Moon-Shong Tang, Ph.D.

Effect of carcinogenic acrolein on DNA repair and mutagenic susceptibility
Wang HT; Hu Y; Tong D; Huang J; Gu L; Wu XR; Chung FL; Li GM; Tang MS
2012 Jan 24;:?-? #, Journal of biological chemistry
Acrolein (Acr), a ubiquitous environmental contaminant, is a human carcinogen. Acr can react with DNA to form mutagenic alpha- and gamma-hydroxy-1, N2-cyclic propano-2-deoxyguanosine adducts (alpha-OH-Acr-dG and gamma-OH-Acr-dG). We demonstrate here that Acr-dG adducts can be efficiently repaired by the nucleotide excision repair (NER) pathway in normal human bronchial epithelia (NHBE) and lung fibroblasts (NHLF). However, the same adducts were poorly processed in cell lysates isolated from Acr-treated NHBE and NHLF, suggesting that Acr inhibits NER. In addition, we show that Acr treatment also inhibits base excision repair (BER) and mismatch repair (MMR). While Acr does not change the expression of XPA, XPC, hOGG1, PMS2 or MLH1 genes, it causes a reduction of XPA, XPC, hOGG1, PMS2 and MLH1 proteins; this effect, however, can be neutralized by the proteasome inhibitor, MG132. Acr treatment further enhances both bulky and oxidative DNA damage-induced mutagenesis. These results indicate that Acr not only damages DNA, but can also modify DNA repair proteins and further causes degradation of these modified repair proteins. We propose that these two detrimental effects contribute to Acr mutagenicity and carcinogenicity
— id: 150550, year: 2012, vol: , page: ?, stat: Journal Article,

Chromium induces chromosomal instability, which is partly due to deregulation of BubR1 and Emi1, two APC/C inhibitors
Hu, Liyan; Liu, Xin; Chervona, Yana; Yang, Feikun; Tang, Moon-Shong; Darzynkiewicz, Zbigniew; Dai, Wei
2011 Jul 15;10(14):2373-2379, Cell cycle
Disruption of cell cycle checkpoints and interference with the normal cell cycle progression frequently result in cell death or malignant transformation. Hexavalent chromium [Cr(VI)] is a well-known carcinogen that has been implicated in the occurrence of many types of human malignancies, including lung cancer. However, the exact mechanism by which Cr(VI) causes malignant transformation in the lung remains unknown. We have demonstrated that chronic exposure to a non-cytotoxic concentration of Cr(VI) induced a variety of chromosomal abnormalities, including premature sister chromatid separation, chromosomal breakage and the presence of lagging/misaligned chromosomes. After treatment with nocodazole, both HeLa and normal lung bronchial epithelial cells were arrested at mitosis. However, Cr(VI) significantly compromised M-phase arrest induced by nocodazole. Cr(VI) suppressed BubR1 activation and reduced expression of Emi1, leading to an unscheduled activation of APC/C. Consistent with this observation, Cr(VI) treatment caused enhanced polyubiquitination of geminin during mitotic release, while it deregulated the activity of Cdt1, a DNA replication licensing factor. Combined, these results suggest that Cr(VI)-induced chromosomal instability is partly due to a perturbation of APC/C activities, leading to chromosomal instability
— id: 135558, year: 2011, vol: 10, page: 2373, stat: Journal Article,

Acrolein induced DNA damage, mutagenicity and effect on DNA repair
Tang, Moon-Shong; Wang, Hsiang-Tsui; Hu, Yu; Chen, Wei-Sheng; Akao, Makoto; Feng, Zhaohui; Hu, Wenwei
2011 Sep;55(9):1291-1300, Molecular nutrition & food research
Acrolein (Acr) is a ubiquitous environmental contaminant; it also can be generated endogenously by lipid peroxidation. Acr contains a carbonyl group and an olefinic double bond; it can react with many cellular molecules including amino acids, proteins and nucleic acids. In this review article we focus on updating information regarding: (i) Acr-induced DNA damage and methods of detection, (ii) repair of Acr-DNA damage, (iii) mutagenicity of Acr-DNA adducts, (iv) sequence specificity and methylation effect on Acr-DNA adduct formation and (v) the role of Acr in human cancer. We have found that Acr can inhibit DNA repair and induces mutagenic Acr-dG adducts and that the binding spectrum of Acr in the p53 gene in normal human bronchial epithelial cells is similar to the p53 mutational spectrum in lung cancer. Since Acr-DNA adduct has been identified in human lung tissue and Acr causes bladder cancer in human and rat models, we conclude that Acr is a major lung and bladder carcinogen, and its carcinogenicity arises via induction of DNA damage and inhibition of DNA repair
— id: 137067, year: 2011, vol: 55, page: 1291, stat: Journal Article,

Downregulation of miR-205 and miR-31 confers resistance to chemotherapy-induced apoptosis in prostate cancer cells
Bhatnagar, N; Li, X; Padi, S K R; Zhang, Q; Tang, M-S; Guo, B
2010 ;1:e105-e105, Cell death & disease
Advanced prostate cancers are known to acquire not only invasive capabilities but also significant resistance to chemotherapy-induced apoptosis. To understand how microRNAs (miRNAs) may contribute to prostate cancer resistance to apoptosis, we compared microRNA expression profiles of a benign prostate cancer cell line WPE1-NA22 and a highly malignant WPE1-NB26 cell line (derived from a common lineage). We found that miR-205 and miR-31 are significantly downregulated in WPE1-NB26 cells, as well as in other cell lines representing advanced-stage prostate cancers. Antiapoptotic genes BCL2L2 (encoding Bcl-w) and E2F6 are identified as the targets of miR-205 and miR-31, respectively. By downregulating Bcl-w and E2F6, miR-205 and miR-31 promote chemotherapeutic agents-induced apoptosis in prostate cancer cells. The promoter region of the miR-205 gene was cloned and was found to be hypermethylated in cell lines derived from advanced prostate cancers, contributing to the downregulation of the gene. Treatment with DNA methylation inhibitor 5-aza-2'-deoxycytidine induced miR-205 expression, downregulated Bcl-w, and sensitized prostate cancer cells to chemotherapy-induced apoptosis. Thus, downregulation of miR-205 and miR-31 has an important role in apoptosis resistance in advanced prostate cancer
— id: 135623, year: 2010, vol: 1, page: e105, stat: Journal Article,

Ultraviolet A light: potential underlying causes of melanoma
Tang, Moon-Shong
2010 Oct;6(10):1523-1526, Future Oncology
— id: 114194, year: 2010, vol: 6, page: 1523, stat: Journal Article,

Melanocytes are deficient in repair of oxidative DNA damage and UV-induced photoproducts
Wang, Hsiang-Tsui; Choi, Bongkun; Tang, Moon-shong
2010 Jul 6;107(27):12180-12185, Proceedings of the National Academy of Sciences of the United States of America
Melanomas occur mainly in sunlight-exposed skin. Xeroderma pigmentosum (XP) patients have 1,000-fold higher incidence of melanoma, suggesting that sunlight-induced 'bulky' photoproducts are responsible for melanomagenesis. Sunlight induces a high level of reactive oxygen species in melanocytes (MCs); oxidative DNA damage (ODD) may thus also contribute to melanomagenesis, and XP gene products may participate in the repair of ODD. We examined the effects of melanin on UVA (320-400 nm) irradiation-induced ODD and UV photoproducts and the repair capacity in MC and XP cells for ODD and UV-induced photoproducts. Our findings indicate that UVA irradiation induces a significantly higher amount of formamidopyrimidine glycosylase-sensitive ODD in MCs than in normal human skin fibroblasts (NHSFs). In contrast, UVA irradiation induces an insignificant amount of UvrABC-sensitive sites in either of these two types of cells. We also found that, compared to NHSFs, MCs have a reduced repair capacity for ODD and photoproducts; H(2)O(2) modified- and UVC-irradiated DNAs induce a higher mutation frequency in MCs than in NHSFs; and, XP complementation group A (XPA), XP complementation group C, and XP complementation group G cells are deficient in ODD repair and ODD induces a higher mutation frequency in XPA cells than in NHSFs. These results suggest that: (i) melanin sensitizes UVA in the induction of ODD but not bulky UV photoproducts; (ii) the high susceptibility to UVA-induced ODD and the reduced DNA repair capacity in MCs contribute to carcinogenesis; and (iii) the reduced repair capacity for ODD contributes to the high melanoma incidence in XP patients
— id: 110876, year: 2010, vol: 107, page: 12180, stat: Journal Article,

Repair of mitomycin C mono- and interstrand cross-linked DNA adducts by UvrABC: a new model
Weng, Mao-Wen; Zheng, Yi; Jasti, Vijay P; Champeil, Elise; Tomasz, Maria; Wang, Yinsheng; Basu, Ashis K; Tang, Moon-Shong
2010 Nov 1;38(20):6976-6984, Nucleic acids research
Mitomycin C induces both MC-mono-dG and cross-linked dG-adducts in vivo. Interstrand cross-linked (ICL) dG-MC-dG-DNA adducts can prevent strand separation. In Escherichia coli cells, UvrABC repairs ICL lesions that cause DNA bending. The mechanisms and consequences of NER of ICL dG-MC-dG lesions that do not induce DNA bending remain unclear. Using DNA fragments containing a MC-mono-dG or an ICL dG-MC-dG adduct, we found (i) UvrABC incises only at the strand containing MC-mono-dG adducts; (ii) UvrABC makes three types of incisions on an ICL dG-MC-dG adduct: type 1, a single 5' incision on 1 strand and a 3' incision on the other; type 2, dual incisions on 1 strand and a single incision on the other; and type 3, dual incisions on both strands; and (iii) the cutting kinetics of type 3 is significantly faster than type 1 and type 2, and all of 3 types of cutting result in producing DSB. We found that UvrA, UvrA + UvrB and UvrA + UvrB + UvrC bind to MC-modified DNA specifically, and we did not detect any UvrB- and UvrB + UvrC-DNA complexes. Our findings challenge the current UvrABC incision model. We propose that DSBs resulted from NER of ICL dG-MC-dG adducts contribute to MC antitumor activity and mutations
— id: 114506, year: 2010, vol: 38, page: 6976, stat: Journal Article,

DNA wrapping is required for DNA damage recognition in the Escherichia coli DNA nucleotide excision repair pathway
Wang, Hailin; Lu, Meiling; Tang, Moon-shong; Van Houten, Bennett; Ross, J B Alexander; Weinfeld, Michael; Le, X Chris
2009 Aug 4;106(31):12849-12854, Proceedings of the National Academy of Sciences of the United States of America
Localized DNA melting may provide a general strategy for recognition of the wide array of chemically and structurally diverse DNA lesions repaired by the nucleotide excision repair (NER) pathway. However, it is not clear what causes such DNA melting and how it is driven. Here, we show a DNA wrapping-melting model supported by results from dynamic monitoring of the key DNA-protein and protein-protein interactions involved in the early stages of the Escherichia coli NER process. Using an analytical technique involving capillary electrophoresis coupled with laser-induced fluorescence polarization, which combines a mobility shift assay with conformational analysis, we demonstrate that DNA wrapping around UvrB, mediated by UvrA, is an early event in the damage-recognition process during E. coli NER. DNA wrapping of UvrB was confirmed by Forster resonance energy transfer and fluorescence lifetime measurements. This wrapping did not occur with readily denaturable damaged DNA substrates ('bubble' DNA), suggesting that DNA wrapping of UvrB plays an important role in the induction of DNA melting around the damage site. Analysis of DNA wrapping of mutant UvrB Y96A further suggests that a cooperative interaction between DNA wrapping of UvrA(2)B and contact of the beta-hairpin of UvrB with the bulky damage moiety may be involved in the local DNA melting at the damage site
— id: 133700, year: 2009, vol: 106, page: 12849, stat: Journal Article,

Mutagenicity and Sequence Specificity of Acrolein-DNA Adducts
Wang, Hsiang-Tsui; Zhang, Siyi; Hu, Yu; Tang, Moon-Shong
2009 Mar 16;22(3):511-517, Chemical research in toxicology
Acrolein (Acr) is a major toxicant in cigarette smoke (CS); it can interact with DNA forming two major adduct isomers: alpha-OH-Acr-dG and gamma-OH-Acr-dG. Previously, we found that the Acr-DNA binding pattern in the human p53 gene coincides with the p53 mutational pattern in CS-related lung cancer; hence, we proposed that Acr is a major lung cancer etiological agent [ Feng , Z. , Hu , W. , Hu , Y. , and Tang , M.-s. ( 2006 ) Acrolein is a major cigarette-related lung cancer agent: Preferential binding at p53 mutational hotspots and inhibition of DNA repair . Proc. Natl. Acad. Sci. U.S.A. 103 , 15404 - 15409 ]. This hypothesis has been brought into question with recent work that failed to detect Acr-induced mutations in the pSP189 system [ Kim , S. I. , Pfeifer , G. P. , and Besaratinia , A. ( 2007 ) Lack of mutagenicity of acrolein-induced DNA adducts in mouse and human cells . Cancer Res. 67 , 11640 - 116472 ]. To resolve this controversy, we determined the level and the type of Acr-dG formation, and the mutagenicity of Acr-dG adducts in the same pSP189 system. We also mapped the Acr-dG adduct distribution at the nucleotide level and the Acr-dG-induced mutational spectrum in this system. We found that (1) gamma-OH-Acr-dG is the major adduct formed in Acr-modified DNA based on the LC-ESI-MS/MS analysis; (2) the mutation frequency is proportional to the extent of Acr modifications, the majority of which are G:C to T:A and G:C to A:T mutations; and (3) sequences with a run of Gs are the mutational hotspots. Using the UvrABC nuclease incision method to map the Acr-dG distribution in the supF gene sequence, we confirmed that Acr-DNA adducts preferentially form in guanine-rich sequences that are also mutational hotspots. These results reaffirm that Acr-dG adducts are mutagenic and support our hypothesis that Acr is a major etiological agent for CS and cooking fume-related lung cancer
— id: 98293, year: 2009, vol: 22, page: 511, stat: Journal Article,

Recognition and incision of Cr(III) ligand-conjugated DNA adducts by the nucleotide excision repair proteins UvrABC: importance of the Cr(III)-purine moiety in the enzymatic reaction
Arakawa, Hirohumi; Tang, Moon-Shong
2008 Jun;21(6):1284-1289, Chemical research in toxicology
Hexavalent chromium [Cr(VI)] is an ubiquitous environmental contaminant and a well-known etiological agent of human lung cancer. Inside human cells, Cr(VI) is reduced to Cr(III), which can conjugate with amino acids, ascorbic acids, and glutathiones in the cytoplasm. Conjugated and unconjugated Cr(III) can enter the nucleus to form adducts with DNA and electrostatically interact with the phosphate group of DNA. It has been found that in both human and Escherichia coli systems, Cr(III) ligand-conjugated DNA ternary adducts are efficiently repaired by the nucleotide excision repair (NER) pathway. In contrast, DNA adducts formed by unconjugated Cr(III) with DNA are repaired significantly less efficiently by the NER system. These results raise the possibility that the NER system repairs Cr(III) ligand-conjugated DNA adducts and biadducts such as Cr(III)-guanine-phosphate adducts but not Cr(III)-phosphate adducts. To test this hypothesis, we determined the cutting efficiency and the mode of cutting of DNA modified with tannin-conjugated Cr(III) by the E. coli NER enzymes UvrABC. Tannin compounds, gallic acid (GA), and ethyl gallate (EGA) can reduce Cr(VI) to Cr(III) to form Cr(III)-GA 2 and Cr(III)-EGA 2, respectively, which can interact with a single guanine or adenine base but not with the DNA phosphate backbone. We found that UvrABC is able to incise Cr(III)-GA 2- and Cr(III)-EGA 2-modified plasmid DNA, and the amount of incision increased as a function of tannin concentration used for modifications. In contrast, UvrABC nuclease does not incise GA- and EGA-modified plasmid DNA. Mapping the sequence specificity of Cr(III)-GA 2- and Cr(III)-EGA 2-DNA formation in the human p53 gene sequence by UvrABC nuclease cutting, we found that the sequence specificity for both adducts is the same but is much more selective than Cr(III)-guanine-DNA adducts. Together, these results suggest that NER proteins from E. coli recognize the purine-Cr(III) adduct but not the Cr(III)-backbone phosphate complex
— id: 93303, year: 2008, vol: 21, page: 1284, stat: Journal Article,

PAH-DNA adducts in a Chinese population: relationship to PAH exposure, smoking and polymorphisms of metabolic and DNA repair genes
Hu, Yu; Li, Gang; Xue, Xiaonan; Zhou, Zongcan; Li, Xiaomei; Fu, Juanling; Cohen, Beverly; Roy, Nirmal; Li, Donghui; Sun, Jianya; Nan, Peihong; Tang, Moon-Shong; Qu, Qingshan
2008 Feb;13(1):27-40, Biomarkers
The present study was conducted in a Chinese population to evaluate the usefulness and sensitivity of PAH-DNA adduct as a biomarker of PAH exposure, and to examine the potential effects of smoking and polymorphisms of responsive genes on DNA adduct formation induced by PAH exposure. The polymorphisms of genes examined include GSTM1, GSTT1, CYP1A1, microsomal epoxide hydrolase (mEH) and excision repair cross-complementary group 2 (ERCC2). A total of 194 subjects with a broad range of PAH exposures were recruited, including 116 occupationally exposed workers, 49 metropolitan residents and 29 suburban gardeners. A significant exposure-response relationship was observed between PAH exposure and DNA adducts in leukocytes across the entire group of subjects (p < 0.0001). The levels of PAH-DNA adducts in the subgroup with lowest occupational exposure to PAHs (< 0.1 microg BaP m(-3)) was significantly higher than that in metropolitan residents and suburban gardeners. However, no significant difference was detected between residents and gardeners, with mean BaP concentrations of 0.028 and 0.011 microg m(-3), respectively. The polymorphisms of genes examined failed to show significant effects on PAH-induced adduct formation except ERCC2 Lys751Gln genotypes. A significantly higher level of PAH-DNA adduct was found in subjects with wild-type ERCC2 than those who have either heterozygous or homozygous variant alleles (p < 0.01). Smoking, age and gender did not substantially contribute to PAH-induced DNA adduct formation in this study. The study suggests that PAH-DNA adducts may serve as a reliable biomarker of PAH exposure in occupational settings but may not be sensitive enough to be used in populations with environmental exposures to PAHs
— id: 79241, year: 2008, vol: 13, page: 27, stat: Journal Article,

Particulate matter inhibits DNA repair and enhances mutagenesis
Mehta, Manju; Chen, Lung-Chi; Gordon, Terry; Rom, William; Tang, Moon-Shong
2008 Dec 8;657(2):116-121, Mutation research
Exposure to ambient air pollution has been associated with adverse health effects including lung cancer. A recent epidemiology study has established that each 10mug/m(3) elevation in long-term exposure to average PM(2.5) ambient concentration was associated with approximately 8% of lung cancer mortality. The underlying mechanisms of how PM contributes to lung carcinogenesis, however, remain to be elucidated. We have recently found that transition metals such as nickel and chromium and oxidative stress induced lipid peroxidation metabolites such as aldehydes can greatly inhibit nucleotide excision repair (NER) and enhance carcinogen-induced mutations. Because PM is rich in metal and aldehyde content and can induce oxidative stress, we tested the effect of PM on DNA repair capacity in cultured human lung cells using in vitro DNA repair synthesis and host cell reactivation assays. We found that PM greatly inhibits NER for ultraviolet (UV) light and benzo(a)pyrene diol epoxide (BPDE) induced DNA damage in human lung cells. We further demonstrated that PM exposure can significantly increase both spontaneous and UV-induced mutagenesis. These results together suggest that the carcinogenicity of PM may act through its combined effect on suppression of DNA repair and enhancement of DNA replication errors
— id: 90028, year: 2008, vol: 657, page: 116, stat: Journal Article,

Using high throughput resequencing microarrays to detect mutations in genes involved in lung cancer
Piao, LC; Gunnison, A; Nadas, A; Chen, WC; Nonaka, D; Spivack, S; Pass, H; Rom, WN; Tang, MS
2008 DEC 11 ;4(3):159-159, Cancer biomarkers : section A of Disease markers
— id: 91475, year: 2008, vol: 4, page: 159, stat: Journal Article,

Gene profiling of normal human bronchial epithelial cells in response to asbestos and benzo(a)pyrene diol epoxide (BPDE)
Belitskaya-Levy, Ilana; Hajjou, Mustapha; Su, Wei-cheng; Yie, Ting-An; Tchou-Wong, Kam-Meng; Tang, Moon-shong; Goldberg, Judith D; Rom, William N
2007 ;26(4):281-294, Journal of environmental pathology, toxicology & oncology
Asbestos and benzo(a)pyrene diol epoxide (BPDE) are pulmonary carcinogens with synergistic interaction in causing lung cancer. We used Affymetrix microarrays to study gene modulation in vitro using normal human bronchial epithelial cells exposed to chrysotile asbestos and/or BPDE for 4 or 24 h. Linear models were used to compare treated cells to controls at each time point to identify statistically significant up- or downregulation of genes. Profiles of genes regulated by chrysotile were dominated by cytokines, growth factors, and DNA damage. Profiles of genes with BPDE and chrysotile regulation were correlated with proliferation, DNA damage recognition and nucleotide-excision repair, cytokines, and apoptosis. Chemokines, growth-regulated oncogene-alpha (Gro-alpha, CXCL-1), and IL-8, were significantly increased, and these had previously been observed in bronchoalveolar lavage from asbestos workers or in animal models. Interestingly, the Hermansky-Pudlak gene, which is mutated in an autosomal recessive form of pulmonary fibrosis, was downregulated threefold by BPDE at 4 h. This is an interesting example of gene (Hermansky-Pudlak syndrome) and environment (BPDE) interaction. Transcription factors, including activating transcription factor 3 and Cbp/p300-interacting transactivator, were upregulated by chrysotile. Real Time PCR for IL-8, ATF-3, GADD45B, CXC Ligand 1, and CTGF compared to GAPDH validated microarray findings at 24 h. These in vitro findings in NHBE cells model environment-gene interaction for asbestos and BPDE, highlighting effects of inflammation, fibrosis, proliferation, and DNA damage recognition and repair
— id: 76391, year: 2007, vol: 26, page: 281, stat: Journal Article,

Sequence specificity of Cr(III)-DNA adduct formation in the p53 gene: NGG sequences are preferential adduct-forming sites
Arakawa, Hirohumi; Wu, Feng; Costa, Max; Rom, William; Tang, Moon-Shong
2006 Mar;27(3):639-645, Carcinogenesis
Hexavalent chromium [Cr(VI)] is a known etiological factor in human lung cancer. Cr(VI) exposure-related lung cancer has a high mutation incidence in the p53 gene. Upon intake in human cells Cr(VI) is reduced to Cr(III), which is able to conjugate with amino acids and consequently form either binary Cr(III)-DNA or ternary Cr(III)-amino acid-DNA adducts. Both binary and ternary Cr(III)-DNA adducts are mutagenic. We have found that the Escherichia coli nucleotide excision enzyme UvrABC nuclease is able to incise Cr(III)- and Cr(III)-histidine-modified plasmid DNA and the extent of incision is proportional to the amount of Cr(III)-DNA adducts in the plasmid. In order to determine the role of Cr(III)-DNA adducts in the mutagenesis of the p53 gene in human cancer using the UvrABC nuclease incision method, we have mapped the Cr(III)-DNA distribution in PCR DNA fragments amplified from exons 5, 7 and 8 of the p53 gene. We have found that the sequence specificities of Cr(III)-DNA and Cr(III)-histidine-DNA adducts in the p53 gene sequence are identical and that both types of adducts are preferentially formed at -NGG- sequences, including codons 245, 248 and 249, the mutational hotspots in human lung cancer. It has been found that Cr(III)-DNA adducts induce mainly G to T mutations. Therefore, these results suggest that Cr(III)-DNA adduct formation contributes to the p53 gene mutations in lung carcinogenesis
— id: 63597, year: 2006, vol: 27, page: 639, stat: Journal Article,

Acrolein is a major cigarette-related lung cancer agent: Preferential binding at p53 mutational hotspots and inhibition of DNA repair
Feng, Zhaohui; Hu, Wenwei; Hu, Yu; Tang, Moon-shong
2006 Oct 17;103(42):15404-15409, Proceedings of the National Academy of Sciences of the United States of America
The tumor suppressor gene p53 is frequently mutated in cigarette smoke (CS)-related lung cancer. The p53 binding pattern of carcinogenic polycyclic aromatic hydrocarbons (PAHs) found in CS coincides with the p53 mutational pattern found in lung cancer, and PAHs have thus been considered to be major culprits for lung cancer. However, compared with other carcinogenic compounds, such as aldehydes, the amount of PAHs in CS is minute. Acrolein (Acr) is abundant in CS, and it can directly adduct DNA. Acr-DNA adducts, similar to PAH-DNA adducts, induce predominantly G-to-T transversions in human cells. These findings raise the question of whether Acr-DNA adducts are responsible for p53 mutations in CS-related lung cancer. To determine the role of Acr-DNA adducts in p53 mutagenesis in CS-related lung cancer we mapped the distribution of Acr-DNA adducts at the sequence level in the p53 gene of lung cells using the UvrABC incision method in combination with ligation-mediated PCR. We found that the Acr-DNA binding pattern is similar to the p53 mutational pattern in human lung cancer. Acr preferentially binds at CpG sites, and this enhancement of binding is due to cytosine methylation at these sequences. Furthermore, we found that Acr can greatly reduce the DNA repair capacity for damage induced by benzo[a]pyrene diol epoxide. Together these results suggest that Acr is a major etiological agent for CS-related lung cancer and that it contributes to lung carcinogenesis through two detrimental effects: DNA damage and inhibition of DNA repair
— id: 70154, year: 2006, vol: 103, page: 15404, stat: Journal Article,

Malondialdehyde, a major endogenous lipid peroxidation product, sensitizes human cells to UV- and BPDE-induced killing and mutagenesis through inhibition of nucleotide excision repair
Feng, Zhaohui; Hu, Wenwei; Marnett, Lawrence J; Tang, Moon-shong
2006 Oct 10;601(1-2):125-136, Mutation research
Aldehydes are ubiquitous contaminants in the human environment. Intracellular aldehydes are mainly derived from the metabolism of polyunsaturated fatty acids and from lipid peroxidation, which is significantly elevated under oxidative stress conditions. Oxidative stress has long been suspected to be involved in many disease processes, including carcinogenesis, neurodegeneration and aging, but its mechanisms are largely unknown. Aldehydes are reactive not only toward nucleic acids but also to many amino acids, and these aldehyde-protein interactions have been suspected of affecting many cellular functions, including DNA repair. To test this possibility we determined the effect of malondialdehyde (MDA), one of the most abundant intracellular aldehyde, on ultraviolet (UV) light- and benzo(a)pyrene diol epoxide (BPDE)-induced cytotoxicity and mutagenesis in human cells. We found that MDA treatment greatly sensitized cells to both UV- and BPDE-induced cell killing and that, MDA pre-treatment significantly enhanced UV-induced mutagenesis. Using in vitro DNA repair synthesis and host cell reactivation assays we found that MDA treatment of cells greatly inhibited nucleotide excision repair for both and UV light- and BPDE-induced DNA damage. Further experiments raise the possibility that the inhibitory effect on nucleotide excision repair is mainly caused by the direct interaction of MDA with cellular repair proteins. Together these results strongly suggest that intracellular aldehydes play an important role in oxidative stress-related mutagenesis and carcinogenesis through their inhibitory effect on DNA repair mechanisms as well as on induction of DNA damage
— id: 69579, year: 2006, vol: 601, page: 125, stat: Journal Article,

Sensitive biomarker of polycyclic aromatic hydrocarbons (PAHs): urinary 1-hydroxyprene glucuronide in relation to smoking and low ambient levels of exposure
Hu, Y; Zhou, Z; Xue, X; Li, X; Fu, J; Cohen, B; Melikian, A A; Desai, M; Tang, M -S; Huang, X; Roy, N; Sun, J; Nan, P; Qu, Q
2006 Jul-Aug;11(4):306-318, Biomarkers
The study was conducted in a Chinese population with occupational or environmental exposures to polycyclic aromatic hydrocarbons (PAHs). A total of 106 subjects were recruited from coke-oven workers (workers), residents in a metropolitan area (residents) and suburban gardeners (gardeners). All subjects were monitored twice for their personal exposures to PAHs. The biological samples were collected for measurements of 1-hydroxypyrene (1-OHP) and cotinine in urine. The geometric means of personal exposure levels of pyrene, benz(a)anthracene (BaA) and benzo(a)pyrene (BaP) in workers were 1.470, 0.978 and 0.805 microg m-3, respectively. The corresponding levels in residents were 0.050, 0.034 and 0.025 microg m-3; and those in gardeners were 0.011, 0.020 and 0.008 microg m-3, respectively. The conjugate of 1-OHP with glucuronide (1-OHP-G) is the predominant form of pyrene metabolite in urine and it showed strong associations with exposures not only to pyrene, but also to BaA, BaP and total PAHs. Most importantly, a significant difference in 1-OHP-G was even detected between the subgroups with exposures to BaP at < 0.010 and > 0.010 but < 0.020 microg m-3, suggesting that 1-OHP-G is a good marker that can be used for the risk assessment of BaP exposure at levels currently encountered in ambient air. Furthermore, multiple regression analyses of 1-OHP-G on PAHs exposure indicated that cigarette smoke was a major confounding factor and should be considered and adjusted for while using 1-OHP to estimate PAHs exposure
— id: 68305, year: 2006, vol: 11, page: 306, stat: Journal Article,

Gene expression studies provide clues to the pathogenesis of uterine leiomyoma: new evidence and a systematic review
Arslan, Alan A; Gold, Leslie I; Mittal, Khushbakhat; Suen, Ting-Chung; Belitskaya-Levy, Ilana; Tang, Moon-Shong; Toniolo, Paolo
2005 Apr;20(4):852-863, Human reproduction
BACKGROUND: Uterine leiomyomas are extremely common and a major cause of pelvic pain, bleeding, infertility, and the leading indication for hysterectomy. Familial and epidemiological studies provide compelling evidence that genetic alterations play an important role in leiomyoma development. METHODS: Using Affymetrix U133A GeneChip we analysed expression profiles of 22,283 genes in paired samples of leiomyoma and adjacent normal myometrium. We compared our results with previously published data on gene expression in uterine leiomyoma and identified the overlapping gene alterations. RESULTS: We detected 80 genes with average differences of > or = 2-fold and false discovery rates of < 5% (14 overexpressed and 66 underexpressed). A comparative analysis including eight previous gene expression studies revealed eight prominent genes (ADH1, ATF3, CRABP2, CYR61, DPT, GRIA2, IGF2, MEST) identified by at least five different studies, eleven genes (ALDH1, CD24, CTGF, DCX, DUSP1, FOS, GAGEC1, IGFBP6, PTGDS, PTGER3, TYMS) reported by four studies, twelve genes (ABCA, ANXA1, APM2, CCL21, CDKN1A, CRMP1, EMP1, ESR1, FY, MAP3K5, TGFBR2, TIMP3) identified by three studies, and 40 genes reported by two different studies. CONCLUSIONS: Review of gene expression data revealed concordant changes in genes regulating retinoid synthesis, IGF metabolism, TGF-beta signaling and extracellular matrix formation. Gene expression studies provide clues to the relevant pathways of leiomyoma development
— id: 55957, year: 2005, vol: 20, page: 852, stat: Journal Article,

Model of transcriptional regulation of the BRCA1-NBR2 bi-directional transcriptional unit
Suen, Ting-Chung; Tang, Moon-Shong; Goss, Paul E
2005 May 1;1728(3):126-134, Biochimica & biophysica acta
In contrast to hundreds of mutations found in familial breast and/or ovarian cancers, somatic mutations of BRCA1 are very rare. However, a high percentage of sporadic breast and ovarian cancers show a reduction in BRCA1 expression, suggesting that defects in transcriptional regulation is a contributing factor. BRCA1 shares a promoter with its neighboring gene, NBR2, which is transcribed in the opposite direction. We have previously shown that the transcription of BRCA1 is negatively regulated by protein factors that interact with a 36-bp segment, located 575 bp into its first intron. We now report the localization of an 18-bp transcriptional repressor element for NBR2, which resides 948 bp into its first intron. The binding of nuclear proteins to this repressor element was detected by electrophoretic mobility shift assays (EMSAs), and it conferred an orientation-dependent functional suppression onto a heterologous thymidine kinase promoter. Combined with our previous studies, a model of transcriptional regulation of the closely aligned BRCA1-NBR2 bi-directional unit is proposed. A minimal 56-bp DNA region is functional in driving transcription in both directions, while uni-directional control is provided by distinct repressors that bind to sequences located in the first intron of the respective genes
— id: 56175, year: 2005, vol: 1728, page: 126, stat: Journal Article,

Trans-4-hydroxy-2-nonenal inhibits nucleotide excision repair in human cells: a possible mechanism for lipid peroxidation-induced carcinogenesis
Feng, Zhaohui; Hu, Wenwei; Tang, Moon-Shong
2004 Jul 8;101(23):8598-8602, Proceedings of the National Academy of Sciences of the United States of America
Lipid peroxidation (LPO) is a cellular process that commonly takes place under normal physiological conditions. Under excessive oxidative stress, the level of LPO becomes very significant, and a growing body of evidence has shown that excessive LPO may be involved in carcinogenesis. Trans-4-hydroxy-2-nonenal (4-HNE) is a major product of LPO, and its level becomes relatively high in cells under oxidative stress. 4-HNE is able to react readily with various cellular components, including DNA and proteins. We previously found that the 4-HNE-DNA adduct is a potent mutagen in human cells and is preferentially formed at codon 249 of the p53 gene, a mutational hotspot in human cancers. To further understand the role of 4-HNE in carcinogenesis, we addressed the question of whether 4-HNE affects DNA repair in human cells. We found that the repair capacity for benzo[a]pyrene diol epoxide and UV light-induced DNA damage was greatly compromised in human cells or human cell extracts treated with 4-HNE, which is mainly through interaction of 4-HNE with cellular repair proteins. We also found that 4-HNE greatly sensitizes cells to benzo[a]pyrene diol epoxide- and UV-induced killing. Together these results strongly suggest that this LPO metabolite damages not only DNA but also DNA repair mechanisms in human cells. We propose that these two detrimental effects of LPO may contribute synergistically to human carcinogenesis
— id: 46002, year: 2004, vol: 101, page: 8598, stat: Journal Article,

Chromium(VI) enhances (+/-)-anti-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydro benzo[a]pyrene-induced cytotoxicity and mutagenicity in mammalian cells through its inhibitory effect on nucleotide excision repair
Hu, Wenwei; Feng, Zhaohui; Tang, Moon-shong
2004 Nov 9;43(44):14282-14289, Biochemistry
Chromium(VI) [Cr(VI)], a ubiquitous environmental contaminant, is a well-known carcinogen to both humans and experimental animals, although it is a weak mutagen by itself. Occupational exposure to Cr(VI) is strongly associated with a high incidence of lung cancer, but the underlying mechanisms remain unclear. Tobacco smoking is the major cause of lung cancer, and polycyclic aromatic hydrocarbons (PAHs) in tobacco smoke are the major etiological agents. Since humans are frequently exposed to both Cr(VI) and PAHs, it is possible that Cr(VI) and PAHs have a synergistic effect on mutagenecity and cytotoxicity that contributes to the high incidence of lung cancer associated with exposure to both agents. In this study, we tested this possibility by determining the effect of Cr(VI) exposure on (+/-)-anti-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydro benzo[a]pyrene (BPDE, an active metabolite of PAHs) induced cytotoxicity, mutagenicity, and DNA adduct formation in Chinese hamster ovary (CHO) cells. Using the adenine phosphoribosyltransferase (APRT(+)) --> APRT(-) forward mutation assay, we found that while Cr(VI) alone induced low mutation frequency, it greatly enhanced BPDE-induced mutations in nucleotide excision repair (NER)-proficient CHO cells. Cr(VI) exposure also greatly enhanced BPDE-induced killing in NER-proficient cells. It is known that the cytotoxicity and mutagenicity of BPDE are mainly caused by the formation of DNA adduct, which are removed by NER. To test the possibility that the enhancement of cytotoxicity and mutagenicity by Cr(VI) is caused by the inhibition of NER, NER-deficient cells were used, and the enhancement effects of Cr(VI) were not observed in those cells. We further found that while Cr(VI) exposure does not change the total BPDE-DNA adduct formation, it significantly inhibited the repair of BPDE-DNA adducts from genomic DNA in NER-proficient cells. Using a host cell reactivation assay, we found that the repair of BPDE-DNA adduct in a luciferase reporter gene is greatly inhibited after Cr(VI) exposure in NER-proficient cells while not in NER-deficient cells. Together these results clearly demonstrate that Cr(VI) exposure can greatly enhance the mutagenicity and cytotoxicity of PAHs by inhibiting the cellular NER pathway, and this may constitute an important mechanism for Cr(VI)-induced human carcinogenesis
— id: 47759, year: 2004, vol: 43, page: 14282, stat: Journal Article,

Nickel (II) enhances benzo[a]pyrene diol epoxide-induced mutagenesis through inhibition of nucleotide excision repair in human cells: a possible mechanism for nickel (II)-induced carcinogenesis
Hu, Wenwei; Feng, Zhaohui; Tang, Moon-Shong
2004 Mar;25(3):455-462, Carcinogenesis
Nickel (II), a ubiquitous environmental and industrial contaminant, is a well-known human carcinogen, particularly in human lung cancer. Although by itself it is a weak mutagen, nickel (II) is able to significantly enhance the genotoxicity of other mutagens and carcinogens, such as polycyclic aromatic hydrocarbons (PAHs) and ultraviolet light. Certain human populations, especially cigarette smokers, are frequently exposed to both nickel (II) and PAHs. To understand the interplay of nickel (II) and PAHs in mutagenesis and human carcinogenesis, we used a shuttle vector mutagenicity assay to examine the effect of nickel (II) on (+/-) anti-7beta, 8alpha-dihydroxy-9alpha, 10alpha-epoxy-7,8,9,10-tetrahydroxybenzo[a]pyrene (BPDE)-induced mutagenesis in human cells. BPDE is an activated metabolite of benzo[a]pyrene (BP), a major carcinogen in cigarette smoke. The shuttle vector pSP189 modified with BPDE was transfected into human cells with and without nickel (II) exposure. We found that nickel (II) exposure significantly enhanced BPDE-induced mutation frequency, but did not change BPDE-induced mutational spectrum in the supF gene of pSP189 plasmids replicated in nucleotide excision repair (NER)-proficient human cells. However, the enhancing effect of nickel (II) on BPDE-induced mutation frequency was not observed in NER-deficient human XPA cells. We also found that nickel (II) exposure of human cells did not change the spontaneous mutation frequency of the supF gene in NER-proficient or NER-deficient human cells, indicating that nickel (II) did not affect the replication fidelity in human cells. Using a plasmid containing a luciferase reporter gene and a host cell reactivation assay, we have found that nickel (II) exposure greatly inhibited the repair of BPDE-DNA adducts in NER-proficient but not in NER-deficient cells. Together these results strongly suggest that nickel (II) can greatly enhance the mutagenicity and genotoxicity of PAHs by inhibiting the NER pathway in human cells, and this may constitute an important mechanism for nickel (II)-induced human carcinogenesis
— id: 48182, year: 2004, vol: 25, page: 455, stat: Journal Article,

Differential effects of polycyclic aromatic hydrocarbons on transactivation of AP-1 and NF-kappaB in mouse epidermal cl41 cells
Li, Jingxia; Chen, Haobin; Ke, Qingdong; Feng, Zhaohui; Tang, Moon-Shong; Liu, Bingci; Amin, Shantu; Costa, Max; Huang, Chuanshu
2004 Jun;40(2):104-115, Molecular carcinogenesis
Polycyclic aromatic hydrocarbons (PAHs) and their derivatives, such as benzo[a]pyrene (B[a]P), (+/-)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE), and 5-methylchrysene-1,2-diol-3,4-epoxide (5-MCDE), are complete carcinogens. However, the tumor promotion effects of PAHs remain unclear. We therefore investigated the possible activation of activator protein-1 (AP-1) and nuclear factor-kappaB (NFkappaB) in mouse epidermal Cl41 cells after different PAHs treatments, including B[a]P, B[a]PDE, chrysene-1,2-diol-3,4-epoxid (CDE), and 5-MCDE. The results showed that B[a]PDE and 5-MCDE were able to activate AP-1 and NF-kappaB, whereas B[a]P showed only marginal effect on AP-1 activation, and B[a]P and CDE had no effect on NF-kappaB activation. Treatment with either B[a]PDE or 5-MCDE also resulted in mitogen-activated protein kinases (MAPKs) activation as well as inhibitory subunit kappa-B (IkappaBalpha) phosphorylation and degradation, whereas B[a]P and CDE had no effect. Pretreatment with PD98059, a specific inhibitor for extracellular signal-regulated protein kinases (ERKs) upstream kinase MEK1/2, or SB202190, a p38 kinase inhibitor, resulted in a dramatic inhibition of B[a]PDE-induced AP-1 transactivation. In addition, B[a]PDE-induced AP-1 activation was also inhibited by overexpressing a dominant negative mutant of JNK1 in the cells. All these suggest ERKs, c-jun N-terminal kinases (JNKs), and p38 kinase signal transduction pathways are required for AP-1 induction by B[a]PDE. Taken together, B[a]PDE and 5-MCDE are the active compounds of PAHs to initiate signaling pathways. Considering the important roles of AP-1 and NF-kappaB in tumor promotion, we speculated the activation of AP-1 and NF-kappaB by B[a]PDE and 5-MCDE may involve in their or their parent compounds' tumor promotion effects. This study may help in better understanding the tumor promotion effects of PAHs
— id: 42690, year: 2004, vol: 40, page: 104, stat: Journal Article,

PI-3K and Akt are mediators of AP-1 induction by 5-MCDE in mouse epidermal Cl41 cells
Li, Jingxia; Chen, Haobin; Tang, Moon-Shong; Shi, Xianglin; Amin, Shantu; Desai, Dhimant; Costa, Max; Huang, Chuanshu
2004 Apr 12;165(1):77-86, Journal of cell biology
5-Methylchrysene has been found to be a complete carcinogen in laboratory animals. However, the tumor promotion effects of (+/-)-anti-5-methylchrysene-1,2-diol-3,4-epoxide (5-MCDE) remain unclear. In the present work, we found that 5-MCDE induced marked activator protein-1 (AP-1) activation in Cl41 cells. 5-MCDE also induced a marked activation of phosphatidylinositol 3-kinase (PI-3K). Inhibition of PI-3K impaired 5-MCDE-induced AP-1 transactivation, suggesting that PI-3K is an upstream kinase involved in AP-1 activation by 5-MCDE. Furthermore, we found that Akt is a PI-3K downstream mediator for 5-MCDE-induced AP-1 transactivation, whereas another PI-3K downstream kinase, p70(S6K), was not involved in AP-1 activation by 5-MCDE. Moreover, inhibition of Akt activation blocked 5-MCDE-induced activation of extracellular signal-regulated protein kinases (ERKs) and c-Jun NH(2)-terminal kinases (JNKs), whereas it did not affect p38K activation. Consistently, overexpression of a dominant-negative mutant of ERK2 or JNK1 blocked the AP-1 activation by 5-MCDE. These results demonstrate that 5-MCDE is able to induce AP-1 activation, and the AP-1 induction is specifically through a PI-3K/Akt-dependent and p70(S6K)-independent pathway
— id: 42691, year: 2004, vol: 165, page: 77, stat: Journal Article,

A critical role of PI-3K/Akt/JNKs pathway in benzo[a]pyrene diol-epoxide (B[a]PDE)-induced AP-1 transactivation in mouse epidermal Cl41 cells
Li, Jingxia; Tang, Moon-shong; Liu, Bingci; Shi, Xianglin; Huang, Chuanshu
2004 May 13;23(22):3932-3944, Oncogene
Mouse skin tumorigenicity studies indicate that benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE) contributes to carcinogenesis as both a tumor initiator and promoter. However, the mechanisms that mediate B[a]PDE tumor promotion effects remain unclear. Our results demonstrated that in mouse epidermal Cl41 cells, B[a]PDE treatment resulted in marked activation of AP-1 and its upstream MAPKs, including ERKs, JNKs and p38K. B[a]PDE exposure also led to activation of phosphotidylinositol 3-kinase (PI-3K), Akt and p70 S6 kinase (p70S6k). B[a]PDE-induced AP-1 transactivation was inhibited by pretreatment of cells with PI-3K inhibitors, wortmannin or Ly294002. In contrast, inhibition of p70S6k with rapamycin did not show any inhibitory effects. An overexpression of dominant-negative mutant of PI-3K, Deltap85, impaired B[a]PDE-induced activation of PI-3K, Akt and AP-1 transactivation. Furthermore, an overexpression of dominant-negative Akt mutant, Akt-T308A/S473A, blocked B[a]PDE-induced activation of Akt, AP-1 and JNKs, while it did not affect the activation of p70S6k, ERKs and p38 kinase. These results demonstrated that B[a]PDE was able to induce AP-1 transactivation and this AP-1 induction was specific through PI-3K/Akt/JNKs-dependent and p70S6k-independent pathways. This study also indicated that Akt-T308A/S473A blocks B[a]PDE-induced AP-1 activation specific through impairing JNK pathway. These findings will help us to understand the signal transduction pathways involved in the carcinogenic effects of B[a]PDE
— id: 42695, year: 2004, vol: 23, page: 3932, stat: Journal Article,

Tobacco carcinogens and co-carcinogens damage the p53 and K-ras genes and reduce DNA repair capacity
Tang, MS
2004 NOV ;13(11):1968S-1968S, Cancer epidemiology biomarkers & prevention
— id: 50167, year: 2004, vol: 13, page: 1968S, stat: Journal Article,

DNA damage, repair, and mutation induction by (+)-Syn and (-)-anti-dibenzo[a,l]pyrene-11,12-diol-13,14-epoxides in mouse cells
Yoon, Jung-Hoon; Besaratinia, Ahmad; Feng, Zhaohui; Tang, Moon-Shong; Amin, Shantu; Luch, Andreas; Pfeifer, Gerd P
2004 Oct 15;64(20):7321-7328, Cancer research
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental carcinogens. PAHs are classified into bay and fjord region compounds according to structural differences in the molecule region where enzymatic epoxidation occurs. Dibenzo[a,l]pyrene (DB[a,l]P), one of the fjord region compounds, has been demonstrated to be the most carcinogenic PAH known to date. DB[a,l]P is activated to fjord region (+)-syn and (-)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyr ene (DB[a,l]PDE) metabolites. In this study, we analyzed mutagenesis induced by (+)-syn- and (-)-anti-DB[a,l]PDE at the cII transgene in Big-Blue mouse cells. The mutant frequency of untreated cells (background level) was 6.53 x 10(-5). This level increased 3.7-fold for 20 nmol/L, 5.3-fold for 50 nmol/L, and 7.9-fold for 100 nmol/L (+)-syn-DB[a,l]PDE, respectively. In the case of (-)-anti-DB[a,l]PDE it increased 4.5-fold for 20 nmol/L, 6.7-fold for 50 nmol/L, and 10.6-fold for 100 nmol/L, respectively, indicating that (-)-anti-DB[a,l]PDE is slightly more mutagenic than (+)-syn-DB[a,l]PDE. The mutational spectra of (+)-syn- and (-)-anti-DB[a,l]PDE were quite similar except for several hotspots, specific for either (+)-syn-DB[a,l]PDE or (-)-anti-DB[a,l]PDE. The most frequently induced mutations were A to T transversions, which were 43.9% for (+)-syn- and 38.8% for (-)-anti-DB[a,l]PDE. In addition, G to T transversions were induced significantly, at frequencies of 18.5% by (+)-syn- and 18.1% by (-)-anti-DB[a,l]PDE. Using UvrABC cleavage and ligation-mediated PCR or the terminal transferase-dependent PCR method, we have determined DB[a,l]PDE-DNA adduct formation sites and repair rates in carcinogen-exposed cells. The mutation hotspots coincided with sites of strong adduct formation, but not all of the adduct hotspots were mutational hotspots. Slow adduct removal occurred for both (+)-syn- and (-)-anti-DB[a,l]PDE adducts over a time period of up to 72 hours. The data suggest that, although the (-)-anti-isomer is slightly more mutagenic, DNA adducts of both DB[a,l]PDE stereoisomers may have similar biological properties. We discuss the implications of these findings for human cancer mutagenesis
— id: 72108, year: 2004, vol: 64, page: 7321, stat: Journal Article,

Formation of trans-4-hydroxy-2-nonenal- and other enal-derived cyclic DNA adducts from omega-3 and omega-6 polyunsaturated fatty acids and their roles in DNA repair and human p53 gene mutation
Chung, Fung-Lung; Pan, Jishen; Choudhury, Sujata; Roy, Rabindra; Hu, Wenwei; Tang, Moon-shong
2003 Oct 29;531(1-2):25-36, Mutation research
The cyclic 1,N(2)-propanodeoxyguanosine adducts, derived from alpha,beta-unsaturated aldehydes or enals, including acrolein (Acr), crotonaldehyde (Cro), and trans-4-hydroxy-2-nonenal (HNE), have been detected as endogenous DNA lesions in rodent and human tissues. Collective evidence has indicated that the oxidative metabolism of polyunsaturated fatty acids (PUFAs) is an important pathway for endogenous formation of these adducts. In a recent study, we examined the specific role of different types of fatty acids, omega-3 and omega-6 PUFAs, in the formation of cyclic adducts of Acr, Cro, and HNE. Our studies showed that the incubation of deoxyguanosine 5'-monophosphate with omega-3 or omega-6 fatty acids under oxidative conditions in the presence of ferrous sulfate yielded different amounts of Acr, Cro, and HNE adducts, depending on the types of fatty acids. We observed that Acr- and Cro-dG adducts are primarily formed from omega-3, and the adducts derived from longer chain enals, such as HNE, were detected exclusively from omega-6 fatty acids. Acr adducts are also formed from omega-6 fatty acids, but to a lesser extent; the yields of Acr adducts are proportional to the number of double bonds present in the PUFAs. Two previously unknown cyclic adducts, one from pentenal and the other from heptenal, were detected as products from omega-3 and omega-6 fatty acids, respectively. Because omega-6 PUFAs are known to be involved in the promotion of tumorigenesis, we investigated the role of HNE adducts in p53 gene mutation by mapping the HNE binding to the human p53 gene with UvrABC nuclease and determined the formation of HNE-dG adducts in the gene. The results showed that HNE-dG adducts are preferentially formed in a sequence-specific manner at the third base of codon 249 in the p53 gene, a mutational hotspot in human cancers. The DNA repair study using plasmid DNA containing HNE-dG adducts as a substrate in HeLa cell extracts showed that HNE adducts are readily repaired, and that nucleotide excision repair appears to be a major pathway involved. Together, results of these studies provide a better understanding of the involvement of different PUFAs in DNA damage and their possible roles in tumorigenesis
— id: 72109, year: 2003, vol: 531, page: 25, stat: Journal Article,

Mutational spectrum and genotoxicity of the major lipid peroxidation product, trans-4-hydroxy-2-nonenal, induced DNA adducts in nucleotide excision repair-proficient and -deficient human cells
Feng, Zhaohui; Hu, Wenwei; Amin, Shantu; Tang, Moon-shong
2003 Jul 1;42(25):7848-7854, Biochemistry
trans-4-Hydroxy-2-nonenal (4-HNE), a major product of lipid peroxidation, is able to interact with DNA to form 6-(1-hydroxyhexanyl)-8-hydroxy-1,N(2)-propano-2'-deoxyguanosine (4-HNE-dG) adducts, but its genotoxicity and mutagenicity remain elusive. It has been reported that 4-HNE treatment in human cells induces a high frequency of G.C to T.A mutations at the third base of codon 249 (AGG*) of the p53 gene, a mutational hot spot in human cancers, particularly in hepatocellular carcinoma. This G.C to T.A transversion at codon 249, however, has been thought to be caused by etheno-DNA adducts induced by the endogenous metabolite of 4-HNE, 2,3-epoxy-4-hydroxynonanal. We have recently found that 4-HNE preferentially forms 4-HNE-dG adducts at the GAGG*C/A sequence in the p53 gene including codon 249 (GAGG*C). Our finding supports the possibility that G.C to T.A mutations at codon 249 may be induced by 4-HNE-dG adducts. To investigate this possibility, we determined the mutational spectrum induced by 4-HNE-dG adducts in the supF gene of shuttle vector pSP189 replicated in human cells. We have found that 4-HNE-dG adducts are mutagenic and genotoxic in human cells, and that G.C to T.A transversions are the most prevalent mutations induced by 4-HNE-dG adducts. Furthermore, 4-HNE-dG adducts induce a significantly higher level of genotoxicity and mutagenicity in nucleotide excision repair (NER)-deficient human and Escherichia coli cells than in NER-proficient cells, indicating that NER is a major pathway for repairing 4-HNE-dG adducts in both human and E. coli cells. Together, these results suggest that 4-HNE-dG adducts may contribute greatly to the G.C to T.A mutation at codon 249 of the p53 gene, and may play an important role in carcinogenesis
— id: 39186, year: 2003, vol: 42, page: 7848, stat: Journal Article,

Effects of genomic context and chromatin structure on transcription-coupled and global genomic repair in mammalian cells
Feng, Zhaohui; Hu, Wenwei; Chasin, Lawrence A; Tang, Moon-shong
2003 Oct 15;31(20):5897-5906, Nucleic acids research
It has been long recognized that in mammalian cells, DNA damage is preferentially repaired in the transcribed strand of transcriptionally active genes. However, recently, we found that in Chinese hamster ovary (CHO) cells, UV-induced cyclobutane pyrimidine dimers (CPDs) are preferentially repaired in both the transcribed and the non-transcribed strand of exon 1 of the dihydrofolate reductase (DHFR) gene. We mapped CPD repair at the nucleotide level in the transcriptionally active DHFR gene and the adjacent upstream OST gene, both of which have been translocated to two chromosomal positions that differ from their normal endogeneous positions. This allowed us to study the role of transcription, genomic context and chromatin structure on repair. We found that CPD repair in the transcribed strand is the same for endogenous and translocated DHFR genes, and the order of repair efficiency is exon 1 > exon 2 > exon 5. However, unlike the endogenous DHFR gene, efficient repair of CPDs in the non-transcribed strand of exon 1 is not observed in the translocated DHFR gene. CPDs are efficiently repaired in the transcribed strand in endogenous and translocated OST genes, which indicates that efficient repair in exon 1 of the non-transcribed strand of the endogenous DHFR gene is not due to the extension of transcription-coupled repair of the OST gene. Using micrococcal nuclease digestion, we probed the chromatin structure in the DHFR gene and found that chromatin structure in the exon 1 region of endogenous DHFR is much more open than at translocated loci. These results suggest that while transcription-coupled repair is transcription dependent, global genomic repair is greatly affected by chromatin structure
— id: 39040, year: 2003, vol: 31, page: 5897, stat: Journal Article,

Chromium(VI) exposure enhances polycyclic aromatic hydrocarbon-DNA binding at the p53 gene in human lung cells
Feng, Zhaohui; Hu, Wenwei; Rom, William N; Costa, Max; Tang, Moon-Shong
2003 Apr;24(4):771-778, Carcinogenesis
Chromium(VI) [Cr(VI)] is a ubiquitous environmental and industrial contaminant. Cr(VI) exposure is strongly associated with a higher incidence of human lung cancer, but the mechanism of Cr(VI) carcinogenicity remains unclear. Cigarette smoking has been known as the prominent cause of lung cancer, and polycyclic aromatic hydrocarbons (PAHs), the major carcinogens in cigarette smoke, have been suggested as being responsible for the initiation and development of lung cancer. It has been reported that lung cancer from workers exposed to Cr(VI) has a high percentage of G to T transversion mutations in the non-transcribed strand of the p53 gene, a hallmark of PAH-induced mutation. Cr(VI) is a weak mutagen although it can induce a high percentage of G to T transversion mutations. These results raise the possibility that Cr(VI) may enhance PAH binding at the p53 gene in lung tissue. To test this possibility, we have determined the effect of Cr(VI) exposure on benzo[a]pyrene diol epoxides (BPDE)-DNA binding at total genomic DNA level and at the p53 gene in normal human lung fibroblast cells. We found that in lung cells Cr(VI) pre-exposure does not affect the BPDE-DNA binding at the total genomic DNA level or at exons 5, 6 and 9 of the p53 gene; however, it greatly enhances BPDE-DNA binding at exons 7 and 8 of the p53 gene, especially at mutational hotspots of lung cancer: codons 248, 273 and 282 of the p53 gene. No enhancement of BPDE-DNA binding in the p53 was observed when naked genomic DNA isolated from Cr(VI)-exposed cells was modified with BPDE in vitro. These results suggest that Cr(VI) exposure may enhance chromatin structure-dependent carcinogen-DNA binding. This effect may contribute to the synergism of Cr(VI) and BPDE on mutagenesis and cell transformation, and may also contribute to the higher incidence of lung cancer in Cr(VI)-exposed populations
— id: 39234, year: 2003, vol: 24, page: 771, stat: Journal Article,

Preferential carcinogen-DNA adduct formation at codons 12 and 14 in the human K-ras gene and their possible mechanisms
Hu, Wenwei; Feng, Zhaohui; Tang, Moon-Shong
2003 Aug 26;42(33):10012-10023, Biochemistry
In the ras gene superfamily, codon 12 (-TGGTG-) of the K-ras gene is the most frequently mutated codon in human cancers. Recently, we have found that bulky chemical carcinogens preferentially form DNA adducts at codons 12 and 14 (-CGTAG-) in the K-ras gene in normal human bronchial epithelial (NHBE) cells. Furthermore, DNA adducts formed at codon 12 of the K-ras gene are poorly repaired compared with those at other codons including codon 14. These results suggest that targeted carcinogen-DNA adduct formation is a major reason for the observed high mutation frequency at codon 12 of the K-ras gene in human cancers. This preferential carcinogen-DNA adduct formation at codons 12 and 14 could result from effects of (1) primary sequences of these codons and their surrounding codons in the K-ras gene, (2) the chromatin structure, and/or (3) epigenetic factors such as C5 cytosine methylation or other DNA modifications at these codons and their surrounding codons. To distinguish these possibilities, we have introduced modifications with benzo[a]pyrene diol epoxide, N-hydroxy-2-aminofluorene, and aflatoxin B1 8,9-epoxide in (1) naked intact genomic DNA isolated from NHBE cells, (2) fragmented genomic DNA digested by restriction enzymes, and (3) in vitro synthesized DNA fragments containing the K-ras gene exon 1 sequence with or without methylation of the cytosines at CpG sites and the cytosines pairing with the guanines of codons 12 and 14. The distribution of carcinogen-DNA adducts in the K-ras gene was mapped at the nucleotide sequence level using the UvrABC nuclease incision method with or without the ligation-mediated polymerase chain reaction technique. We have found that carcinogens preferentially form adducts at codons 12 and 14 in the K-ras gene exon 1 in intact as well as in fragmented genomic DNA. In contrast, this preferential DNA adduct formation at codons 12 and 14 was not observed in PCR-amplified DNA fragments containing the K-ras gene exon 1 sequence. Methylation of the cytosine at the CpG site of codon 14, or the cytosine pairing with guanine of codon 14, greatly enhanced carcinogen-DNA adduct formation at codon 14 but did not affect carcinogen-DNA adduct formation at codon 12. Methylation of the cytosine pairing with the guanine of codon 12 also did not enhance carcinogen-DNA adduct formation at codon 12. Furthermore, we found that the cytosine at the CpG site of codon 14 is highly methylated in NHBE cells. These results suggest that cytosine methylation at the CpG site is the major reason for the preferential DNA damage at codon 14 and that epigenetic modification(s) other than cytosine methylation may contribute to the preferential DNA damage at codon 12 of the K-ras gene
— id: 39105, year: 2003, vol: 42, page: 10012, stat: Journal Article,

Gene expression of primary human bronchial epithelial cells in response to coal dusts with different prevalence of coal workers' pneumoconiosis
Hu, Wenwei; Zhang, Qi; Su, Wei Cheng; Feng, Zhaohui; Rom, William; Chen, Lung Chi; Tang, Moonshong; Huang, Xi
2003 Jul 11;66(13):1249-1265, Journal of toxicology & environmental health. Pt. A
Striking regional differences in the prevalence of coal workers' pneumoconiosis (CWP) have been observed but not fully understood. This study investigated the early biological responses of primary lung cells to treatment with coal dusts from various seams. High-density oligoarray technology (GeneChip, Affymetrix, Santa Clara, CA) was used to compile gene expression profiles of primary human bronchial epithelial cells to low concentrations (2 microg/cm(2)) of coals for 6 h or 24 h of treatment. Data showed that a total of 1050 out of 12,000 genes on the chip were altered by 2 coal dusts. The coal from the Pennsylvania (PA) coal-mine region with a high prevalence of CWP altered 908 genes, many more than the coal from Utah (UT) with a low prevalence of CWP, which affected 356 genes. Many genes decreased their expression levels in response to the PA coal at 6 h and/or 24 h of treatment. For example, transferrin receptor, a gene known to control cellular iron uptake, was downregulated in the cells treated with the iron-containing PA coal in order to protect cells from iron overload. The UT coal without bioavailable iron had no such effect. The downregulation patterns of genes were confirmed by reverse-transcription polymerase chain reaction (RT-PCR). This study is one of the first in profiling gene expressions of primary bronchial epithelial cells treated with coals from various seams, which may set stages for future studies on specific genes
— id: 39145, year: 2003, vol: 66, page: 1249, stat: Journal Article,

The action of a dietary retinoid on gene expression and cancer induction in electron-irradiated rat skin
Burns, Fredric J; Chen, Shuaili; Xu, Guijuan; Wu, Feng; Tang, Moon-Shong
2002 Dec;43 Suppl:S229-S232, Journal of radiation research
Current models of radiation carcinogenesis generally assume that the DNA is damaged in a variety of ways by the radiation and that subsequent cell divisions contribute to the conversion of the damage to heritable mutations. Cancer may seem complex and intractable, but its complexity provides multiple opportunities for preventive interventions. Mitotic inhibitors are among the strongest cancer preventive agents, not only slowing the growth rate of preneoplasias but also increasing the fidelity of DNA repair processes. Ionizing radiation, including electrons, is a strong inducer of cancer in rat skin, and dietary retinoids have shown potent cancer preventive activity in the same system. A non-toxic dietary dose of retinyl acetate altered gene expression levels 24 hours after electron irradiation of rat skin. Of the 8740 genes on an Affymetrix rat expression array, the radiation significantly (5 fold or higher) altered 188, while the retinoid altered 231, including 16 radiation-altered genes that were reversely altered. While radiation strongly affected the expression of stress response, immune/inflammation and nucleic acid metabolism genes, the retinoid most strongly affected proliferation-related genes, including some significant reversals, such as, keratin 14, retinol binding protein, and calcium binding proteins. These results point to reversal of proliferation-relevant genes as a likely basis for the anti-radiogenic effects of dietary retinyl acetate
— id: 72110, year: 2002, vol: 43 Suppl, page: S229, stat: Journal Article,

Molecular responses of mammalian cells to nickel and chromate exposure
Costa M; Salnikow K; Sutherland JE; Klutz T; Peng W; Tang M-S
Biomarkers of environmentally associated disease : technologies, concepts, and perspectives Boca Raton FL : Lewis Publishers, 2002,
— id: 4434, year: 2002, vol: , page: 261, stat: Chapter,

Preferential DNA damage and poor repair determine ras gene mutational hotspot in human cancer
Feng, Zhaohui; Hu, Wenwei; Chen, James X; Pao, Annie; Li, Haiying; Rom, William; Hung, Mien-Chie; Tang, Moon-shong
2002 Oct 16;94(20):1527-1536, Journal of the National Cancer Institute
BACKGROUND: Mutations in ras genes are commonly found in human cancers and in animal models. Although mutations at codons 12, 13, and 61 of H-, N- and K-ras genes can activate their oncogenic function, mutations at codon 12 of K-ras are the most common mutations found among the three ras genes in human cancers. To investigate whether codon 12 of human K-ras is especially susceptible to carcinogens and/or whether carcinogen-DNA adducts at this codon are repaired less efficiently, we examined tobacco smoke carcinogen-induced DNA damage in normal human bronchial epithelial and fibroblast cells. METHODS: We used the UvrABC nuclease incision method in combination with ligation-mediated polymerase chain reaction to map the distribution of DNA adducts induced by benzo[a]pyrene diol epoxide (BPDE) and other bulky carcinogens within exons 1 and 2 in H-ras, N-ras, and K-ras. We also analyzed BPDE-DNA adduct repair efficiency in these three genes using the same method. RESULTS: Codons 12 and 14 of the K-ras gene were hotspots for carcinogen-DNA adduct formation, with little and no adduct formation at codons 13 and 61, respectively. The BPDE-DNA adducts formed at codon 14 were repaired almost twice as quickly as those formed at codon 12. There was some BPDE-DNA adduct formation at codons 12 of H-ras and N-ras, but this codon was not a hotspot. Furthermore, no substantial difference in repair rates between codon 12 and the other codons analyzed (codons 3 and 18) was observed in either the H-ras or N-ras genes. CONCLUSION: These findings link the human cancer mutational hotspot at codon 12 of K-ras to preferential DNA damage and poor repair
— id: 39575, year: 2002, vol: 94, page: 1527, stat: Journal Article,

Transcription-coupled DNA repair is genomic context-dependent
Feng, Zhaohui; Hu, Wenwei; Komissarova, Elena; Pao, Annie; Hung, Mien-Chie; Adair, Gerald M; Tang, Moon-shong
2002 Apr 12;277(15):12777-12783, Journal of biological chemistry
DNA damage is preferentially repaired in the transcribed strand of many active genes. Although the concept of DNA repair coupled with transcription has been widely accepted, its mechanisms remain elusive. We recently reported that in Chinese hamster ovary cells while ultraviolet light-induced cyclobutane pyrimidine dimers (CPDs) are preferentially repaired in the transcribed strand of dihydrofolate reductase gene, CPDs are efficiently repaired in both strands of adenine phosphoribosyltransferase (APRT) locus, in either a transcribed or nontranscribed APRT gene (1). These results suggested that the transcription dependence of repair may depend on genomic context. To test this hypothesis, we constructed transfectant cell lines containing a single, actively transcribed APRT gene, integrated at different genomic sites. Mapping of CPD repair in the integrated APRT genes in three transfectant cell lines revealed two distinct repair patterns, either preferential repair of CPDs in the transcribed strand or very poor repair in both strands. Similar kinetics of micrococcal nuclease digestion were seen for all three transfectant APRT gene domains and endogenous APRT locus. Our results suggest that both the efficiency and strand-specificity of repair of an actively transcribed gene are profoundly affected by genomic context but do not reflect changes in first order nucleosomal structure
— id: 39720, year: 2002, vol: 277, page: 12777, stat: Journal Article,

4-aminobiphenyl is a major etiological agent of human bladder cancer: evidence from its DNA binding spectrum in human p53 gene
Feng, Zhaohui; Hu, Wenwei; Rom, William N; Beland, Frederick A; Tang, Moon-shong
2002 Oct;23(10):1721-1727, Carcinogenesis
4-aminobiphenyl (4-ABP) is a major etiological agent of human bladder cancer, and its metabolites are able to form DNA adducts that may induce mutation and initiate bladder carcinogenesis. Thirty to sixty percent of human bladder cancer has a mutation in the p53 gene, and the mutational spectrum bears two characteristics: compared with other cancers, the pattern of mutations is more evenly distributed along the p53 gene, and the mutational hotspots occur at both CpG sites, such as codons 175, 248 and 273, and non-CpG sites, such as codons 280 and 285, the latter two being unique mutational hotspots for bladder and other urinary tract cancers. These findings raise the possibility that the special p53 mutational features in human bladder cancer are due to the unique binding spectrum of metabolically activated 4-ABP in bladder cells. To address this question, here we have mapped the 4-ABP-DNA adduct distribution in the p53 gene at the nucleotide sequence level in human bladder cells. We found that, unlike benzo[a]pyrene trans-7,8-dihydrodiol-9,10-epoxide-DNA adduction, which preferentially occurs at CpG sites, 4-ABP-DNA adduction is not biased for CpG sites, and the adducts are more evenly distributed along the p53 gene; nonetheless, the p53 mutational hotspots in bladder cancer at codons 175, 248, 280 and 285 are also the preferential sites for 4-ABP adduct formation. These results strongly suggest that the unique binding spectrum of 4-ABP contributes greatly to the unique mutational spectrum in the p53 gene of human bladder cancer, and provide further molecular evidence to directly link 4-ABP to bladder cancer
— id: 39577, year: 2002, vol: 23, page: 1721, stat: Journal Article,

N-hydroxy-4-aminobiphenyl-DNA binding in human p53 gene: sequence preference and the effect of C5 cytosine methylation
Feng, Zhaohui; Hu, Wenwei; Rom, William N; Beland, Frederick A; Tang, Moon-shong
2002 May 21;41(20):6414-6421, Biochemistry
4-Aminobiphenyl (4-ABP) is a major etiological agent for human bladder cancer. Metabolically activated 4-ABP is able to interact with DNA to form adducts that may induce mutations and initiate carcinogenesis. Thirty to sixty percent of bladder cancer has a mutation in the tumor suppressor p53 gene, and the mutational spectrum bears unique features. To date the DNA binding spectrum of 4-ABP in the p53 gene is not known due to the lack of methodology to detect 4-ABP-DNA adducts at nucleotide sequence level. We have found that UvrABC nuclease, a nucleotide excision repair complex isolated from Escherichia coli, is able to incise specifically and quantitatively DNA fragments modified with N-hydroxy-4-aminobiphenyl (N-OH-4-ABP), an activated intermediate of 4-ABP. Using the UvrABC nuclease incision method, we mapped the binding spectrum of N-OH-4-ABP in DNA fragments containing exons 5, 7, and 8 of the human p53 gene and also determined the effect of C5 cytosine methylation on N-OH-4-ABP-DNA binding. We found that codon 285, a mutational hotspot at a non-CpG site in bladder cancer, is the preferential binding site for N-OH-4-ABP. We also found that C5 cytosine methylation greatly enhanced N-OH-4-ABP binding at CpG sites, and that two mutational hotspots at CpG sites, codons 175 and 248, became preferential binding sites for N-OH-4-ABP only after being methylated. These results suggest that both the unique DNA binding specificity of 4-ABP and cytosine methylation contribute to the mutational spectrum of the p53 gene in human bladder cancer
— id: 32120, year: 2002, vol: 41, page: 6414, stat: Journal Article,

Transcription-coupled and transcription-independent repair of cyclobutane pyrimidine dimers in the dihydrofolate reductase gene
Hu, Wenwei; Feng, Zhaohui; Chasin, Lawrence A; Tang, Moon-shong
2002 Oct 11;277(41):38305-38310, Journal of biological chemistry
Using a ligation-mediated polymerase chain reaction technique, we have mapped the repair of ultraviolet light-induced cyclobutane pyrimidine dimers (CPDs) at the nucleotide level in exons 1, 2, and 5 of the dihydrofolate reductase (DHFR) gene in Chinese hamster ovary cells. We found that CPDs are preferentially repaired in the transcribed strand (T strand) and that the order of repair efficiency is exon 1 > exon 2 > exon 5. In the cells with a deletion of the DHFR gene encompassing the promoter region and the first four exons, CPDs are not repaired in the T strand of the residual DHFR gene. These results substantiate the idea that the preferential repair of CPDs in the T strand is transcription dependent. However, in the wild type gene we have found that CPDs are efficiently repaired in the nontranscribed strand (NT strand) of exon 1 but not in the NT strand of exons 2 and 5. Probing the chromatin structure of exons 1, 2, and 5 of the DHFR gene with micrococcal nuclease revealed that the exon 1 region is much more sensitive to micrococcal nuclease digestion than the exon 2 and exon 5 regions, suggesting that the chromatin structure in the exon 1 region is much more open. These results suggest that, although preferential repair of the T strand of the DHFR gene is transcription dependent, repair of the NT strand is greatly affected by chromatin structure
— id: 39609, year: 2002, vol: 277, page: 38305, stat: Journal Article,

The major lipid peroxidation product, trans-4-hydroxy-2-nonenal, preferentially forms DNA adducts at codon 249 of human p53 gene, a unique mutational hotspot in hepatocellular carcinoma
Hu, Wenwei; Feng, Zhaohui; Eveleigh, Jamie; Iyer, Ganesh; Pan, Jishen; Amin, Shantu; Chung, Fung-Lung; Tang, Moon-Shong
2002 Nov;23(11):1781-1789, Carcinogenesis
Trans-4-hydroxy-2-nonenal (4-HNE), a major electrophilic by-product of lipid peroxidation, is able to interact with DNA to form exocyclic guanine adducts. 4-HNE is a mutagen and a significant amount of 4-HNE-guanine adduct has been detected in normal cells. Recently, it has been reported that exposure of the wild-type p53 human lymphoblastoid cell line to 4-HNE causes a high frequency of G to T transversion mutations at the third base of codon 249 (-AGG*-) in the p53 gene, a mutational hotspot in human cancers, particularly hepatocellular carcinoma. These findings raise a possibility that 4-HNE could be an important etiological agent for human cancers that have a mutation at codon 249 of the p53 gene. However, to date, the sequence specificity of 4-HNE-DNA binding remains unclear due to the lack of methodology. To address this question, we have developed a method, using UvrABC nuclease, a nucleotide excision repair enzyme complex isolated from Escherichia coli, to map the distribution of 4-HNE-DNA adducts in human p53 gene at the nucleotide sequence level. We found that 4-HNE-DNA adducts are preferentially formed at the third base of codon 249 in the p53 gene. The preferential binding of 4-HNE was also observed at codon 174, which has the same sequence and the same nearest neighbor sequences (-GAGG*C-) as codon 249. These results suggest that 4-HNE may be an important etiological agent for human cancers that have a mutation at codon 249 of the p53 gene
— id: 39567, year: 2002, vol: 23, page: 1781, stat: Journal Article,

Inhibition of benzo(a)pyrene diol-epoxide-induced transactivation of activated protein 1 and nuclear factor kappaB by black raspberry extracts
Huang, Chuanshu; Huang, Yi; Li, Jingxia; Hu, Wenwei; Aziz, Robeena; Tang, Moon-shong; Sun, Nanjun; Cassady, John; Stoner, Gary D
2002 Dec 1;62(23):6857-6863, Cancer research
Freeze-dried black raspberries have been shown to inhibit the development of chemically induced esophageal and colon cancer in rodents.In addition, organic extracts of black raspberries inhibit benzo(a)pyrene (BaP)-induced cell transformation in vitro. The molecular mechanisms through which black raspberries inhibit carcinogenesis remain unclear. We investigated the effects of black raspberry extracts on transactivation of activated protein 1 (AP-1) and nuclear factor kappaB (NFkappaB) induced by BaP diol-epoxide (BPDE), the ultimate carcinogen of BaP, in mouse epidermal JB6 Cl 41 (Cl 41) cells. Black raspberries were extracted with methanol, and the methanol extract was partitioned and chromatographed into several fractions designated RU-F003, RU-F004, RU-DM, and RU-ME. Pretreatment of Cl 41 cells with RU-F003, RU-DM, or RU-ME resulted in an inhibition of BPDE-induced AP-1 and NFkappaB activities. The RU-ME fraction was the most potent inhibitor among the fractions tested. In contrast, fraction RU-F004 did not inhibit BPDE-induced AP-1 or NFkappaB activities in Cl 41 cells. The inhibitory effects of RU-ME on BPDE-induced activation of AP-1 and NFkappaB appear to be mediated via inhibition of mitogen activated protein kinase activation and inhibitory subunit kappaB phosphorylation, respectively. Pretreatment of cells with berry fractions did not result in an inhibition of BPDE binding to DNA; thus, this was not a mechanism of reduced AP-1 and NFkappaB activities. None of the fractions was found to affect p53-dependent transcription activity. In view of the important roles of AP-1 and NFkappaB in tumor promotion/progression, these results suggest that the ability of black raspberries to inhibit tumor development may be mediated by impairing signal transduction pathways leading to activation of AP-1 and NFkappaB. The RU-ME fraction appears to be the major fraction responsible for the inhibitory activity of black raspberries
— id: 38406, year: 2002, vol: 62, page: 6857, stat: Journal Article,

Mapping polycyclic aromatic hydrocarbon and aromatic amine-induced DNA damage in cancer-related genes at the sequence level
Tang, Moon-shong; Pfeifer, Gerd P; Denissenko, Mikhail F; Feng, Zhaohui; Hu, Wenwei; Pao, Annie; Zheng, Yi; Zheng, Jessica B; Li, Haiying; Chen, James X
2002 Mar;205(1-2):103-113, International journal of hygiene & environmental health
Genomic injury induced by environmental carcinogens, such as polycyclic aromatic hydrocarbons and aromatic amines, is the initial step that can trigger mutagenesis and carcinogenesis. In addition to the physico-chemical property of DNA damaging agents, several important factors such as primary sequence, chromatin structure, methylation, protein association, and transcriptional activity can affect not only the initial level and distribution of DNA damage but also the efficiency of repair. Therefore, mapping the DNA damage induced by environmental agents in cancer-related genes such as p53 and ras at the sequence level provides essential information for assessing their carcinogenic potential. Recently, using the E. coli nucleotide excision enzyme complex, UvrABC nucleases in combination with ligation-mediated polymerase chain reaction, we developed a method to map DNA damage in the p53 and ras genes. These studies led us to conclude that targeted DNA damage, in combination with growth selection, contributes greatly in shaping the mutation spectrum in these genes in human cancer. Here we present the rationale and details of this approach, typical experimental results and necessary precautions
— id: 39640, year: 2002, vol: 205, page: 103, stat: Journal Article,

The effect of C(5) cytosine methylation at CpG sequences on mitomycin-DNA bonding profiles
Li, VS; Tang, MS; Kohn, H
2001 APR ;9(4):863-873, Bioorganic & medicinal chemistry
Recent studies have documented that cytosine C(5) methylation of CpG sequences enhances mitomycin C(1) adduction. The reports differ on the extent and uniformity oft modification at the nucleotide level. We have determined the bonding profiles for mitomycin monoalkylation in two DNA restriction fragments where the CpG sequences were methylated. Three mitomycin substrates were used and two different enzymatic assays employed to monitor the extent of drug modification at the individual base sites. Drug-DNA modification was accomplished with 1 and 10-decarbamoylmitomycin C (2) under reductive (Na2S2O4) conditions and with N-methyl-7-methoxyaziridinomitosene (3) under nonreductive conditions. The UvrABC incision assay permitted us to quantitate the sites of drug adduction, and the lambda -exonuclease stop assay provided a qualitative estimation of drug-DNA modification consistent with the UvrABC data. We learned that C(5) cytosine methylation (m(5)C) enhanced the extent of overall DNA modification. Using the UvrABC endonuclease assay, we found that modification by 1 increased 2.0 and 7.4 times for the two DNA restriction fragments. Analysis of the modification sires at the nucleotide sequence level revealed that guanine (G) was the: only base modified and that the overall increased level of DNA adduction was due to enhanced modification of select m(5)CpG* (G* = mitomycin (mitosene) adduction sites) loci compared with CpG* sites: the largest differences reached two orders of magnitude. Significantly, not all CpG* sites underwent increased drug adduction upon C(5) cytosine methylation. The effect of C(5) cytosine methylation on the drug adduction profiles was less pronounced for G* sites located within dinucleotide sequences other than CpG*. We observed that DNA methylation often led to slightly diminished adduction levels at these sites. The different m(5)CpC* adduction patterns provided distinctive sequence-selective bonding profiles for 1-3. We have attributed the large differences in guanine reactivity to DNA structural factors created, in part. by C(5) cytosine methylation. The significance of these findings in cancer chemotherapy is briefly discussed. (C) 2001 Published by Elsevier Science Ltd
— id: 98282, year: 2001, vol: 9, page: 863, stat: Journal Article,

Sequence-dependent interactions of two forms of UvrC with DNA helix-stabilizing CC-1065-N3-adenine adducts
Nazimiec, M; Lee, CS; Tang, YL; Ye, XC; Case, R; Tang, M
2001 SEP 18 ;40(37):11073-11081, Biochemistry
The uvrA, uvrB, and uvrC genes of Escherichia coli control the initial steps of nucleotide excision repair. The uvrC gene product is involved in at least one of the dual incisions produced by the UvrABC complex. Using single-stranded (ss) DNA affinity chromatography, we have separated two forms of UvrC from both wild-type E. coli cells and overproducing cells. UvrCI elutes at 0.4 M KCl, and UvrCII elutes at 0.6 M KCI. In general, both forms, in the presence of UvrA and UvrB, actively incise UV-irradiated and CC-1065-modified DNA in the same fashion; i.e., they incise six to eight nucleotides 5 ' to and three to five nucleotides 3 ' to a photoproduct or a CC-1065-N3-adenine adduct. They produce different incisions, however, at a CC-1065-N3-adenine adduct in the sequence 5 ' -GATTA*CG- present in the MspI-BstNI 117 bp fragment of M13mpl. UvrABCI incises at both the 5 ' and 3 ' sides of the adduct (UvrABCI cut), while UvrABCII incises only at the 5 ' side (UvrABCII cut). Mixing UvrCI and UvrCII results in both UvrABCI and UvrABCII cuts, and the levels of these two types of cutting are proportional to the amount of UvrCI and UvrCII. DNase I footprints of the MspI-BstNI 117 bp DNA fragment containing a site-directed CC-1065-adenine adduct at the 5 ' -GATTA*CG- site show that UvrCII, but not UvrCI, binds to the adduct site. Furthermore, the pattern of DNase I footprints induced by UvrCII binding differs from the pattern of the footprints induced by UvrA, UvrAB, and UvrABCI binding. Interestingly, while the presence of unirradiated DNA enhances the efficiency of UvrABCII in incising UV-irradiated DNA, it does not enhance UvrABCII incision of the CC-1065-N3-adenine adduct formed at 5 ' -GATTA*CG-. These results show that two different forms of UvrC differ in DNA binding properties as well as incision modes at some kinds of DNA damage
— id: 54893, year: 2001, vol: 40, page: 11073, stat: Journal Article,

Two forms of UvrC protein with different double-stranded DNA binding affinities
Tang Ms; Nazimiec M; Ye X; Iyer GH; Eveleigh J; Zheng Y; Zhou W; Tang YY
2001 Feb 9;276(6):3904-3910, Journal of biological chemistry
Using phosphocellulose followed by single-stranded DNA-cellulose chromatography for purification of UvrC proteins from overproducing cells, we found that UvrC elutes at two peaks: 0.4 m KCl (UvrCI) and 0.6 m KCl (UvrCII). Both forms of UvrC have a major peptide band (>95%) of the same molecular weight and identical N-terminal amino acid sequences, which are consistent with the initiation codon being at the unusual GTG site. Both forms of UvrC are active in incising UV-irradiated, supercoiled phiX-174 replicative form I DNA in the presence of UvrA and UvrB proteins; however, the specific activity of UvrCII is one-fourth that of UvrCI. The molecular weight of UvrCII is four times that of UvrCI on the basis of results of size exclusion chromatography and glutaraldehyde cross-linking reactions, indicating that UvrCII is a tetramer of UvrCI. Functionally, these two forms of UvrC proteins can be distinguished under reaction conditions in which the protein/nucleotide molar ratio is >0.06 by using UV-irradiated, (32)P-labeled DNA fragments as substrates; under these conditions UvrCII is inactive in incision, but UvrCI remains active. The activity of UvrCII in incising UV-irradiated, (32)P- labeled DNA fragments can be restored by adding unirradiated competitive DNA, and the increased level of incision corresponds to a decreased level of UvrCII binding to the substrate DNA. The sites of incision at the 5' and 3' sides of a UV-induced pyrimidine dimer are the same for UvrCI and UvrCII. Nitrocellulose filter binding and gel retardation assays show that UvrCII binds to both UV-irradiated and unirradiated double-stranded DNA with the same affinity (K(a), 9 x 10(8)/m) and in a concentration-dependent manner, whereas UvrCI does not. These two forms of UvrC were also produced by the endogenous uvrC operon. We propose that UvrCII-DNA binding may interfere with Uvr(A)(2)B-DNA damage complex formation. However, because of its low copy number and low binding affinity to DNA, UvrCII may not interfere with Uvr(A)(2)B-DNA damage complex formation in vivo, but instead through double-stranded DNA binding UvrCII may become concentrated at genomic areas and therefore may facilitate nucleotide excision repair
— id: 21266, year: 2001, vol: 276, page: 3904, stat: Journal Article,

Methylated CpG dinucleotides are the preferential targets for G-to-T transversion mutations induced by benzo[a]pyrene diol epoxide in mammalian cells: Similarities with the p53 mutation spectrum in smoking-associated lung cancers
Yoon, JH; Smith, LE; Feng, ZH; Tang, MS; Lee, CS; Pfeifer, GP
2001 OCT 1 ;61(19):7110-7117, Cancer research
A large fraction of the p53 mutations in lung cancers from smokers are G-to-T transversions, a type of mutation that is infrequent in lung cancers from nonsmokers and in most other tumors. Previous studies have indicated that there is an association between G-to-T transversion hotspots in lung cancers and sites of preferential formation of polycyclic aromatic hydrocarbon adducts along the p53 gene. p53 codons containing methylated CpG sequences are preferential targets for formation of adducts by (+/-) anti-7 beta ,8 alpha -dihydroxy-9 alpha 10 alpha -epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). To assess the role of CpG methylation in induction of mutations by BPDE, we analyzed BPDE mutagenesis in three CpG methylated target genes: a supF shuttle vector and the ell and lacI transgenes in embryonic mouse fibroblasts. After methylation of the shuttle vector at all CpG sequences, 42% of all G-to-T transversions were at CpG sites compared with 23% in unmethylated DNA. In the cII transgene, which is methylated at CpG sequences in vivo, 83 of 147 (56%) of the BPDE-induced mutations were G-to-T transversions, and 58% (48 of 83) of all G-to-T transversions occurred at methylated CpG sequences. In the lacI gene, 68% (75 of 111) of the BPDE-induced mutations were G-to-T events, and 58 of 75 (77%) of these occurred at methylated CpG sequences. The occurrence of transversion hotspots at methylated CpGs correlated with high levels of BPDE adducts formed at such sites. This situation mirrors the one in the p53 gene in lung cancers from smokers where 236 of 465 (51%) of the G-to-T transversions occurred at methylated CpG sites. These findings further strengthen a link between polycyclic aromatic hydrocarbons present in cigarette smoke and lung cancer mutations and provide evidence that mutational processes other than C-to-T transition mutations can occur selectively at methylated CpG sequences
— id: 98274, year: 2001, vol: 61, page: 7110, stat: Journal Article,

Cyclobutane pyrimidine dimers and bulky chemical DNA adducts are efficiently repaired in both strands of either a transcriptionally active or promoter-deleted APRT gene
Zheng, Y; Pao, A; Adair, GM; Tang, MS
2001 MAY 18 ;276(20):16786-16796, Journal of biological chemistry
Both prokaryotic and eukaryotic cells have the capacity to repair DNA damage preferentially in the transcribed strand of actively expressed genes. However, we have found that several types of DNA damage, including cyclobutane pyrimidine dimers (CPDs) are repaired with equal efficiency in both the transcribed and nontranscribed strands of the adenine phosphoribosyltransferase (APRT) gene in Chinese hamster ovary cells. We further found that, in two mutant cell lines in which the entire APRT promoter region has been deleted, CPDs are still efficiently repaired in both strands of the promoterless APRT gene, even though neither strand appears to be transcribed. These results suggest that efficient repair of both strands at this locus does not require transcription of the APRT gene. We have also mapped CPD repair in exon 3 of the APRT gene in each cell line at single nucleotide resolution. Again, we found similar rates of CPD repair in both strands of the APRT gene domain in both APRT promoter-deletion mutants and their parental cell line. Our findings suggest that current models of transcription-coupled repair and global genomic repair may underestimate the importance of factors other than transcription in governing the efficiency of nucleotide excision repair
— id: 55079, year: 2001, vol: 276, page: 16786, stat: Journal Article,

C5 cytosine methylation at CpG sites enhances sequence selectivity of mitomycin C-DNA bonding
Li, V S; Reed, M; Zheng, Y; Kohn, H; Tang, M
2000 Mar 14;39(10):2612-2618, Biochemistry
We have established that UvrABC nuclease is equally efficient in cutting mitomycin C (MC)-DNA monoadducts formed at different sequences and that the degree of UvrABC cutting represents the extent of drug-DNA bonding. Using this method we determined the effect of C5 cytosine methylation on the DNA monoalkylation by MC and the related analogues N-methyl-7-methoxyaziridinomitosene (MS-NMA) and 10-decarbamoylmitomycin C (DC-MC). We have found that C5 cytosine methylation at CpG sites greatly enhances MC and MS-NMA DNA adduct formation at those sites while reducing adduct formation at non-CpG sequences. In contrast, although DC-MC DNA bonding at CpG sites is greatly enhanced by CpG methylation, its bonding at non-CpG sequences is not appreciably affected. These cumulative results suggest that C5 cytosine methylation at CpG sites enhances sequence selectivity of drug-DNA bonding. We propose that the methylation pattern and status (hypo- or hypermethylation) of genomic DNA may determine the cells' susceptibility to MC and its analogues, and these effects may, in turn, play a crucial role in the antitumor activities of the drugs
— id: 133304, year: 2000, vol: 39, page: 2612, stat: Journal Article,

Mutation hotspots and DNA methylation
Pfeifer, GP; Tang, MS; Denissenko, MF
2000 JUN ;249(4):1-19, Current topics in microbiology & immunology
— id: 98298, year: 2000, vol: 249, page: 1, stat: Journal Article,

Both (+/-)syn- and (+/-)anti-7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxides initiate tumors in mouse skin that possess -CAA- to -CTA- mutations at Codon 61 of c-H-ras
Tang MS; Vulimiri SV; Viaje A; Chen JX; Bilolikar DS; Morris RJ; Harvey RG; Slaga TJ; DiGiovanni J
2000 Oct 15;60(20):5688-5695, Cancer research
We have determined the tumor-initiating activity of (+/-)syn- and (+/-)anti-7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide (syn- and anti-DMBADE), the two metabolically formed bay-region diol epoxides of DMBA, and we have also analyzed mutations in the H-ras gene from tumors induced by these compounds. Using a two-stage, initiation-promotion protocol for tumorigenesis in mouse skin, we have found that both syn- and anti-DMBADE are active tumor initiators, and that the occurrence of papillomas is carcinogen dose dependent. All of the papillomas induced by syn-DMBADE (a total of 40 mice), 96% of those induced by anti-DMBADE (a total of 25 mice), and 94% of those induced by DMBA (a total of 16 mice) possessed a -CAA- to -CTA- mutation at codon 61 of H-ras. No mutations in codons 12 or 13 were detected in any tumor. Topical application of syn- and anti-DMBADE produced stable adducts in mouse epidermal DNA, most of which comigrated with stable DNA adducts formed after topical application of DMBA. Further analysis of the data showed that levels of the major syn- and anti-DMBADE-deoxyadenosine adducts formed after topical application of DMBA are sufficient to account for the tumor-initiating activity of this carcinogen on mouse skin. Previously, we showed that both the syn- and anti-DMBADE bind to the adenine (A182) at codon 61 of H-ras. Collectively, these results indicate that the adenine adducts induced by both bay-region diol epoxides of DMBA lead to the mutation at codon 61 of H-ras and, consequently, initiate tumorigenesis in mouse skin
— id: 39526, year: 2000, vol: 60, page: 5688, stat: Journal Article,

Influence of APOE genotype on familial aggregation of AD in an urban population
Devi, G; Ottman, R; Tang, M; Marder, K; Stern, Y; Tycko, B; Mayeux, R
1999 Sep 11;53(4):789-794, Neurology
OBJECTIVE: To examine the influence of the proband's APOE genotype on AD among first-degree relatives in a community-based study of African Americans, whites, and Caribbean Hispanics. METHODS: History of AD and demographic information were obtained on 1,073 siblings and parents of 312 patients with AD and 2,722 siblings and parents of 802 nondemented controls. APOE genotyping was performed on all 1,114 patients and controls. RESULTS: A higher proportion of patients with AD (35%) than controls (27%) had one or more APOE-epsilon4 alleles (p = 0.03). When compared with relatives of controls without an APOE-epsilon4 allele, the risk for AD was increased in first-degree relatives of both patients (rate ratio [RR] = 1.9, 95% confidence interval [CI] = 1.2 to 3.1) and controls (RR = 1.8, 95% CI = 1.2 to 2.6) with one or more APOE-epsilone alleles, regardless of ethnic group. There was a similar trend of increased risk in relatives of patients without an APOE-epsilon4 allele, but this was limited to Hispanics and African Americans. CONCLUSIONS: The presence of an APOE-epsilon4 allele increases risk for AD among first-degree relatives, regardless of the probands' disease status, among all ethnic groups. Relatives of patients without an APOE-epsilon4 allele were also at increased risk for AD among Hispanics and African Americans, suggesting that other genes or risk factors may influence risk
— id: 74313, year: 1999, vol: 53, page: 789, stat: Journal Article,

A preliminary study of apolipoprotein E genotype and psychiatric manifestations of Alzheimer's disease
Ramachandran, G; Marder, K; Tang, M; Schofield, P W; Chun, M R; Devanand, D P; Stern, Y; Mayeux, R
1996 Jul;47(1):256-259, Neurology
We evaluated the frequency of depression and psychosis in 46 patients with AD and 135 control subjects with the apolipoprotein (APO) E3/3 or E3/4 genotype. Patients with AD and the APOE3/4 genotype had a more than threefold increase in the signs of depression and psychosis when compared with either patients with the APOE3/3 genotype or to control subjects. Our preliminary study suggests that the phenotype of AD associated with the epsilon 4 allele is more likely to include psychiatric manifestations
— id: 74324, year: 1996, vol: 47, page: 256, stat: Journal Article,