to remain noncycling for 24 h after treatment, the number of exchanges decreased in noncycling cells, which may indicate their ability to recover from damage. These results illustrate an experimental protocol that permits simultaneous assessment of cell cycle compartment recruitment and sister chromatid exchange induction in cells derived from a cytokinetically complex population containing both cycling and noncycling cells
— id: 83272, year: 1990, vol: 50, page: 3551, stat: Journal Article,

Mammary Epithelial Cells As A Model For Studies Of The Regulation Of Gene Expression
Barcellos-hoff MH; Bissell MJ
Functional epithelial cells in culture New York : Liss, 1989,
— id: 5235, year: 1989, vol: , page: 399, stat: Chapter,

Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane
Barcellos-Hoff, M H; Aggeler, J; Ram, T G; Bissell, M J
1989 Feb;105(2):223-235, Development
An essential feature of mammary gland differentiation during pregnancy is the formation of alveoli composed of polarized epithelial cells, which, under the influence of lactogenic hormones, secrete vectorially and sequester milk proteins. Previous culture studies have described either organization of cells polarized towards lumina containing little or no demonstrable tissue-specific protein, or establishment of functional secretory cells exhibiting little or no glandular architecture. In this paper, we report that tissue-specific vectorial secretion coincides with the formation of functional alveoli-like structures by primary mammary epithelial cells cultured on a reconstituted basement membrane matrix (derived from Engelbreth-Holm-Swarm murine tumour). Morphogenesis of these unique three-dimensional structures was initiated by cell-directed remodelling of the exogenous matrix leading to reorganization of cells into matrix-ensheathed aggregates by 24 h after plating. The aggregates subsequently cavitated, so that by day 6 the cells were organized into hollow spheres in which apical cell surfaces faced lumina sealed by tight junctions and basal surfaces were surrounded by a distinct basal lamina. The profiles of proteins secreted into the apical (luminal) and basal (medium) compartments indicated that these alveoli-like structures were capable of an appreciable amount of vectorial secretion. Immunoprecipitation with a broad spectrum milk antiserum showed that more than 80% of caseins were secreted into the lumina, whereas iron-binding proteins (both lactoferrin and transferrin) were present in comparable amounts in each compartment. Thus, these mammary cells established protein targeting pathways directing milk-specific proteins to the luminal compartment. A time course monitoring secretory activity demonstrated that establishment of tissue-specific vectorial secretion and increased total and milk protein secretion coincided with functional alveolar-like multicellular architecture. This culture system is unique among models of epithelial cell polarity in that it demonstrates several aspects of epithelial cell polarization: vectorial secretion, apical junctions, a sequestered compartment and formation of a basal lamina. These lumina-containing structures therefore reproduce the dual role of mammary epithelia to secrete vectorially and to sequester milk proteins. Thus, in addition to maintaining tissue-specific cytodifferentiation and function, a basement membrane promotes the expression of tissue-like morphogenesis
— id: 83275, year: 1989, vol: 105, page: 223, stat: Journal Article,

Measurement of sister chromatid exchanges and their relationship to DNA damage, repair and cell killing
Deen, D F; Morgan, W F; Tofilon, P J; Barcellos-Hoff, M H
1989 ;42(3):349-360, Pharmacology & therapeutics
We have concentrated on how SCE is measured, how it correlates with other measures of cellular damage, and how SCE can be used as a tool to study cell cycle kinetics. We have not discussed the biological significance of SCEs, i.e. why they occur spontaneously, and what role, if any, in cellular preservation they play. Similarly, our discussion of the mechanisms of formation of SCE has been deliberately brief. These are important considerations, but the answers to them are still largely only speculative. What is clear is that SCE is an extremely interesting biological phenomenon from a variety of points of view, and even though the mechanisms underlying SCE are not yet well understood, measurement of these events has produced much useful and provocative information
— id: 83273, year: 1989, vol: 42, page: 349, stat: Journal Article,

COMPARTMENTALIZATION AND VECTORIAL SECRETION OF MILK PROTEINS BY PRIMARY MAMMARY EPITHELIAL CELLS CULTURED ON RECONSTITUTED BASEMENT MEMBRANE
BARCELLOS-HOFF M H; RAM T G; AGGELER J; BISSELL M J
1988 ;107(6 PART 3):795A-795A, Journal of cell biology
— id: 104707, year: 1988, vol: 107, page: 795A, stat: Journal Article,

Cell-cycle, phase-specific cell killing by carmustine in sensitive and resistant cells
Linfoot, P A; Barcellos-Hoff, M H; Brent, T P; Marton, L J; Deen, D F
1988 ;(6):183-186, NCI monographs
Cell-cycle, phase-specific cell kill caused by carmustine (BCNU) was measured in 4 cell lines with different sensitivities to the drug. Cells were treated with BCNU for 1 hour after which enriched subpopulations in various phases of the cell cycle were obtained by centrifugal elutriation and were assayed for cell survival. Levels of activity of guanine O6-alkyltransferase were measured for each line; intracellular levels of this repair protein have been correlated with cellular resistance to chloroethylnitrosoureas. Only BTRC-19, a clone of the 9L line, had significant levels of alkyltransferase activity and exhibited a relatively flat age-response curve to BCNU. Alkyltransferase activity was not detected in the other 3 cell lines, all of which displayed a similar age response in which G1- and G2/M-phase cells were relatively sensitive to BCNU compared with the response of S-phase cells. We conclude that alkyltransferase activity may overwhelm other determinants that cause cell-cycle phase specificity to BCNU
— id: 83277, year: 1988, vol: , page: 183, stat: Journal Article,

POLARIZED SECRETION BY MAMMARY EPITHELIAL CELL CULTURES ON EHS-MATRIX
BARCELLOS-HOFF M H; NEVILLE P N; AGGELER J; BISSELL M J
1987 ;105(4 PART 2):220A-220A, Journal of cell biology
— id: 104708, year: 1987, vol: 105, page: 220A, stat: Journal Article,

The influence of extracellular matrix on gene expression: is structure the message?
Bissell, M J; Barcellos-Hoff, M H
1987 ;8:327-343, Journal of cell science. Supplement
The study of the regulation of gene expression in cultured cells, particularly in epithelial cells, has been both hampered and facilitated by the loss of function that accompanies culture on traditional plastic substrata. Initially, investigations of differentiated function were thwarted by the inadequacy of tissue culture methods developed to support growth of mesenchymal cells. However, with the recognition that the unit of function in higher organisms is larger than the cell itself, and that gene expression is dependent upon cell interactions with hormones, substrata and other cells, came the understanding that the epithelial cell phenotype is profoundly influenced by the extracellular environment. In the last decade research on epithelial cells has centred on culture conditions that recreate the appropriate environment for function with very promising and important results. The investigations into the modulation of phenotype in culture produced not only a better model, but also contributed to a better understanding of the regulation of normal function. Using cultured mammary gland epithelial cells as a primary model of these interactions, our studies of gene expression are based on three premises. (1) That the extracellular matrix (ECM) on which the cells sit is an extension of the cells and an active participant in the regulation of cellular function; i.e. the ECM is an 'informational' entity in the sense that it receives, imparts and integrates structural and functional signals. (2) That ECM-induced functional differentiation in the mammary gland is mediated through changes in cell shape, i.e. that the structure is in large part 'the message' required to maintain differentiated gene expression. (3) That the unit of function includes the cell plus its extracellular matrix; in a larger context, the unit is the organ itself. These tenets and the data presented below are consistent with a model of 'Dynamic Reciprocity', where the ECM is postulated to exert an influence on gene expression via transmembrane proteins and cytoskeletal components. In turn, cytoskeletal association with polyribosomes affects mRNA stability and rates of protein synthesis, while its interaction with the nuclear matrix could affect mRNA processing and, possibly, rates of transcription
— id: 83276, year: 1987, vol: 8, page: 327, stat: Journal Article,

Transferrin is a major mouse milk protein and is synthesized by mammary epithelial cells
Lee, E Y; Barcellos-Hoff, M H; Chen, L H; Parry, G; Bissell, M J
1987 Mar;23(3):221-226, In vitro cellular & developmental biology
We have identified a major mouse milk protein as transferrin (Tf) using immunoprecipitation, 2-dimensional electrophoresis, Ouchterlony diffusion and V-8 protease digests. We show that Tf is synthesized by mammary epithelial cells themselves and that its synthesis and secretion is regulated distinctly from that of other milk proteins. In culture, the kinetics of Tf synthesis and secretion are distinct from that of beta-casein; furthermore, Tf is relatively insensitive to lactogenic hormones whereas beta-casein is hormone-dependent. In vivo, however, Tf is regulated by pregnancy. While the virgin gland produces small amounts of Tf, its production is greatly increased during pregnancy and lactation. Thus, Tf synthesis in the mammary gland is modulated by as yet unknown factors in vivo. These observations are discussed in terms of Tf's possible role in mammary gland growth, differentiation and function
— id: 83278, year: 1987, vol: 23, page: 221, stat: Journal Article,

A SCANNING ELECTRON MICROSCOPE STUDY OF THE MORPHOGENESIS OF MOUSE MAMMARY CULTURES ON COLLAGEN GELS AND EHS MATRIX
NEVILLE M C; STAHL L; LUTES V; BISSELL M J; BARCELLOS-HOFF M H
1987 ;105(4 PART 2):139A-139A, Journal of cell biology
— id: 104709, year: 1987, vol: 105, page: 139A, stat: Journal Article,

Cycling and noncycling cancer cells : expression of differential sensitivity to 1,3 bis(2-chloroethyl)-1-nitrosourea (BCNU) and X-rays
Barcellos-Hoff, Mary Helen
[S.l. : s.n.], 1986,
Thesis (Ph.D.) - University of California, San Francisco, 1986
— id: 2093, year: 1986, vol: , page: , stat: ,

"DIFFERENTIAL INDUCTION OF SISTER CHROMATID EXCHANGE (SCE) BY ""1,3BIS-(2CHLOROETHYL)-1-NITROSOUREA (BCNU) IN CYCLING AND NONCYCLING CELLS OF 9L SPHEROIDS"
BARCELLOSHOFF, MH; MARTON, LJ; DEEN, DF
1986 ;27(4 PART 2):386-386, Proceedings (American Association for Cancer Research)
— id: 104710, year: 1986, vol: 27, page: 386, stat: Journal Article,

CELL CYCLE CHARACTERISTICS OF 9L SPHEROIDS DETERMINED FLOW CYTOMETRICALLY WITH MONOCLONAL ANTIBODIES TO BROMODEOXYURIDINE-SUBSTITUTED DNA
BARCELLOS-HOFF M H; GRAY J W; MARTON L J; DEEN D F
1985 ;101(5 PART 2):322A-322A, Journal of cell biology
— id: 104711, year: 1985, vol: 101, page: 322A, stat: Journal Article,

Growth delay in 9L rat brain tumor spheroids after irradiation with single and split doses of X rays
Jostes, R F; Williams, M E; Barcellos-Hoff, M H; Hoshino, T; Deen, D F
1985 May;102(2):182-189, Radiation research
The response of 9L spheroids to irradiation with single and split doses of X rays has been investigated. Irradiation with single doses caused a dose-dependent decrease in spheroid growth rate, which eventually returned to the growth rate for unirradiated spheroids. This delay appeared to be related to cell survival. When spheroids were irradiated with two 4-Gy doses of X rays separated by various times the amount of growth delay was intermediate between that observed with single doses of 4 and 8 Gy. For relatively short times (15-90 min), recovery probably resulted from repair processes, but for longer times (up to 24 hr), recovery also appeared to depend on cellular redistribution and repopulation effects
— id: 83283, year: 1985, vol: 102, page: 182, stat: Journal Article,

The effect of combination treatment with cis-platinum and low dose rate 125I radiation in a murine brachytherapy model
da Silva, V; Gutin, P H; Barcellos-Hoff, M H; Bernstein, M A; Deen, D F; Weaver, K A
1984 Aug;10(8):1471-1472, International journal of radiation oncology biology physics
We studied the effect of combination treatment with cis-platinum during low dose rate irradiation from 125I sources in the RIF-1 murine flank tumor model. Cell survival measured with a colony forming efficiency assay was used as the experimental endpoint. Sources implanted into tumors delivered 25 Gy of radiation to isodosed annuli of tissue at a dose rate of 1.3 Gy/hr. When 3 mg/kg of cis-platinum was administered 24, 12, and 1 hr before or 0, 12 and 24 hr after irradiation, the effect on cell kill was additive. Thus in the RIF-1 model there is no particular advantage gained by treatment with the combinations of cis-platinum and 125I radiation used in these studies
— id: 83285, year: 1984, vol: 10, page: 1471, stat: Journal Article,

Radiosensitization of the RIF-1 murine flank tumor by desmethylmisonidazole (Ro 05 9963) during interstitial brachytherapy
Bernstein, M; Gutin, P H; Deen, D F; Weaver, K A; Levin, V A; Barcellos, M H
1982 Mar-Apr;8(3-4):487-490, International journal of radiation oncology biology physics
We have investigated the interaction of the nitroimidazole radiosensitizer desmethylmisonidazole (Ro 05 9963) with interstitial radiation using a clonogenic cell assay as the endpoint. Removable I-125 sources were implanted at right angles to the outer surface of globular RIF-1 tumors grown in the flanks of C3H/He mice. Desmethylmisonidazole was administered by continuous intraperitoneal infusion at a rate of 2.7 mg/gm body weight /24 hr. Tumor and serum drug levels were measured by high performance liquid chromatography. Tumor drug levels of 40--100 mugm/gm tumor tissue were achieved consistently. No appreciable cytotoxicity was produced by desmethylmisonidazole alone, but a radiosensitization effect with a dose modifying factor of 1.6 was found
— id: 105534, year: 1982, vol: 8, page: 487, stat: Journal Article,

125I interstitial implants in the RIF-1 murine flank tumor: an animal model for brachytherapy
Bernstein, M; Gutin, P H; Weaver, K A; Deen, D F; Barcellos, M H
1982 Sep;91(3):624-637, Radiation research
— id: 105533, year: 1982, vol: 91, page: 624, stat: Journal Article,

Further evidence for the absence of a hypoxic fraction in the 9L rat tumour multicellular spheroid system
Gutin, P H; Barcellos, M H; Shrieve, D C; Sano, Y; Bernstein, M; Deen, D F
1982 Sep;55(657):688-690, British journal of radiology
— id: 105532, year: 1982, vol: 55, page: 688, stat: Journal Article,