Wei Dai, Ph.D.

Regulation of Pax6 by CTCF during Induction of Mouse ES Cell Differentiation
Gao, Jie; Wang, Jie; Wang, Yumei; Dai, Wei; Lu, Luo
2011 ;6(6):e20954-e20954, PLoS ONE
Pax6 plays an important role in embryonic cell (ES) differentiation during embryonic development. Expression of Pax6 undergoes from a low level to high levels following ES cell differentiation to neural stem cells, and then fades away in most of the differentiated cell types. There is a limited knowledge concerning how Pax6 is regulated in ES cell differentiation. We report that Pax6 expression in mouse ES cells was controlled by CCCTC binding factor (CTCF) through a promoter repression mechanism. Pax6 expression was significantly enhanced while CTCF activity was kept in the constant during ES cell differentiation to radial glial cells. Instead, the interaction of CTCF with Pax6 gene was regulated by decreased CTCF occupancy in its binding motifs upstream from Pax6 P0 promoter following the course of ES cell differentiation. Reduced occupancy of CTCF in the binding motif region upstream from the P0 promoter was due to increased DNA methylations in the CpG sites identified in the region. Furthermore, changes in DNA methylation levels in vitro and in vivo effectively altered methylation status of these identified CpG sites, which affected ability of CTCF to interact with the P0 promoter, resulting in increases in Pax6 expression. We conclude that there is an epigenetic mechanism involving regulations of Pax6 gene during ES cell differentiation to neural stem cells, which is through increases or decreases in methylation levels of Pax6 gene to effectively alter the ability of CTCF in control of Pax6 expression, respectively
— id: 134709, year: 2011, vol: 6, page: e20954, 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,

Effect of a structurally modified human granulocyte colony stimulating factor, G-CSFa, on leukopenia in mice and monkeys
Jiang, Yongping; Jiang, Wenhong; Qiu, Yuchang; Dai, Wei
2011 ;4:28-28, Journal of hematology & oncology
ABSTRACT: BACKGROUND: Granulocyte colony stimulating factor (G-CSF) regulates survival, proliferation, and differentiation of neutrophilic granulocyte precursors, Recombinant G-CSF has been used for the treatment of congenital and therapy-induced neutropenia and stem cell mobilization. Due to its intrinsic instability, recombinant G-CSF needs to be excessively and/or frequently administered to patients in order to maintain a plasma concentration high enough to achieve therapeutic effects. Therefore, there is a need for the development of G-CSF derivatives that are more stable and active in vivo. METHODS: Using site-direct mutagenesis and recombinant DNA technology, a structurally modified derivative of human G-CSF termed G-CSFa was obtained. G-CSFa contains alanine 17 (instead of cysteine 17 as in wild-type G-CSF) as well as four additional amino acids including methionine, arginine, glycine, and serine at the amino-terminus. Purified recombinant G-CSFa was tested for its in vitro activity using cell-based assays and in vivo activity using both murine and primate animal models. RESULTS: In vitro studies demonstrated that G-CSFa, expressed in and purified from E. coli, induced a much higher proliferation rate than that of wild-type G-CSF at the same concentrations. In vivo studies showed that G-CSFa significantly increased the number of peripheral blood leukocytes in cesium-137 irradiated mice or monkeys with neutropenia after administration of clyclophosphamide. In addition, G-CSFa increased neutrophil counts to a higher level in monkeys with a concomitant slower declining rate than that of G-CSF, indicating a longer half-life of G-CSFa. Bone marrow smear analysis also confirmed that G-CSFa was more potent than G-CSF in the induction of granulopoiesis in bone marrows of myelo-suppressed monkeys. CONCLUSION: G-CSFa, a structurally modified form of G-CSF, is more potent in stimulating proliferation and differentiation of myeloid cells of the granulocytic lineage than the wild-type counterpart both in vitro and in vivo. G-CSFa can be explored for the development of a new generation of recombinant therapeutic drug for leukopenia
— id: 134915, year: 2011, vol: 4, page: 28, stat: Journal Article,

Hyperosmotic stress-induced corneal epithelial cell death through activation of Polo-like kinase 3 and c-Jun
Wang, Ling; Dai, Wei; Lu, Luo
2011 May;52(6):3200-3206, Investigative ophthalmology & visual science. IOVS
PURPOSE: Hyperosmotic stress causes cell shrinkage, perturbs cell function, and damages DNA, resulting in cell cycle arrest and apoptosis. In the present study, the authors explore the mechanism involving hyperosmotic stress-induced activation of c-Jun/AP-1 through a novel Plk3 pathway in human corneal epithelial cells. METHODS: Human primary corneal epithelial cells and cell line were cultured in a serum-free keratinocyte medium and DMEM/F12 medium containing 10% FBS in a 37 degrees C incubator supplied with 5% CO(2), respectively. Western blot analysis was used to determine protein expression and phosphorylation levels. Protein kinase activities were measured by immunocomplex kinase assay. Cell viability and apoptosis were determined by MTT assay and caspase-3 (DEVDase) activity. RESULTS: It was found that hyperosmotic stress-induced increases in the phosphorylation of c-Jun, resulting in apoptosis through the activation of Plk3 in human corneal epithelial cells. Plk3 was activated by extracellular hyperosmotic stress to directly phosphorylate c-Jun in the serine 63 and 73 residues. Hyperosmotic stress-induced c-Jun phosphorylation was enhanced by overexpression of constitutively positive Plk3 mutants and suppressed by the knockdown of Plk3 mRNA with Plk3-specific siRNA. Further studies indicated that the phosphorylation of c-Jun by Plk3 was responsible for hyperosmotic stress-induced apoptosis, which was independent from activation of the JNK signaling pathway in human corneal epithelial cells. CONCLUSIONS: These results, for the first time, provide a novel and alternative signaling mechanism that involves hyperosmotic stress-induced activation of the Plk3 pathway in addition to JNK/p38 MAPK pathways to regulate the c-Jun/AP-1 transcriptional complex and human corneal epithelial cell fate
— id: 135200, year: 2011, vol: 52, page: 3200, stat: Journal Article,

Hyperosmotic stress-induced ATF-2 activation through Polo-like kinase 3 in human corneal epithelial cells
Wang, Ling; Payton, Reid; Dai, Wei; Lu, Luo
2011 Jan 21;286(3):1951-1958, Journal of biological chemistry
Elevated extracellular solute concentration (hyperosmotic stress) perturbs cell function and stimulates cell responses by evoking MAPK cascades and activating AP-1 transcription complex resulting in alterations of gene expression, cell cycle arrest, and apoptosis. The results presented here demonstrate that hyperosmotic stress elicited increases in ATF-2 phosphorylation through a novel Polo-like kinase 3 (Plk3) pathway in human corneal epithelial (HCE) cells. We found in hyperosmotic stress-induced HCE cells that Plk3 transferred to the nuclear compartment and was colocalized with ATF-2 in nuclei. Kinase activity of Plk3 was significantly activated by hyperosmotic stimulation. Further downstream, active Plk3 phosphorylated ATF-2 at the Thr-71 site in vivo and in vitro. Overexpression of Plk3 and its mutants enhanced hyperosmotic stress-induced ATF-2 phosphorylation. In contrast, suppression of Plk3 by knocking down Plk3 mRNA effectively diminished the effect of hyperosmotic stress-induced ATF-2 phosphorylation. The effect of hyperosmotic stress-induced activation of Plk3 on ATF-2 transcription factor function was also examined in CRE reporter-overexpressed HCE cells. Our results for the first time reveal that hyperosmotic stress can activate the Plk3 signaling pathway that subsequently regulates the AP-1 complex by directly phosphorylating ATF-2 independent from the effects of JNK and p38 activation
— id: 132720, year: 2011, vol: 286, page: 1951, stat: Journal Article,

The function of mammalian Polo-like kinase 1 in microtubule nucleation
Xu, Dazhong; Dai, Wei
2011 Jul 12;108(28):11301-11302, Proceedings of the National Academy of Sciences of the United States of America
— id: 135188, year: 2011, vol: 108, page: 11301, stat: Journal Article,

Germinal Cell Aplasia in Kif18a Mutant Male Mice Due to Impaired Chromosome Congression and Dysregulated BubR1 and CENP-E
Liu, Xue-Song; Zhao, Xu-Dong; Wang, Xiaoxing; Yao, Yi-Xin; Zhang, Liang-Liang; Shu, Run-Zhe; Ren, Wei-Hua; Huang, Ying; Huang, Lei; Gu, Ming-Min; Kuang, Ying; Wang, Long; Lu, Shun-Yuan; Chi, Jun; Fen, Jing-Sheng; Wang, Yi-Fei; Fei, Jian; Dai, Wei; Wang, Zhu-Gang
2010 Jan;1(1):26-39, Genes & cancer
Chromosomal instability during cell division frequently causes cell death or malignant transformation. Orderly chromosome congression at the metaphase plate, a paramount process to vertebrate mitosis and meiosis, is controlled by a number of molecular regulators, including kinesins. Kinesin-8 (Kif18A) functions to control mitotic chromosome alignment at the mid-zone by negative regulation of kinetochore oscillation. Here the authors report that disrupting Kif18a function results in complete sterility in male but not in female mice. Histological examination reveals that Kif18a(-/-) testes exhibit severe developmental impairment of seminiferous tubules. Testis atrophy in Kif18a(-/-) mice is caused by perturbation of microtubule dynamics and spindle pole integrity, leading to chromosome congression defects during mitosis and meiosis. Depletion of KIF18A via RNAi causes mitotic arrest accompanied by unaligned chromosomes and increased microtubule nucleating centers in both GC-1 and HeLa cells. Prolonged depletion of KIF18A causes apoptosis due to perturbed microtubule dynamics. Further studies reveal that KIF18A silencing results in degradation of CENP-E and BubR1, which is accompanied by premature sister chromatid separation. KIF18A physically interacts with BubR1 and CENP-E, and this interaction is modulated during mitosis. Combined, the studies indicate that KIF18A is essential for normal chromosome congression during cell division and that the absence of KIF18A function causes severe defects in microtubule dynamics, spindle integrity, and checkpoint activation, leading to germinal cell aplasia in mice
— id: 138226, year: 2010, vol: 1, page: 26, stat: Journal Article,

Effect of hypoxic stress-activated Polo-like kinase 3 on corneal epithelial wound healing
Lu, Jiawei; Wang, Ling; Dai, Wei; Lu, Luo
2010 Oct;51(10):5034-5040, Investigative ophthalmology & visual science. IOVS
PURPOSE: Hypoxia/reoxygenation conditions can generate oxidative stresses resulting in the suppression of cell proliferation and the delay of corneal epithelial wound healing. The purpose of this study was to investigate the cellular mechanism involving the role of the stress-responsive Polo-like kinase 3 (Plk3) in hypoxic stress-induced delay of corneal epithelial wound healing. METHODS: Plk3 activities were determined by immunochemistry and immunocomplex kinase assay approaches. Corneal epithelial wound healing was evaluated by a whole-eye organ culture model and by scratch-induced wound closure assay. Corneal epithelial layer was removed by using a corneal rust-ring-remover in wild-type and Plk3(-/-) mice. Wound healing was analyzed using a confocal imaging system. Cell growth was measured by MTT assays. RESULTS: The effect of hypoxic stress on early stages of corneal epithelial wound healing was compared with other oxidative stresses, including UV, CoCl(2), and H(2)O(2) treatments. Hypoxic stress-induced delay of corneal epithelial wound healing was further evaluated in human corneal epithelial cells and in the corneas of wild-type and Plk3 knockout (Plk3(-/-)) mice. Hypoxic stress-induced Plk3 activation resulted in growth attenuation and delay of wound healing. Further evidence demonstrated that the increase in Plk3 activity in constitutively active Plk3-expressed cells significantly enhanced stress-induced delay of wound healing. In contrast, hypoxic stress-induced delay of wound healing was markedly diminished in the corneas of Plk3 deficient Plk3(-/-) mice. CONCLUSIONS: These results provide for the first time important evidence that Plk3 plays a significant role in hypoxic stress-induced attenuation of cell growth and delay of corneal epithelial wound healing
— id: 133854, year: 2010, vol: 51, page: 5034, stat: Journal Article,

Regulation of PTEN Stability and Activity by Plk3
Xu, Dazhong; Yao, Yixin; Jiang, Xuejun; Lu, Luo; Dai, Wei
2010 Dec 17;285(51):39935-39942, Journal of biological chemistry
By studying primary isogenic murine embryonic fibroblasts (MEFs), we have shown that PLK3 null MEFs contain a reduced level of phosphatase and tensin homolog (PTEN) and increased Akt1 activation coupled with decreased GSK3beta activation under normoxia and hypoxia. Purified recombinant Plk3, but not a kinase-defective mutant, efficiently phosphorylates PTEN in vitro. Mass spectrometry identifies threonine 366 and serine 370 as two putative residues that are phosphorylated by Plk3. Immunoblotting using a phosphospecific antibody confirms these sites as Plk3 phosphorylation sites. Moreover, treatment of MEFs with LiCl, an inhibitor of GSK3beta and CK2, only partially suppresses the phosphorylation, suggesting Plk3 as an additional kinase that phosphorylates these sites in vivo. Plk3-targeting mutants of PTEN are expressed at a reduced level in comparison with the wild-type counterpart, which is associated with an enhanced activity of PDK1, an upstream activator of Akt1. Furthermore, the reduced level of PTEN in PLK3 null MEFs is stabilized by treatment with MG132, a proteosome inhibitor. Combined, our study identifies Plk3 as a new player in the regulation of the PI3K/PDK1/Akt signaling axis by phosphorylation and stabilization of PTEN
— id: 115421, year: 2010, vol: 285, page: 39935, stat: Journal Article,

Plk3 Functions as an Essential Component of the Hypoxia Regulatory Pathway by Direct Phosphorylation of HIF-1{alpha}
Xu, Dazhong; Yao, Yixin; Lu, Luo; Costa, Max; Dai, Wei
2010 Dec 10;285(50):38944-38950, Journal of biological chemistry
Polo-like kinase 3 (Plk3) plays an important role in the regulation of cell cycle progression and stress responses. Plk3 also has a tumor-suppressing activity as aging PLK3-null mice develop tumors in multiple organs. The growth of highly vascularized tumors in PLK3-null mice suggests a role for Plk3 in angiogenesis and cellular responses to hypoxia. By studying primary isogenic murine embryonic fibroblasts, we tested the hypothesis that Plk3 functions as a component in the hypoxia signaling pathway. PLK3(-/-) murine embryonic fibroblasts contained an enhanced level of HIF-1alpha under hypoxic conditions. Immunoprecipitation and pulldown analyses revealed that Plk3 physically interacted with HIF-1alpha under hypoxia. Purified recombinant Plk3, but not a kinase-defective mutant, phosphorylated HIF-1alpha in vitro, resulting in a major mobility shift. Mass spectrometry identified two unique serine residues that were phosphorylated by Plk3. Moreover, ectopic expression followed by cycloheximide or pulse-chase treatment demonstrated that phospho-mutants exhibited a much longer half-life than the wild-type counterpart, strongly suggesting that Plk3 directly regulates HIF-1alpha stability in vivo. Combined, our study identifies Plk3 as a new and essential player in the regulation of the hypoxia signaling pathway
— id: 115272, year: 2010, vol: 285, page: 38944, stat: Journal Article,

Pharicin A, a novel natural ent-kaurene diterpenoid, induces mitotic arrest and mitotic catastrophe of cancer cells by interfering with BubR1 function
Xu, Han-Zhang; Huang, Ying; Wu, Ying-Li; Zhao, Yong; Xiao, Wei-Lie; Lin, Qi-Shan; Sun, Han-Dong; Dai, Wei; Chen, Guo-Qiang
2010 JUL 15 ;9(14):2897-2907, Cell cycle
In this study, we report the functional characterization of a new ent-kaurene diterpenoid termed pharicin A, which was originally isolated from Isodon, a perennial shrub frequently used in Chinese folk medicine for tumor treatment. Pharicin A induces mitotic arrest in leukemia and solid tumor-derived cells identified by their morphology, DNA content and mitotic marker analyses. Pharicin A-induced mitotic arrest is associated with unaligned chromosomes, aberrant BubR1 localization and deregulated spindle checkpoint activation. Pharicin A directly binds to BubR1 in vitro, which is correlated with premature sister chromatid separation in vivo. Pharicin A also induces mitotic arrest in paclitaxel-resistant Jurkat and U2OS cells. Combined, our study strongly suggests that pharicin A represents a novel class of small molecule compounds capable of perturbing mitotic progression and initiating mitotic catastrophe, which merits further preclinical and clinical investigations for cancer drug development
— id: 112194, year: 2010, vol: 9, page: 2897, stat: Journal Article,

A genome-wide deletion mutant screen identifies pathways affected by nickel sulfate in Saccharomyces cerevisiae
Arita, Adriana; Zhou, Xue; Ellen, Thomas P; Liu, Xin; Bai, Jingxiang; Rooney, John P; Kurtz, Adrienne; Klein, Catherine B; Dai, Wei; Begley, Thomas J; Costa, Max
2009 ;10:524-524, BMC genomics
BACKGROUND: The understanding of the biological function, regulation, and cellular interactions of the yeast genome and proteome, along with the high conservation in gene function found between yeast genes and their human homologues, has allowed for Saccharomyces cerevisiae to be used as a model organism to deduce biological processes in human cells. Here, we have completed a systematic screen of the entire set of 4,733 haploid S. cerevisiae gene deletion strains (the entire set of nonessential genes for this organism) to identify gene products that modulate cellular toxicity to nickel sulfate (NiSO(4)). RESULTS: We have identified 149 genes whose gene deletion causes sensitivity to NiSO(4) and 119 genes whose gene deletion confers resistance. Pathways analysis with proteins whose absence renders cells sensitive and resistant to nickel identified a wide range of cellular processes engaged in the toxicity of S. cerevisiae to NiSO(4). Functional categories overrepresented with proteins whose absence renders cells sensitive to NiSO(4) include homeostasis of protons, cation transport, transport ATPases, endocytosis, siderophore-iron transport, homeostasis of metal ions, and the diphthamide biosynthesis pathway. Functional categories overrepresented with proteins whose absence renders cells resistant to nickel include functioning and transport of the vacuole and lysosome, protein targeting, sorting, and translocation, intra-Golgi transport, regulation of C-compound and carbohydrate metabolism, transcriptional repression, and chromosome segregation/division. Interactome analysis mapped seven nickel toxicity modulating and ten nickel-resistance networks. Additionally, we studied the degree of sensitivity or resistance of the 111 nickel-sensitive and 72 -resistant strains whose gene deletion product has a similar protein in human cells. CONCLUSION: We have undertaken a whole genome approach in order to further understand the mechanism(s) regulating the cell's toxicity to nickel compounds. We have used computational methods to integrate the data and generate global models of the yeast's cellular response to NiSO(4). The results of our study shed light on molecular pathways associated with the cellular response of eukaryotic cells to nickel compounds and provide potential implications for further understanding the toxic effects of nickel compounds to human cells
— id: 105506, year: 2009, vol: 10, page: 524, stat: Journal Article,

Suppression of genomic instabilities caused by chromosome mis-segregation: a perspective from studying BubR1 and Sgo1
Dai, Wei
2009 Dec;108(12):904-911, Journal of the Formosan Medical Association = Taiwan yi zhi
Aneuploidy is a major manifestation of chromosomal instability, which is defined as a numerical abnormality of chromosomes in diploid cells. It is highly prevalent in a variety of human malignancies. Increased chromosomal instability is the major driving force for tumor development and progression. To suppress genomic stability during cell division, eukaryotic cells have evolved important molecular mechanisms, commonly referred to as checkpoints. The spindle checkpoint ensures that cells with defective mitotic spindles or a defective interaction between the spindles and kinetochores do not initiate chromosomal segregation during mitosis. Extensive studies have identified and characterized more than a dozen genes that play important roles in the regulation of the spindle checkpoint in mammalian cells. During the past decade, we have carried out extensive investigation of the role of BubR1 (Bub1-related kinase) and Sgo1 (shugoshin 1), two important gene products that safeguard accurate chromosome segregation during mitosis. This mini-review summarizes our studies, as well as those by other researchers in the field, on the functions of these two checkpoint proteins and their molecular regulation during mitosis. Further elucidation of the molecular mechanisms of the spindle checkpoint regulation has the potential to identify important mitotic targets for rational anticancer drug design
— id: 106098, year: 2009, vol: 108, page: 904, stat: Journal Article,

Quantitative detection of the expression of mitochondrial cytochrome c oxidase subunits mRNA in the cerebral cortex after experimental traumatic brain injury
Dai, Wei; Cheng, Hui-lin; Huang, Ren-qiang; Zhuang, Zong; Shi, Ji-Xin
2009 Jan 28;1251:287-295, Brain research
Secondary brain damage plays a critical role in the outcome of patients with traumatic brain injury (TBI). The multiple mechanisms underlying secondary brain damage, including posttraumatic cerebral ischemia, glutamate excitotoxicity, oxidative stress, calcium overload and inflammation, are associated with increased mortality and morbidity after head injury. TBI is documented to have detrimental effects on mitochondria, such as alterations in glucose utilization and the depression of mitochondrial oxidative phosphorylation. Studies on mitochondrial metabolism have provided evidence for dysfunction of the cytochrome oxidase complex of the electron transport chain (complex IV) after TBI. A growing body of evidence indicates that cytochrome c oxidase is vital for mitochondrial oxidative phosphorylation. Therefore, this study aimed to detect the expression of cytochrome c oxidase (CO) mRNA in a rat weight-dropping trauma model and to clarify the differences between injured cortex (IC) and contralateral cortex (CC) after TBI. A total of forty-four rats were randomly assigned to 7 groups: control groups (n=4), sham-operated group (n=20), 6 h, 1 d, 3 d, 5 d and 7 d postinjury groups (n=4 for each group). The group consisted of sham-operated animals underwent parietal craniotomy without TBI. The rats in postinjury groups were subjected to TBI. The rats of control group were executed immediately without TBI or craniotomy after anesthesia. The brain-injured and sham-operated animals were killed on 6 h, 1 d, 3 d, 5 d and 7 d, respectively. Tissue sections from IC and CC were obtained and the expression of cytochrome c oxidase I, II, and III (CO I, II, III) mRNA, three mitochondrial encoded subunits of complex IV, were assessed by Real-time quantitative PCR. A reduction of CO I, II, and III mRNA expression was detected from IC and reduced to the lowest on 3 d. By contrast, the mRNA expression from CC suggested a slight elevation. The differences may indicate the degree of metabolic and physiologic dysfunction. Our results will better define the roles of gene expression and metabolic function in long-term prognosis and outcome after TBI. With a considerable understanding of post-injury mitochondrial dysfunction, therapeutic interventions targeted to the mitochondria may prevent secondary brain damage that leads to long-term cell death and neurobehavioral disability
— id: 96274, year: 2009, vol: 1251, page: 287, stat: Journal Article,

Effects of dietary Pb on accumulation, histopathology, and digestive enzyme activities in the digestive system of tilapia (Oreochromis niloticus)
Dai, Wei; Du, Huahua; Fu, Linglin; Jin, Chengguan; Xu, Zirong; Liu, Huitao
2009 Feb;127(2):124-131, Biological trace element research
With the increasing occurrence of dietary lead (Pb) contamination in aquatic environment, threat of the dietary Pb toxicity to aquatic organisms attracted more attention. In this study, after being exposed to dietary Pb at concentrations of 0, 100, 400, and 800-microg/g dry weight for 60 days, the groups of tilapia (Oreochromis niloticus) were sacrificed and sampled to analyze the effects of dietary Pb on accumulation, histopathology, and digestive enzyme activities in tissues of the digestive system. The results showed that the Pb accumulation in tissues increased with the dietary Pb concentrations. Moreover, Pb accumulated in sampled tissues in the following order: intestine > stomach > liver. By observation of liver histological sections in optical microscope, lesions could be detected in the Pb-contaminated groups. It was also demonstrated that the inhibitory effect of dietary Pb on digestive enzyme activities was dietary Pb concentration dependent. Different degrees of inhibition of enzyme activities were exhibited in sampled tissues. It was indicated that digestive enzyme activities in the digestive system might be considered as the potential biomarkers of dietary Pb contamination in tilapia
— id: 96277, year: 2009, vol: 127, page: 124, stat: Journal Article,

Changes in Growth Performance, Metabolic Enzyme Activities, and Content of Fe, Cu, and Zn in Liver and Kidney of Tilapia (Oreochromis niloticus) Exposed to Dietary Pb
Dai, Wei; Fu, Linglin; Du, Huahua; Jin, Chengguan; Xu, Zirong
2009 May;128(2):176-183, Biological trace element research
Tilapia (Oreochromis niloticus) were exposed to 0, 100, 400, and 800 mug/g concentrations of Pb in diet for 60 days, and changes in growth performance, metabolic enzyme activities, and essential trace elements (Fe, Cu, and Zn) content in liver and kidney were investigated. Daily weight gain, feed conversation ratio, and survival of tilapia were not significantly affected by dietary Pb. Alanine transaminase (ALT), aspartate transaminase (AST), and lactate dehydrogenase (LDH) activities in liver and kidney were affected by dietary Pb in a dissimilar way: Pb concentration-related decreases in ALT, AST, and LDH activities were observed in kidney, while these enzyme activities in liver were stimulated in a Pb concentration-dependent manner. It was demonstrated that the inhibitory effects of dietary Pb on alkaline phosphatase, Na, K-adenosine triphosphatase (ATPase), Ca, and Mg-ATPase activities in both liver and kidney were Pb concentration-dependent. It was also indicated that the content of Fe, Cu, and Zn in liver and kidney decreased with the increasing dietary Pb concentrations. The results suggested that long-term dietary Pb exposure could affect metabolic enzyme activities and the content of Fe, Cu, and Zn in liver and kidney, whereas growth impairment was not observed in tilapia
— id: 96276, year: 2009, vol: 128, page: 176, stat: Journal Article,

Compressive sensing DNA microarrays
Dai, Wei; Sheikh, Mona A; Milenkovic, Olgica; Baraniuk, Richard G
2009 ;:162824-162824, EURASIP journal on bioinformatics & systems biology
Compressive sensing microarrays (CSMs) are DNA-based sensors that operate using group testing and compressive sensing (CS) principles. In contrast to conventional DNA microarrays, in which each genetic sensor is designed to respond to a single target, in a CSM, each sensor responds to a set of targets. We study the problem of designing CSMs that simultaneously account for both the constraints from CS theory and the biochemistry of probe-target DNA hybridization. An appropriate cross-hybridization model is proposed for CSMs, and several methods are developed for probe design and CS signal recovery based on the new model. Lab experiments suggest that in order to achieve accurate hybridization profiling, consensus probe sequences are required to have sequence homology of at least 80% with all targets to be detected. Furthermore, out-of-equilibrium datasets are usually as accurate as those obtained from equilibrium conditions. Consequently, one can use CSMs in applications in which only short hybridization times are allowed
— id: 96272, year: 2009, vol: , page: 162824, stat: Journal Article,

Arsenic trioxide suppresses paclitaxel-induced mitotic arrest
Duan, Q; Komissarova, E; Dai, W
2009 JUN ;42(3):404-411, Cell proliferation
To understand if there exists a functional interaction between arsenic trioxide and paclitaxel in vitro. HeLa and HCT116 (rho 53(+/+) and rho 53(-/-)) cells were treated with As2O3 and/or paclitaxel for various times. Treated cells were collected for analyses using a combination of flow cytometry, fluorescence microscopy and Western blotting. Because As2O3 is capable of inhibiting tubulin polymerization and inducing mitotic arrest, we examined whether there existed any functional interaction between As2O3 and paclitaxel, a well-known microtubule poison. Flow cytometry and fluorescence microscopy revealed that although As2O3 alone caused a moderate level of mitotic arrest, it greatly attenuated paclitaxel-induced mitotic arrest in cells with p53 deficiency. Western blot analysis showed that As2O3 significantly blocked phosphorylation of BubR1, Cdc20, and Cdc27 in cells treated with paclitaxel, suggesting that arsenic compromised the activation of the spindle checkpoint. Our further studies revealed that the attenuation of paclitaxel-induced mitotic arrest by As2O3 resulted primarily from sluggish cell cycle progression at S phase but not enhanced mitotic exit. The observations that As2O3 has a negative impact on the cell cycle checkpoint activation by taxol should have significant clinical implications because the efficacy of taxol in the clinics is associated with its ability to induce mitotic arrest and subsequent mitotic catastrophe
— id: 98846, year: 2009, vol: 42, page: 404, stat: Journal Article,

Defects in chromosome congression and mitotic progression in KIF18A-deficient cells are partly mediated through impaired functions of CENP-E
Huang, Ying; Yao, Yixin; Xu, Han-Zhang; Wang, Zhu-Gang; Lu, Luo; Dai, Wei
2009 Aug 15;8(16):2643-2649, Cell cycle
KIF18A, a molecular motor, is an essential component in the regulation of orderly chromosome congression by attenuation of the kinetochore oscillation amplitude at the midzone during mitosis in vertebrate cells. Here we report that KIF18A depletion resulted in mitotic arrest which was accompanied by the presence of unaligned chromosomes in HeLa cells. This resembles the phenotype induced by an impaired function of CENP-E, also a mitotic kinesin essential for the formation of the mitotic spindles. Our further analysis showed that KIF18A depletion caused specific downregulation of CENP-E. Downregulation of CENP-E as the result of KIF18A silencing was not due to reduced transcription but primarily due to the enhanced protein degradation. Co-immunoprecipitation revealed that KIF18A physically interacted with CENP-E and BubR1 during mitosis. Ectopic expression of the wild-type tail domain of CENP-E, but not a corresponding mutant, significantly suppressed chromosome congression defects in mitotic cells. Together, our studies strongly suggest that chromosome congression defects as the result of KIF18A depletion is at least in part mediated through destabilizing kinetochore CENP-E
— id: 101639, year: 2009, vol: 8, page: 2643, stat: Journal Article,

Enhanced genomic instabilities caused by deregulated microtubule dynamics and chromosome segregation: a perspective from genetic studies in mice
Rao, CV; Yamada, HY; Yao, YX; Dai, W
2009 SEP ;30(9):1469-1474, Carcinogenesis
Aneuploidy is defined as numerical abnormalities of chromosomes and is frequently (> 90%) present in solid tumors. In general, tumor cells become increasingly aneuploid with tumor progression. It has been proposed that enhanced genomic instability at least contributes significantly to, if not requires, tumor progression. Two major modes for genomic instability are microsatellite instability (MIN) and chromosome instability (CIN). MIN is associated with DNA-level defects (e.g. mismatch repair defects), and CIN is associated with mitotic errors such as chromosome mis-segregation. The mitotic spindle assembly checkpoint (SAC) ensures that cells with defective mitotic spindles or defective interaction between the spindles and kinetochores do not initiate chromosomal segregation during mitosis. Thus, the SAC functions to protect the cell from chromosome mis-segregation and anueploidy during cell division. A loss of the SAC function results in gross aneuploidy, a condition from which cells with an advantage for proliferation will be selected. During the past several years, a flurry of genetic studies in mice and humans strongly support the notion that an impaired SAC causes enhanced genomic instabilities and tumor development. This review article summarizes the roles of key spindle checkpoint proteins {i.e. Mad1/Mad1L1, Mad2/Mad2L1, BubR1/Bub1B, Bub3/Bub3 [conventional protein name (yeast or human)/mouse protein name]} and the modulators (i.e. Chfr/Chfr, Rae1/Rae1, Nup98/Nup98, Cenp-E/CenpE, Apc/Apc) in genomic stability and suppression of tumor development, with a focus on information from genetically engineered mouse model systems. Further elucidation of molecular mechanisms of the SAC signaling has the potential for identifying new targets for rational anticancer drug design
— id: 102303, year: 2009, vol: 30, page: 1469, stat: Journal Article,

Covalent modifications of histones during mitosis and meiosis
Xu, Dazhong; Bai, Jingxiang; Duan, Qing; Costa, Max; Dai, Wei
2009 Nov 15;8(22):3688-3694, Cell cycle
Higher order chromosome structures are the hallmark of mitotic and meiotic cells. Chromatin condensation and compaction are essential for rapid chromosome congression and accurate chromosome segregation during cell division. The core histones possess tails at their amino-termini. These tails, which extend from the surface of the nucleosomes, are highly dynamic and subject to an extensive array of covalent modifications. Modified histone tails play an important role, not only in the folding of nucleosomal arrays into higher order chromatin structures but also in gene regulation. The combination of these distinct covalent modifications of histones constitutes 'the histone code' that regulates various cellular processes, including mitotic and meiotic progression
— id: 105250, year: 2009, vol: 8, page: 3688, stat: Journal Article,

A genome-wide screen in Saccharomyces cerevisiae reveals pathways affected by arsenic toxicity
Zhou, Xue; Arita, Adriana; Ellen, Thomas P; Liu, Xin; Bai, Jingxiang; Rooney, John P; Kurtz, Adrienne D; Klein, Catherine B; Dai, Wei; Begley, Thomas J; Costa, Max
2009 Nov;94(5):294-307, Genomics
We have used Saccharomyces cerevisiae to identify toxicologically important proteins and pathways involved in arsenic-induced toxicity and carcinogenicity in humans. We performed a systemic screen of the complete set of 4733 haploid S. cerevisiae single-gene-deletion mutants to identify those that have decreased or increased growth, relative to wild type, after exposure to sodium arsenite (NaAsO(2)). IC(50) values for all mutants were determined to further validate our results. Ultimately we identified 248 mutants sensitive to arsenite and 5 mutants resistant to arsenite exposure. We analyzed the proteins corresponding to arsenite-sensitive mutants and determined that they belonged to functional categories that include protein binding, phosphate metabolism, vacuolar/lysosomal transport, protein targeting, sorting, and translocation, cell growth/morphogenesis, cell polarity and filament formation. Furthermore, these data were mapped onto a protein interactome to identify arsenite-toxicity-modulating networks. These networks are associated with the cytoskeleton, ubiquitination, histone acetylation and the MAPK signaling pathway. Our studies have potential implications for understanding toxicity and carcinogenesis in arsenic-induced human conditions, such as cancer and aging
— id: 104719, year: 2009, vol: 94, page: 294, stat: Journal Article,

Unique structures in a tumor herpesvirus revealed by cryo-electron tomography and microscopy
Dai, Wei; Jia, Qingmei; Bortz, Eric; Shah, Sanket; Liu, Jun; Atanasov, Ivo; Li, Xudong; Taylor, Kenneth A; Sun, Ren; Zhou, Z Hong
2008 Mar;161(3):428-438, Journal of structural biology
Gammaherpesviruses, including the human pathogens Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, are causative agents of lymphomas and other malignancies. The structural characterization of these viruses has been limited due to difficulties in obtaining adequate amount of virion particles. Here we report the first three-dimensional structural characterization of a whole gammaherpesvirus virion by an emerging integrated approach of cryo-electron tomography combined with single-particle cryo-electron microscopy, using murine gammaherpesvirus-68 (MHV-68) as a model system. We found that the MHV-68 virion consists of distinctive envelope and tegument compartments, and a highly conserved nucleocapsid. Two layers of tegument are identified: an inner tegument layer tethered to the underlying capsid and an outer, flexible tegument layer conforming to the overlying, pleomorphic envelope, consistent with the sequential viral tegumentation process inside host cells. Surprisingly, comparison of the MHV-68 virion and capsid reconstructions shows that the interactions between the capsid and inner tegument proteins are completely different from those observed in alpha and betaherpesviruses. These observations support the notion that the inner layer tegument across different subfamilies of herpesviruses has evolved significantly to confer specific characteristics related to viral-host interactions, in contrast to a highly conserved capsid for genome encapsidation and protection
— id: 96284, year: 2008, vol: 161, page: 428, stat: Journal Article,

Methylation Linear Discriminant Analysis (MLDA) for identifying differentially methylated CpG islands
Dai, Wei; Teodoridis, Jens M; Graham, Janet; Zeller, Constanze; Huang, Tim H M; Yan, Pearlly; Vass, J Keith; Brown, Robert; Paul, Jim
2008 ;9:337-337, BMC bioinformatics
BACKGROUND: Hypermethylation of promoter CpG islands is strongly correlated to transcriptional gene silencing and epigenetic maintenance of the silenced state. As well as its role in tumor development, CpG island methylation contributes to the acquisition of resistance to chemotherapy. Differential Methylation Hybridisation (DMH) is one technique used for genome-wide DNA methylation analysis. The study of such microarray data sets should ideally account for the specific biological features of DNA methylation and the non-symmetrical distribution of the ratios of unmethylated and methylated sequences hybridised on the array. We have therefore developed a novel algorithm tailored to this type of data, Methylation Linear Discriminant Analysis (MLDA). RESULTS: MLDA was programmed in R (version 2.7.0) and the package is available at CRAN 1. This approach utilizes linear regression models of non-normalised hybridisation data to define methylation status. Log-transformed signal intensities of unmethylated controls on the microarray are used as a reference. The signal intensities of DNA samples digested with methylation sensitive restriction enzymes and mock digested are then transformed to the likelihood of a locus being methylated using this reference. We tested the ability of MLDA to identify loci differentially methylated as analysed by DMH between cisplatin sensitive and resistant ovarian cancer cell lines. MLDA identified 115 differentially methylated loci and 23 out of 26 of these loci have been independently validated by Methylation Specific PCR and/or bisulphite pyrosequencing. CONCLUSION: MLDA has advantages for analyzing methylation data from CpG island microarrays, since there is a clear rational for the definition of methylation status, it uses DMH data without between-group normalisation and is less influenced by cross-hybridisation of loci. The MLDA algorithm successfully identified differentially methylated loci between two classes of samples analysed by DMH using CpG island microarrays
— id: 96278, year: 2008, vol: 9, page: 337, stat: Journal Article,

Phosphorylation of H3S10 blocks the access of H3K9 by specific antibodies and histone methyltransferase. Implication in regulating chromatin dynamics and epigenetic inheritance during mitosis
Duan, Qing; Chen, Haobin; Costa, Max; Dai, Wei
2008 Nov 28;283(48):33585-33590, Journal of biological chemistry
Post-translational modifications of histones play a critical role in regulating genome structures and integrity. We have focused on the regulatory relationship between covalent modifications of histone H3 lysine 9 (H3K9) and H3S10 during the cell cycle. Immunofluorescence microscopy revealed that H3S10 phosphorylation in HeLa, A549, and HCT116 cells was high during prophase, prometaphase, and metaphase, whereas H3K9 monomethylation (H3K9me1) and dimethylation (H3K9me2), but not H3K9 trimethylation (H3K9me3), were significantly suppressed. When H3S10 phosphorylation started to diminish during anaphase, H3K9me1 and H3K9me2 signals reemerged. Western blot analyses confirmed that mitotic histones, extracted in an SDS-containing buffer, had little H3K9me1 and H3K9me2 signals but abundant H3K9me3 signals. However, when mitotic histones were extracted in the same buffer without SDS, the difference in H3K9me1 and H3K9me2 signals between interphase and mitotic cells disappeared. Removal of H3S10 phosphorylation by pretreatment with lambda-phosphatase unmasked mitotic H3K9me1 and H3K9me2 signals detected by both fluorescence microscopy and Western blotting. Further, H3S10 phosphorylation completely blocked methylation of H3K9 but not demethylation of the same residue in vitro. Given that several conserved motifs consisting of a Lys residue immediately followed by a Ser residue are present in histone tails, our studies reveal a potential new mechanism by which phosphorylation not only regulates selective access of methylated lysines by cellular factors but also serves to preserve methylation patterns and epigenetic programs during cell division
— id: 91974, year: 2008, vol: 283, page: 33585, stat: Journal Article,

High-level secretion of a chimeric thermostable lichenase from Bacillus subtilis by screening of site-mutated signal peptides with structural alterations
Fu, Ling-lin; Xu, Zi-rong; Shuai, Jiang-bing; Hu, Chun-xia; Dai, Wei; Li, Wei-fen
2008 Mar;56(3):287-292, Current microbiology
A chimeric gene mHG (669 bp) was constructed by substitution of Clostridium thermocellum ZJL4 lichenase (CG) N-terminal fragment (except its signal sequence) for the counterpart of Bacillus sp. A3 lichenase (BG). To acquire high-level secretion of the chimeric lichenase (mHG) in Bacillus subtilis, a series of site-mutated signal peptides were designed. The activity of mHG, which was directed by an artificial hydrophobic signal peptide H1 (MMARKIAGMATSLLVIFSSSAVA) from cytoplasm into growth medium, reached 80.56 U/ml after 22-h incubation, indicating that signal peptide hydrophobicity appears to be critical for early stages in mHG export. By purification of the mHG (approximately 25.3 kDa) from cultures of B. subtilis (pBSG-H1), enzymatic property assays showed that the common optima for mHG were 70 degrees C and pH 5.0. The residual activity of mHG at 90 degrees C for 10 min was 83.45% of its maximum activity, which was almost similar to that of CG (90 degrees C, 10 min, 84.33%). This constructed shuttle expression vector with a novel signal peptide exhibited its applicability for high-level production of heterologous proteins from B. subtilis. Moreover, the high-level secreted mHG with relatively high thermostability could be a potential candidate for feed industrial applications
— id: 96283, year: 2008, vol: 56, page: 287, stat: Journal Article,

Preparation and identification of scFv and bsFv against transferrin receptor
Liu, Jing; Xiao, Daiwen; Zhou, Xiaoou; Wen, Xue; Dai, Hong; Wang, Zhihua; Shen, Xin; Dai, Wei; Yang, Daofeng; Shen, Guanxin
2008 Dec;28(6):621-625, Journal of Huazhong University of Science & Technology. Medical sciences = Hua zhong ke ji da xue xue bao. Yi xue Ying De wen ban = Huazhong keji daxue xuebao. Yixue Yingdewen ban
To obtain single chain variable fragment (scFv) and bivalent single chain variable fragment (bsFv) against transferrin receptor, up-stream and down-stream primers were designed according to the complementary sequences of FR1 region of variable heavy (VH) and FR4 of variable light (VL), respectively, which contained inter-linker G4S and the restriction endonuclease SfiI, AscI and NotI. Two pieces of scFv fragments were first amplified through PCR and then inserted into plasmid pAB1, which could express scFv protein once induced by IPTG in the host bacteria. To express scFv and bsFv, E. coli TG1 was cultured in LB broth and was induced by IPTG. The restriction enzyme digestion map and DNA sequencing demonstrated that scFv and bsFv genes were successfully inserted into the expression plasmid. SDS-PAGE and Western blotting revealed the protein band at 35kD and 60kD, which were consistent with the molecular weight of scFv and bsFv respectively. Flow cytometry showed that scFv and bsFv harbored the specific binding activity with TfR expressed in various tumor cells, and the avidity of bsFv was higher than that of the parent scFv
— id: 96273, year: 2008, vol: 28, page: 621, stat: Journal Article,

Comparison of receptor models for source apportionment of volatile organic compounds in Beijing, China
Song, Yu; Dai, Wei; Shao, Min; Liu, Ying; Lu, Sihua; Kuster, William; Goldan, Paul
2008 Nov;156(1):174-183, Environmental pollution
Identifying the sources of volatile organic compounds (VOCs) is key to reducing ground-level ozone and secondary organic aerosols (SOAs). Several receptor models have been developed to apportion sources, but an intercomparison of these models had not been performed for VOCs in China. In the present study, we compared VOC sources based on chemical mass balance (CMB), UNMIX, and positive matrix factorization (PMF) models. Gasoline-related sources, petrochemical production, and liquefied petroleum gas (LPG) were identified by all three models as the major contributors, with UNMIX and PMF producing quite similar results. The contributions of gasoline-related sources and LPG estimated by the CMB model were higher, and petrochemical emissions were lower than in the UNMIX and PMF results, possibly because the VOC profiles used in the CMB model were for fresh emissions and the profiles extracted from ambient measurements by the two-factor analysis models were 'aged'
— id: 96282, year: 2008, vol: 156, page: 174, stat: Journal Article,

Identifying anthropogenic and natural influences on extreme pollution of respirable suspended particulates in Beijing using backward trajectory analysis
Song, Yu; Miao, Weijie; Liu, Bing; Dai, Wei; Cai, Xuhui
2008 Jun 15;154(1-3):459-468, Journal of hazardous materials
In China, daily respirable suspended particulate (RSP, particles with aerodynamic diameters less than 10 microm) concentrations exceeding 420 microg m(-3) are considered 'hazardous' to health. These can lead to the premature onset of certain diseases and premature death of sick and elderly people; even healthy people are warned to avoid outdoor activity when RSP concentrations are high. Such high pollution levels are defined as extreme RSP pollution events. Recent epidemiological studies have shown that a distinct difference exists between the health effects caused by natural sources and anthropogenic sources, mandating knowledge of the source of extreme RSP pollution. Twenty-six extreme RSP pollution events were recorded in Beijing from January 2003 to December 2006. The HYSPLIT4 (Hybrid Single Particle Lagrangian Integrated Trajectory) model (Version 4) was used to discriminate the sources of these extreme RSP pollution events. The model found that twelve events were caused from natural sources (dust storms), nine events from anthropogenic sources (e.g., vehicles and industrial activities, etc.) under quasi-quiescent weather, and five events were from mixed causes. Identifying such events will be valuable in epidemiological studies on air pollution in Beijing
— id: 96285, year: 2008, vol: 154, page: 459, stat: Journal Article,

Activation of Polo-like kinase 3 by hypoxic stresses
Wang, Ling; Gao, Jie; Dai, Wei; Lu, Luo
2008 Sep 19;283(38):25928-25935, Journal of biological chemistry
Hypoxia/reoxygenation stress induces the activation of specific signaling proteins and activator protein 1 (AP-1) to regulate cell cycle regression and apoptosis. In the present study, we report that hypoxia/reoxygenation stress activates AP-1 by increasing c-Jun phosphorylation and DNA binding activity through activation of Polo-like-kinase 3 (Plk3) resulting in apoptosis. The specific effect of hypoxia/reoxygenation stress on Plk3 activation resulting in c-Jun phosphorylation was the opposite of UV irradiation-induced responses that are meanly independent on activation of the stress-induced JNK signaling pathway in human corneal epithelial (HCE) cells. The effect of hypoxia/reoxygenation stress-induced Plk3 activation on increased c-Jun phosphorylation and apoptosis was also mimicked by exposure of cells to CoCl(2). Hypoxia/reoxygenation activated Plk3 in HCE cells to directly phosphorylate c-Jun proteins at phosphorylation sites Ser-63 and Ser-73, and to increase DNA binding activity of c-Jun, detected by EMSA. Further evidence demonstrated that Plk3 and phospho-c-Jun were immunocolocalized in the nuclear compartment of hypoxia/reoxygenation stress-induced cells. Increased Plk3 activity by overexpression of wild-type and dominantly positive Plk3 enhanced the effect of hypoxia/reoxygenation on c-Jun phosphorylation and cell death. In contrast, knocking-down Plk3 mRNA suppressed hypoxia-induced c-Jun phosphorylation. Our results provide a new mechanism indicating that hypoxia/reoxygenation induces Plk3 activation instead of the JNK effect to directly phosphorylate and activate c-Jun, subsequently contributing to apoptosis in HCE cells
— id: 96279, year: 2008, vol: 283, page: 25928, stat: Journal Article,

sSgo1, a major splice variant of Sgo1, functions in centriole cohesion where it is regulated by Plk1
Wang, Xiaoxing; Yang, Yali; Duan, Qing; Jiang, Ning; Huang, Ying; Darzynkiewicz, Zbigniew; Dai, Wei
2008 Mar;14(3):331-341, Developmental cell
Shugoshin 1 (Sgo1) functions as a protector of centromeric cohesion of sister chromatids in higher eukaryotes. Here, we provide evidence for a previously unrecognized role for Sgo1 in centriole cohesion. Sgo1 depletion via RNA interference induces the formation of multiple centrosome-like structures in mitotic cells that result from the separation of paired centrioles. Sgo1+/- mitotic murine embryonic fibroblasts display split centrosomes. Localization study of two major endogenous splice variants of Sgo1 indicates that the smaller variant, sSgo1, is found at the centrosome in interphase and at spindle poles in mitosis. sSgo1 interacts with Plk1 and its spindle pole localization is Plk1 dependent. Centriole splitting induced by Sgo1 depletion or expression of a dominant negative mutant is suppressed by ectopic expression of sSgo1 or by Plk1 knockdown. Our studies strongly suggest that sSgo1 plays an essential role in protecting centriole cohesion, which is partly regulated by Plk1
— id: 78024, year: 2008, vol: 14, page: 331, stat: Journal Article,

Polo-like kinase 3 functions as a tumor suppressor and is a negative regulator of hypoxia-inducible factor-1 alpha under hypoxic conditions
Yang, Yali; Bai, Jingxiang; Shen, Rulong; Brown, Sharron A N; Komissarova, Elena; Huang, Ying; Jiang, Ning; Alberts, Gregory F; Costa, Max; Lu, Luo; Winkles, Jeffrey A; Dai, Wei
2008 Jun 1;68(11):4077-4085, Cancer research
Polo-like kinase 3 (Plk3) is an important mediator of the cellular responses to genotoxic stresses. In this study, we examined the physiologic function of Plk3 by generating Plk3-deficient mice. Plk3(-/-) mice displayed an increase in weight and developed tumors in various organs at advanced age. Many tumors in Plk3(-/-) mice were large in size, exhibiting enhanced angiogenesis. Plk3(-/-) mouse embryonic fibroblasts were hypersensitive to the induction of hypoxia-inducible factor-1 alpha (HIF-1 alpha) under hypoxic conditions or by nickel and cobalt ion treatments. Ectopic expression of the Plk3-kinase domain (Plk3-KD), but not its Polo-box domain or a Plk3-KD mutant, suppressed the nuclear accumulation of HIF-1 alpha induced by nickel or cobalt ions. Moreover, hypoxia-induced HIF-1 alpha expression was tightly associated with a significant down-regulation of Plk3 expression in HeLa cells. Given the importance of HIF-1 alpha in mediating the activation of the 'survival machinery' in cancer cells, these studies strongly suggest that enhanced tumorigenesis in Plk3-null mice is at least partially mediated by a deregulated HIF-1 pathway
— id: 81058, year: 2008, vol: 68, page: 4077, stat: Journal Article,

Pharmacokinetic and toxicity study of intravitreal erythropoietin in rabbits
Zhang, Jing-fa; Wu, Ya-lan; Xu, Jing-ying; Ye, Wen; Zhang, Yu; Weng, Huan; Shi, Wo-dong; Xu, Guo-xu; Lu, Luo; Dai, Wei; Sinclair, Stephen H; Li, Wei-ye; Xu, Guo-tong
2008 Nov;29(11):1383-1390, Acta pharmacologica Sinica
AIM: To study the pharmacokinetics and toxicity of intravitreal erythropoietin (EPO) for potential clinical use. METHODS: For toxicity study, 4 groups (60 rabbits) with intravitreal injection (IVit) of EPO were studied (10 U, 100 U, or 1,000 U) per eye for single injection and 0.6 U/eye (the designed therapeutic level in rabbits) for monthly injections (6X). Eye examination, flash electroretinogram (ERG), and fluorescein angiography (FA) were carried out before and after injection. The rabbits were killed for histological study at different intervals. For the pharmacokinetic study, after IVit of 5 U EPO into left eyes, 44 rabbits were killed at different intervals, and the EPO levels in vitreous, aqueous, retina and serum were analyzed by enzyme-linked immunosorbent assay. RESULTS: At all of the time points examined, the eyes were within normal limits. No significant ERG or FA change was observed. The histology of retina remained unchanged. The pharmacokinetic profile of EPO in ocular compartments was summarized as follows. The half-life times of EPO in vitreous, aqueous and serum were 2.84, 3.24 and 2.12 d, respectively; and Cmax were 4615.75, 294.31 and 1.60 U/L, respectively. EPO concentrations in the retina of the injected eye peaked at 1.36 U/g protein at 6 h following injection, with the half-life observed to be 3.42 d. CONCLUSIONS: IVit of EPO in a wide range is well tolerated and safe for rabbit eyes. At doses up to 10-fold higher than therapeutic levels, EPO has a pharmacokinetic profile with faster clearance, which is favorable for episodic IVit
— id: 96275, year: 2008, vol: 29, page: 1383, stat: Journal Article,

Intravitreal injection of erythropoietin protects both retinal vascular and neuronal cells in early diabetes
Zhang, Jingfa; Wu, Yalan; Jin, Ying; Ji, Fei; Sinclair, Stephen H; Luo, Yan; Xu, Guoxu; Lu, Luo; Dai, Wei; Yanoff, Myron; Li, Weiye; Xu, Guo-Tong
2008 Feb;49(2):732-742, Investigative ophthalmology & visual science. IOVS
PURPOSE: To explore and evaluate the protective effect of erythropoietin (EPO) on retinal cells of chemically induced diabetic rats after EPO was injected intravitreally at the onset of diabetes. METHODS: Diabetes was induced in Sprague-Dawley rats by intraperitoneal injection of streptozotocin (STZ). At the onset of diabetes, a single intravitreal injection of EPO (0.05-200 ng/eye) was performed. In the following 6 weeks, the blood retinal barrier (BRB) was evaluated by Evans blue permeation (EBP). Retinal cell death in different layers was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. The retinal thickness and cell counts were examined at the light microscopic level. Electron microscopy (EM) was used to scrutinize retinal vascular and neuronal injury. Neurosensory retinas of normal and diabetic rats were used as the sources of reverse transcription-polymerase chain reaction (RT-PCR) and Western blot for the detection of EPO, EPO receptor (EpoR), and products of the extracellular signal-regulated kinase (ERK) and the signal transducers and activators of transcription 5 (STAT5) pathways. The distribution of EpoR in retinal layers was demonstrated by immunohistochemistry (IHC). RESULTS: In the diabetic rats, BRB breakdown was detected soon after the onset of diabetes, peaked at 2 weeks, and reached a plateau at 2 to 4 weeks. The number of TUNEL-positive cells increased in the neurosensory retina, especially, the outer nuclear layer (ONL) at 1 week after diabetes onset and reached a peak at 4 to 6 weeks. The retinal thickness and the number of cells in the ONL were reduced significantly. EM observations demonstrated vascular and photoreceptor cell death starting soon after the onset of diabetes. All these changes were largely prevented by EPO treatment. Upregulation of EpoR in the neurosensory retina was detected at both the transcriptional and protein levels 4 to 8 weeks after the onset of diabetes, whereas, the endogenous EPO levels of neurosensory retinas were essentially unchanged during the same period observed. In EPO-treated diabetic groups, EpoR expression remained at upregulated levels. Within 2 weeks of the onset of diabetes, activation of the ERK but not the STAT5 pathway was detected in the diabetic retina treated with EPO. CONCLUSIONS: These data demonstrate that apoptosis is an major contributor to neuronal cell death in the early course of diabetic retinopathy (DR). The upregulation of EpoR may be a compensatory response of retinal cells and tissue to diabetic stresses. The EPO/EpoR system as a maintenance-survival mechanism of retinal neurons responds to the insults of early diabetes other than ischemia. The protective function of EPO/EpoR at the least acts through the EpoR-mediated ERK pathway. Exogenous EPO administration by intravitreal injection in early diabetes may prevent retinal cell death and protect the BRB function. Therefore, this is a novel approach for treatment of early DR
— id: 96281, year: 2008, vol: 49, page: 732, stat: Journal Article,

[Evidence of bacterial biofilms in chronic rhinosinusitis]
Zhang, Zi; Li, Yun-chuan; Han, Ye-hua; Dai, Wei; Zhang, Sheng-zhong; Zhou, Bing; Zhang, Luo; Wang, De-yun; Han, De-min; Zhang, Yong-jie
2008 Nov;43(11):840-844, Zhonghua er bi yan hou tou jing wai ke za zhi = Chinese journal of otorhinolaryngology head & neck surgery
OBJECTIVE: To observe the presence of bacterial biofilms in mucosal specimens in patients operated for chronic rhinosinusitis. METHODS: A total of 12 subjects undergoing endoscopic sinus surgery were included. The control group was 6 patients with obstructive sleep apnea-hypopnea syndrome. Six patients with chronic rhinosinusitis were enrolled in the study group. Mucosa of uncinate process, ethmoid bulla or maxillary sinus was obtained during endoscopic sinus surgery. All the samples were prepared using standard methods for scanning electron microscopy. Patients' information such as age, gender, symptoms, sinus CT, endoscopic examination, skin prick test were recorded in detail. RESULTS: Standard-preparation scanning electron microscopy displaed denuded epithelium and disarrayed cilia in chronic rhinosinusitis patient's mucosa. In the study group, bacterial biofilms of different morphology were seen in five samples; One sample showed filamentous structure like fungi. Using strict scanning electron microscopy morphologic criteria, 83.3% samples in the study group were found to have micrographic evidence of biofilms. No bacterial biofilms were detected in the control group. CONCLUSIONS: In the patients undergoing surgery for chronic rhinosinusitis, different degree of mucosal injury could be found. Bacteria biofilms of different life stages were demonstrated to be present. No bacterial biofilms were detected in the control group
— id: 96271, year: 2008, vol: 43, page: 840, stat: Journal Article,

Intensified reaction of dilute thiophenes in nanoreactor
Zhou, Li; Wang, Shengqiang; Dai, Wei; Zhou, Yaping
2008 Mar 6;112(9):1887-1890, Journal of physical chemistry. A
A discovery that catalytic reactivity is intensified on reducing the size of reactor toward several nanometers scale was applied for the separation of dilute species, for example, the thiophenic compounds in transportation fuels, in the present study. A characteristic reaction of the dilute species was selected as the separation mechanism, and a nanoreactor was formed with preloading reactant/catalyst in volumes of mesopore dimension. Probability for the dilute species to contact the nanoreactor radically enlarged due to the integration of such volumes in a porous material. Because all reagents and the reaction product stayed inside the nanoreactor, separation of fuel from sulfuric compounds and the surplus chemicals used in reaction becomes simple. It was experimentally shown that the nanoreaction exhibited first-order kinetics, and all thiophenes and benzothiophenes contained in different types of model fuels were completely removed at moderate conditions
— id: 96280, year: 2008, vol: 112, page: 1887, stat: Journal Article,

Study on olfactory function in GABAC receptor/channel rho1 subunit knockout mice
Chen, Yan; Zhou, Dongping; Zhou, Kun; Ren, Yiping; Dai, Wei; Xu, Ming; Lu, Luo; Lu, Zhenyu
2007 Oct 29;427(1):10-15, Neuroscience letters
The GABAC receptor/channel rho1 subunit plays an important role in the inhibitory pathway and sensory processing in the retina and spinal cord. Although it was suggested that the rho1 subunit plays a role in olfactory sensations, the precise role of the rho1 subunit in olfactory sensory function is still not clear. In the present study, we report that olfactory function was significantly altered in rho1 subunit knockout (rho1-/-) mice compared to its wildtype counterpart. The rho1 subunit mRNA, detected by reverse transcription (RT)-PCR experiments, was expressed in the olfactory bulb of wild-type mice. Expression of rho1 subunit proteins in the olfactory bulb was detected by immunohistochemistry in mitral cells in the mitral cell layer. Neither mRNA nor proteins of the rho1 subunit were found in olfactory bulb neurons in rho1-/- mice. Alterations of olfactory function in rho1-/- mutant mice compared to their wildtype littermates were examined by olfactory behavioral test. We found that sensitivity to the smell of citral odorant in rho1-/- mice was significantly greater compared to that of wildtype mice. Our results indicate that the GABAC rho1 subunit acts in olfactory bulb neurons as an inhibitory modulator that affects the process of olfactory signaling transmission
— id: 76371, year: 2007, vol: 427, page: 10, stat: Journal Article,

Polo-like Kinases Inhibited by Wortmannin: LABELING SITE AND DOWNSTREAM EFFECTS
Liu, Yongsheng; Jiang, Ning; Wu, Jiangyue; Dai, Wei; Rosenblum, Jonathan S
2007 Jan 26;282(4):2505-2511, Journal of biological chemistry
Polo-like kinases play crucial roles throughout mitosis. We previously reported that wortmannin potently inhibits Polo-like kinase 1 (Plk1). In this study, we show that wortmannin also strongly inhibits Polo-like kinase 3 (Plk3). To further characterize this inhibition, we identified the sites of labeling on Plk1 and Plk3 targeted by AX7503, a tetramethylrhodamine-wortmannin conjugate. AX7503 labeling on Plk1 and Plk3 was found to occur on a conserved ATP binding site residue. In addition, we show that wortmannin inhibits Plk3 activity in live cells at concentrations commonly used to inhibit the more well known targets of wortmannin, the phosphoinositide 3-kinases. Importantly, we found that inhibition of Plk3 by wortmannin lead to a decrease in phosphorylation of p53 on serine 20 induced by DNA damage, demonstrating the effect of wortmannin on a downstream Plk3 target. Taken together, our results suggest that wortmannin can affect multiple functions of Plk3 in cell cycle progression and at the DNA damage check point. The identification of the labeling sites of Plk1 and Plk3 by AX7503 may be useful in designing more effective compounds to target Polo-like kinases for cancer treatment and also may be useful for the structural study of Plk domains
— id: 70085, year: 2007, vol: 282, page: 2505, stat: Journal Article,

Cytometry of ATM activation and histone H2AX phosphorylation to estimate extent of DNA damage induced by exogenous agents
Tanaka, Toshiki; Huang, Xuan; Halicka, H Dorota; Zhao, Hong; Traganos, Frank; Albino, Anthony P; Dai, Wei; Darzynkiewicz, Zbigniew
2007 Sep;71(9):648-661, Cytometry A
This review covers the topic of cytometric assessment of activation of Ataxia telangiectasia mutated (ATM) protein kinase and histone H2AX phosphorylation on Ser139 in response to DNA damage, particularly the damage that involves formation of DNA double-strand breaks. Briefly described are molecular mechanisms associated with activation of ATM and the downstream events that lead to recruitment of DNA repair machinery, engagement of cell cycle checkpoints, and activation of apoptotic pathway. Examples of multiparameter analysis of ATM activation and H2AX phosphorylation vis-a-vis cell cycle phase position and induction of apoptosis that employ flow- and laser scanning-cytometry are provided. They include cells treated with a variety of exogenous genotoxic agents, such as ionizing and UV radiation, DNA topoisomerase I (topotecan) and II (mitoxantrone, etoposide) inhibitors, nitric oxide-releasing aspirin, DNA replication inhibitors (aphidicolin, hydroxyurea, thymidine), and complex environmental carcinogens such as present in tobacco smoke. Also presented is an approach to identify DNA replicating (BrdU incorporating) cells based on selective photolysis of DNA that triggers H2AX phosphorylation. Listed are strategies to distinguish ATM activation and H2AX phosphorylation induced by primary DNA damage by genotoxic agents from those effects triggered by DNA fragmentation that takes place during apoptosis. While we review most published data, recent new findings also are included. Examples of multivariate analysis of ATM activation and H2AX phosphorylation presented in this review illustrate the advantages of cytometric flow- and image-analysis of these events in terms of offering a sensitive and valuable tool in studies of factors that induce DNA damage and/or affect DNA repair and allow one to explore the linkage between DNA damage, cell cycle checkpoints and initiation of apoptosis
— id: 76367, year: 2007, vol: 71, page: 648, stat: Journal Article,

Stress-induced c-Jun activation mediated by Polo-like kinase 3 in corneal epithelial cells
Wang, Ling; Dai, Wei; Lu, Luo
2007 Nov 2;282(44):32121-32127, Journal of biological chemistry
Polo-like kinase 3 (Plk3) activation occurs after exposure to environmental or genotoxic stresses. Plk3 regulates cell fate through regulating cell cycle progression. UV irradiation is one of the major environmental stresses that affect corneal epithelial wound healing. In the present study, we report that UV irradiation activated Plk3 and that Plk3 interacts with AP-1 and c-Jun, which appears to be important to mediate corneal epithelial cell apoptosis after UV irradiation. Recombinant Plk3, as well as Plk3 immunoprecipitated from UV-irradiated cells, phosphorylated c-Jun in vitro. The phosphorylation of c-Jun by Plk3 immunoprecipitates was not altered by the pre-removal of JNK from the cell lysates. In addition, the effect of UV irradiation-induced phosphorylation of c-Jun and apoptosis were not significantly affected by knockdown of JNK mRNA. Co-immunoprecipitation reveals that Plk3 and c-Jun directly interacted with each other. Consistently, Plk3 co-localized with c-Jun to the nucleus after UV irradiation. Further, modulating Plk3 activities by overexpressing Plk3 or its mutants significantly affected UV irradiation-induced c-Jun activity and subsequent apoptosis. Our results thus provide for the first time that Plk3 mediates UV irradiation-induced c-Jun activation by phosphorylating c-Jun, suggesting that Plk3 plays an important role in mediating programmed cell death of corneal epithelial cells after UV irradiation
— id: 76361, year: 2007, vol: 282, page: 32121, stat: Journal Article,

Rig-I-/- mice develop colitis associated with downregulation of G alpha i2
Wang, Yi; Zhang, Hong-Xin; Sun, Yue-Ping; Liu, Zi-Xing; Liu, Xue-Song; Wang, Long; Lu, Shun-Yuan; Kong, Hui; Liu, Qiao-Ling; Li, Xi-Hua; Lu, Zhen-Yu; Chen, Sai-Juan; Chen, Zhu; Bao, Shi-San; Dai, Wei; Wang, Zhu-Gang
2007 Oct;17(10):858-868, Cell research
RIG-I (retinoid acid-inducible gene-I), a putative RNA helicase with a cytoplasmic caspase-recruitment domain (CARD), was identified as a pattern-recognition receptor (PRR) that mediates antiviral immunity by inducing type I interferon production. To further study the biological function of RIG-I, we generated Rig-I(-/-) mice through homologous recombination, taking a different strategy to the previously reported strategy. Our Rig-I(-/-) mice are viable and fertile. Histological analysis shows that Rig-I(-/-) mice develop a colitis-like phenotype and increased susceptibility to dextran sulfate sodium-induced colitis. Accordingly, the size and number of Peyer's patches dramatically decreased in mutant mice. The peripheral T-cell subsets in mutant mice are characterized by an increase in effector T cells and a decrease in naive T cells, indicating an important role for Rig-I in the regulation of T-cell activation. It was further found that Rig-I deficiency leads to the downregulation of G protein alpha i2 subunit (G alpha i2) in various tissues, including T and B lymphocytes. By contrast, upregulation of Rig-I in NB4 cells that are treated with ATRA is accompanied by elevated G alpha i2 expression. Moreover, G alpha i2 promoter activity is increased in co-transfected NIH3T3 cells in a Rig-I dose-dependent manner. All these findings suggest that Rig-I has crucial roles in the regulation of G alpha i2 expression and T-cell activation. The development of colitis may be, at least in part, associated with downregulation of G alpha i2 and disturbed T-cell homeostasis
— id: 76360, year: 2007, vol: 17, page: 858, stat: Journal Article,

BubR1 deficiency results in enhanced activation of MEK and ERKs upon microtubule stresses
Yang, Y L; Duan, Q; Guo, T B; Wang, X X; Ruan, Q; Xu, G T; Zhang, J W; Lu, Z Y; Xu, M; Lu, L; Dai, W
2007 Jun;40(3):397-410, Cell proliferation
Disruption of microtubules activates the spindle checkpoint, of which BubR1 is a major component. Our early studies show that BubR1 haplo-insufficiency results in enhanced mitotic slippage in vitro and tumorigenesis in vivo. OBJECTIVE: Given that both MAPKs/ERKs and MEK play an important role during mitosis, we investigated whether there existed regulatory relationship between the MAPK signalling pathway and BubR1. METHOD AND RESULTS: Here, we have demonstrated that BubR1 deficiency is correlated with enhanced activation of MEK and ERKs after disruption of microtubule dynamics. Specifically, treatment with nocodazole and paclitaxel resulted in hyper-activation of ERKs and MEK in BubR1(+/-) murine embryonic fibroblasts (MEF) compared to that of wild-type MEFs. This enhanced activation of ERKs and MEK was at least partly responsible for more successful proliferation completion when cells were treated with nocodazole. BubR1 knockdown via RNAi resulted in enhanced activation of ERKs and MEK in HeLa cells, correlating with inhibition of PP1, a negative regulator of MEK. Moreover, when BubR1 was partially inactivated due to premature missegregation of chromosomes after Sgo1 depletion, phosphorylation of ERKs and MEK was enhanced in mitotic cells; in contrast, little, if any activated ERKs and MEK were detected in mitotic cells induced by nocodazole. Furthermore, BubR1, activated ERKs and activated MEK all localized to spindle poles during mitosis, and also, the proteins physically interacted with each other. CONCLUSION: Our studies suggest that there exists a cross-talk between spindle checkpoint components and ERKs and MEK and that BubR1 may play an important role in mediating the cross-talk
— id: 76364, year: 2007, vol: 40, page: 397, stat: Journal Article,

Role of c-Fos/JunD in protecting stress-induced cell death
Zhou, H; Gao, J; Lu, Z Y; Lu, L; Dai, W; Xu, M
2007 Jun;40(3):431-444, Cell proliferation
OBJECTIVE: The exposure of mammalian cells to extracellular stress induces the expression of immediate early genes such as c-fos and c-jun and activates transcription factor activator protein-1 (AP-1). The purpose of the current study was to investigate the role of c-Fos and JunD in stress-induced cell death. MATERIALS AND METHODS: We exposed cultured primary mouse embryonic fibroblasts (MEF) to ultraviolet light (UV-C) or hydrogen peroxide (H(2)O(2)). Induction of c-Fos and JunD and activation of MAPK/ERK1/2 signalling in the presence or absence of a MAPK inhibitor were analyzed by western blotting. Activation of AP-1 transcription factors was detected by the electrophoretic mobility shift assay and immunoprecipitation. Cell death was measured by changes in caspase 3 activities and nuclear morphology. Effects of c-Fos and JunD expression on cell death were investigated by transfection. RESULTS: We found that the exposure of cultured primary MEF cells to UV or H(2)O(2) caused a significant increase in c-Fos and JunD protein levels. In addition, these two proteins formed complexes with each other and contributed to activation of AP-1 transcription complexes. More importantly, under both stress conditions, overexpression of JunD alone or overexpression of both c-Fos and JunD reduced caspase 3 activity and cell death. At the same time, UV irradiation activated the MAPK/ERK1/2 signalling pathway. The suppression of MEK1/ERK1/2 activation inhibited UV-induced expression of c-Fos and JunD and increased caspase 3 activity and cell death. CONCLUSION: Our results suggest that both UV and H(2)O(2 )induce the activation of c-Fos/JunD AP-1 complexes resulting in the prevention of cell death. Moreover, UV irradiation-induced increases in c-Fos/JunD expression in primary MEF cells are mediated through the activation of the MAPK/ERK1/2 signalling pathway
— id: 76363, year: 2007, vol: 40, page: 431, stat: Journal Article,

Aging in check
Dai, Wei; Wang, Xiaoxing
2006 Mar 15;2006(7):pe9-pe9, Science of aging knowledge environment
The spindle checkpoint monitors the interaction between spindle microtubules and kinetochores to prevent precocious entry into anaphase, delaying this stage of mitosis until all condensed chromosomes have been attached to the mitotic spindle in a bi-oriented manner (so that the two kinetochores associated with a pair of sister chromatids are oriented toward opposite poles of the spindle). In addition to conserved Bub and Mad family members, which are known to function in the spindle checkpoint pathway in organisms ranging from yeast to mammals, two mRNA transport genes, Rae1 and Nup9, are also involved in the spindle checkpoint function in mammals. Biochemically, activated spindle checkpoint components have been shown to suppress the activity of the anaphase promoting complex/cyclosome. It is generally thought that decreased activity of the checkpoint components predisposes cells to chromosomal instability, aneuploidy, and malignant transformation. Interestingly, a recent study has shed light on a new function of the spindle checkpoint components Bub3 and Rae1 in the regulation of aging. Mice with haploinsufficiency of Bub3 and Rae1 have a short life span that is associated with the early onset of aging-related features. The progeroid phenotypes caused by deficiency of Bub3 and Rae1 are tightly linked to precocious activation of cellular senescence, but not apoptotic, programs. Therefore, premature aging, rather than neoplastic transformation, may be the major manifestation of a compromised spindle checkpoint in vivo
— id: 70086, year: 2006, vol: 2006, page: pe9, stat: Journal Article,

Grabbing Plk1 by the PBD
Dai, Wei; Wang, Xiaoxing
2006 Nov 17;24(4):489-490, Molecular cell
A new centromeric protein termed PBIP1 was identified that recruits Plk1 to the kinetochores. In the November 3 issue of Molecular Cell, show that Plk1 phosphorylates PBIP1 on threonine 78, creating its own high-affinity docking site for the polo-box domain (PBD)
— id: 70160, year: 2006, vol: 24, page: 489, stat: Journal Article,

BubR1 is involved in regulation of DNA damage responses
Fang, Y; Liu, T; Wang, X; Yang, Y-M; Deng, H; Kunicki, J; Traganos, F; Darzynkiewicz, Z; Lu, L; Dai, W
2006 Jun 15;25(25):3598-3605, Oncogene
Defective mitotic spindles or an impaired spindle-kinetochore interaction activates the spindle checkpoint. We have previously shown that BubR1 haplo-insufficiency results in enhanced genomic instability and tumorigenesis in mice. Here we report that BubR1 deficiency also leads to a compromised response to DNA damage. Following treatment with doxorubicin, BubR1(+/-) murine fibroblast cells (MEF) were defective in undergoing G(2)/M arrest. Thus, whereas in the presence of DNA damage BubR1(+/+) MEF cells remained arrested in mitosis, BubR1(+/-) MEFs rapidly exited from mitosis and divided. The impaired mitotic arrest of BubR1(+/-) MEFs was associated with low levels of phospho-histone H2AX, p53, and p21 after DNA damage caused by treatment with both doxorubicin and ultraviolet light (UV). The impaired expression of p53 and p21 was also confirmed in human cell lines with BubR1 knockdown via RNA interference. Affinity pull-down coupled with mass spectrometry identified Poly(ADP-ribose) polymerase 1 (PARP-1) as one of the proteins interacting with BubR1. Reciprocal co-immunoprecipitation analysis confirmed the physical interaction between BubR1 and PARP-1. Our further study revealed that the ability of retaining intact PARP-1 or its cleavage product p89 was compromised in BubR1(+/-) MEFs upon treatment with doxorubicin or UV. Given that PARP-1 mediates DNA damage responses and regulates the activity of p53, our studies suggest that there exists a cross-talk between the spindle checkpoint and the DNA damage checkpoint and that BubR1 may play an important role in mediating the cross-talk
— id: 70084, year: 2006, vol: 25, page: 3598, stat: Journal Article,

Sequential phosphorylation of Ser-10 on histone H3 and ser-139 on histone H2AX and ATM activation during premature chromosome condensation: relationship to cell-cycle phase and apoptosis
Huang, Xuan; Kurose, Akira; Tanaka, Toshiki; Traganos, Frank; Dai, Wei; Darzynkiewicz, Zbigniew
2006 Apr;69(4):222-229, Cytometry A
BACKGROUND: Histone H1 and H3 phosphorylation associated with chromatin condensation during mitosis has been studied extensively. Less is known on histone modifications that occur during premature chromosome condensation (PCC). The aim of the present study was to reveal the status of histone H3 and H2AX phosphorylation on Ser-10 and Ser-139, respectively, as well as ATM activation through phosphorylation on Ser-1981, during PCC, and relate these events to cell-cycle phase and to initiation of apoptosis. MATERIALS AND METHODS: To induce PCC, A549 and HL-60 cells were exposed to the phosphatase inhibitor calyculin A (Cal A). Phosphorylation of histone H3 and H2AX as well as ATM activation were detected immunocytochemically concurrent with analysis of cellular DNA content and activation of caspase-3, a marker of apoptosis. The intensity of cellular fluorescence was measured by flow- or laser scanning cytometry. RESULTS: Induction of PCC led to rapid histone H3 phosphorylation, followed by activation of ATM and then H2AX phosphorylation in both, HL-60 and A549 cells. All these events occurred sequentially, prior to caspase-3 activation, and affected cells in all phases of the cell cycle. ATM activation and H2AX phosphorylation was seen during mitosis of A549 but not HL-60 cells. CONCLUSIONS: Because the Cal A-induced phosphorylation of histone H3 and H2AX, and of ATM, precede caspase-3 activation these modifications are pertinent to PCC and not to apoptosis-associated chromatin condensation. The sequence of histone H3 and H2AX phosphorylation and ATM activation during PCC is compatible with a role of ATM in mediating phosphorylation of H2AX but not H3. Mitosis in some cell types may proceed without ATM activation and H2AX phosphorylation
— id: 76368, year: 2006, vol: 69, page: 222, stat: Journal Article,

Polo box domain of Plk3 functions as a centrosome localization signal, overexpression of which causes mitotic arrest, cytokinesis defects, and apoptosis
Jiang, Ning; Wang, Xiaoxing; Jhanwar-Uniyal, Meena; Darzynkiewicz, Zbigniew; Dai, Wei
2006 Apr 14;281(15):10577-10582, Journal of biological chemistry
Polo-like kinase 3 (Plk3), an immediate early response gene product, plays an important role in the regulation of mitosis, DNA damage checkpoint activation, and Golgi dynamics. Similar to other members of the Plk family, Plk3 has a conserved kinase domain at the N terminus and a Polo box domain consisting of two Polo boxes at the C terminus. In this study, we demonstrate that the Polo box domain of Plk3 is sufficient for subcellular localization of this kinase to the centrosomes, the spindle poles, and the midbody when ectopically expressed in HeLa and U2OS cells. Both Polo boxes are required for the subcellular localization. Overexpression of the Polo box domain, not the kinase domain, of Plk3 causes significant cell cycle arrest and cytokinesis defects, eventually leading to mitotic catastrophe/apoptosis. Interestingly, the Polo box domain of Plk3 is more potent in inhibiting cell proliferation and inducing apoptosis than that of Plk1, suggesting that this domain can provide an additional structural basis for discovery of new anticancer drugs given the current emphasis on Plk1 as a therapeutic target
— id: 70083, year: 2006, vol: 281, page: 10577, stat: Journal Article,

Effects of hydroxyurea and aphidicolin on phosphorylation of ataxia telangiectasia mutated on Ser 1981 and histone H2AX on Ser 139 in relation to cell cycle phase and induction of apoptosis
Kurose, Akira; Tanaka, Toshiki; Huang, Xuan; Traganos, Frank; Dai, Wei; Darzynkiewicz, Zbigniew
2006 Apr;69(4):212-221, Cytometry A
BACKGROUND: DNA replication stress often induces DNA damage. The antitumor drug hydroxyurea (HU), a potent inhibitor of ribonucleotide reductase that halts DNA replication through its effects on cellular deoxynucleotide pools, was shown to damage DNA inducing double-strand breaks (DSBs). Aphidicolin (APH), an inhibitor of alpha-like DNA polymerases, was also reported to cause DNA damage, but the evidence for induction of DSBs by APH is not straightforward. Histone H2AX is phosphorylated on Ser 139 in response to DSBs and one of the protein kinases that phosphorylate H2AX is ataxia telangiectasia mutated (ATM); activation of ATM is through its phosphorylation of Ser 1981. The present study was undertaken to reveal whether H2AX is phosphorylated in cells exposed to HU or APH and whether its phosphorylation is mediated by ATM. MATERIALS AND METHODS: HL-60 cells were treated in cultures with 0.1-5.0 mM HU or 1-4 muM APH for up to 5 h. Activation of ATM and H2AX phosphorylation was detected immunocytochemically using Ab specific to Ser1981-ATM or Ser 139-H2AX epitopes, respectively, concurrent with measurement of cellular DNA content. RESULTS: While exposure of cells to HU led to H2AX phosphorylation selectively during S phase and the cells progressing through the early portion of S (DI = 1.1-1.4) were more affected than late-S phase (DI = 1.6-1.9) cells, ATM was not activated by HU. In fact, the level of constitutive ('programmed') ATM phosphorylation was distinctly suppressed, in all phases of the cell cycle, at 0.1-5.0 mM HU. Cells' exposure to APH also resulted in H2AX phosphorylation at Ser139 with no evidence of ATM activation, and as in the case of HU, the early-S cells were more affected than the late-S phase cells. The rise in frequency of apoptotic cells became apparent after 2 h of exposure to HU or APH, and all apoptotic cells had markedly elevated levels of both H2AX-Ser139 and ATM-Ser1981 phosphorylation. CONCLUSIONS: The lack of correlation between H2AX phosphorylation and ATM activation indicates that protein kinase(s) other than ATM (ATR and/or DNA-dependent protein kinase) are activated by DSBs induced by replication stress. Interestingly, HU inhibits the constitutive ('programmed') level of ATM phosphorylation in untreated cells. However, DNA fragmentation during apoptosis activates ATM and dramatically increases level of H2AX phosphorylation
— id: 76369, year: 2006, vol: 69, page: 212, stat: Journal Article,

ATM activation and histone H2AX phosphorylation as indicators of DNA damage by DNA topoisomerase I inhibitor topotecan and during apoptosis
Tanaka, T; Kurose, A; Huang, X; Dai, W; Darzynkiewicz, Z
2006 Feb;39(1):49-60, Cell proliferation
Damage that engenders DNA double-strand breaks (DSBs) activates ataxia telangiectasia mutated (ATM) kinase through its auto- or trans-phosphorylation on Ser1981 and activated ATM is one of the mediators of histone H2AX phosphorylation on Ser139. The present study was designed to explore: (i) whether measurement of ATM activation combined with H2AX phosphorylation provides a more sensitive indicator of DSBs than each of these events alone, and (ii) to reveal possible involvement of ATM activation in H2AX phosphorylation during apoptosis. Activation of ATM and/or H2AX phosphorylation in HL-60 or Jurkat cells treated with topotecan (Tpt) was detected immunocytochemically in relation to cell cycle phase, by multiparameter cytometry. Exposure to Tpt led to concurrent phosphorylation of ATM and H2AX in S-phase cells, whereas G1 cells were unaffected. Immunofluorescence (IF) of the S-phase cells immunostained for ATM-S1981P and gammaH2AX combined was distinctly stronger compared to that of the cells stained for each of these proteins alone. However, because of the relatively high ATM-S1981P IF of G1 cells, the ratio of IF of S to G1 cells, that is, the factor that determines competence of the assay in distinction of cells with DSBs, was 2- to 3-fold lower for ATM-S1981P alone, or for ATM-S1981P and gammaH2AX IF combined, than for gammaH2AX alone. ATM activation concurrent with H2AX phosphorylation, likely triggered by induction of DSBs during DNA fragmentation, occurred during apoptosis. The data suggest that frequency of activated ATM and phosphorylated H2AX molecules, per apoptotic cell, is comparable
— id: 76366, year: 2006, vol: 39, page: 49, stat: Journal Article,

Extent of constitutive histone H2AX phosphorylation on Ser-139 varies in cells with different TP53 status
Tanaka, T; Kurose, A; Huang, X; Traganos, F; Dai, W; Darzynkiewicz, Z
2006 Aug;39(4):313-323, Cell proliferation
In response to DNA damage by genotoxic agents, histone H2AX is phosphorylated on Ser-139. However, during the cell cycle, predominantly in S and G(2)M phase, histone H2AX is also phosphorylated in untreated normal and tumour cells. This constitutive H2AX phosphorylation is markedly reduced by exposure of cells to the reactive oxygen species scavenger N-acetyl-L-cysteine. Therefore, it appears likely that constitutive H2AX phosphorylation reflects the ongoing oxidative DNA damage induced by the reactive oxygen species during progression through the cell cycle. Because the tumour suppressor p53 (tumour protein p53) is known to induce transcription of genes associated with cell response to oxidative stress, we have compared the intensity of constitutive H2AX phosphorylation, and the effect of N-acetyl-L-cysteine on it, in cells with different tumour protein p53 status. These were human lymphoblastoid cell lines derived from WIL2 cells: TK6, a p53 wt line, NH32, a tumour protein p53 knock-out derived from TK6, and WTK1, a WIL2-derived line that expresses a homozygous mutant of tumour protein p53. Also tested were the tumour protein p53-null promyelocytic HL-60 cells. The degree of constitutive H2AX phosphorylation was distinctly lower in NH32, WTK1 and HL-60 compared to TK6 cells in all phases of the cell cycle. Also, the degree of attenuation of constitutive H2AX phosphorylation by N-acetyl-L-cysteine was less pronounced in NH32, WTK1, and HL-60, compared to TK6 cells. However, the level of reactive oxygen species detected by the cells' ability to oxidize carboxyl-dichlorodihydrofluorescein diacetate was not significantly different in the cell lines studied, which would suggest that regardless of tumour protein p53 status, the level of oxidative DNA damage was similar. The observed higher level of constitutive H2AX phosphorylation in cells harbouring wt tumour protein p53 may thus indicate that tumour protein p53 plays a role in facilitating histone H2AX phosphorylation, an important step in the mobilization of the DNA repair machinery at the site of DNA double-strand breaks
— id: 76365, year: 2006, vol: 39, page: 313, stat: Journal Article,

Assessment of ATM phosphorylation on Ser-1981 induced by DNA topoisomerase I and II inhibitors in relation to Ser-139-histone H2AX phosphorylation, cell cycle phase, and apoptosis
Kurose, Akira; Tanaka, Toshiki; Huang, Xuan; Halicka, H Dorota; Traganos, Frank; Dai, Wei; Darzynkiewicz, Zbigniew
2005 Nov;68(1):1-9, Cytometry A
BACKGROUND: The ATM kinase regulates cell-cycle checkpoints by phosphorylating multiple proteins, including histone H2AX, CHK1, and CHK2 kinases and p53. ATM is activated through auto- or trans- phosphorylation of Ser-1981 in response to DNA damage, particularly induction of DNA double-strand breaks (DSBs). The aim of the present study was to reveal a possible correlation between activation of ATM vis-a-vis H2AX phosphorylation, cell cycle phase, and apoptosis in cells treated with DNA topoisomerase (topo) I (topotecan; Tpt) or topo2 (mitoxantrone; Mtx) inhibitor. MATERIALS AND METHODS: Cultures of HL-60 cells were treated with Tpt or Mtx for various time intervals. ATM or H2AX phosphorylation was detected immunocytochemically, using Ab specific for ATM phosphorylated on Ser-1981 (ATM-S1981(P)) or for H2AX (gammaH2AX) phosphorylated on Ser-139, respectively, concurrent with the analysis of cellular DNA content. Cellular fluorescence was measured by flow cytometry. RESULTS: Untreated cells showed a modest but variable level of labeling with ATM-S1981(P) Ab across the cell cycle, with exception of mitotic cells that were strongly labeled. Exposure of cells to 150 nM Tpt induced ATM phosphorylation concurrent with phosphorylation of H2AX within 10 min; phosphorylation of both proteins was essentially limited to S-phase and was suppressed by caffeine and wortmannin, inhibitors of PI-3-like kinases. Exposure of cells to Mtx also led to ATM and H2AX phosphorylation, which, compared to Tpt, occurred later and was not cell-cycle-phase specific. Apoptosis of HL-60 cells in Tpt or Mtx treated cultures was detected after 2 or 4 h, respectively, and was limited to S-phase cells. CONCLUSIONS: The data are consistent with the role of ATM as a mediator of H2AX phosphorylation in response to DNA damage by topo1 (Tpt) or topo 2 (Mtx) inhibitor. The observed cell-cycle-phase related differences in response to Tpt vs Mtx suggest that while the collisions of DNA replication forks with the 'cleavable complexes' stabilized by topo1 inhibitor are the primary cause of DSBs induced by Tpt, the collisions of RNA polymerase molecules with the complexes stabilized by the topo2 inhibitor play a major role for induction of DSBs by Mtx. The present report is the first that (i) describes cytometric analysis of ATM activation and (ii) demonstrates activation of the enzyme (kinase) and its consequence (substrate phoshorylation), both in relation to cell-cycle phase and onset of apoptosis within the same cells
— id: 76370, year: 2005, vol: 68, page: 1, stat: Journal Article,

Colonic tumorigenesis in BubR1+/-ApcMin/+ compound mutant mice is linked to premature separation of sister chromatids and enhanced genomic instability
Rao, Chinthalapally V; Yang, Yang-Ming; Swamy, Malisetty V; Liu, Tongyi; Fang, Yuqiang; Mahmood, Radma; Jhanwar-Uniyal, Meena; Dai, Wei
2005 Mar 22;102(12):4365-4370, Proceedings of the National Academy of Sciences of the United States of America
Faithful chromosome segregation is essential for the maintenance of genetic stability during cell division and it is at least partly monitored by the spindle checkpoint, a surveillance mechanism preventing the cell from prematurely entering anaphase. The adenomatous polyposis coli (Apc) gene also plays an important role in regulating genomic stability, as mutations of Apc cause aneuploidy. Here we show that whereas Apc(Min)(/+) mice developed many adenomatous polyps, mostly in the small intestine, by 3 mo of age; BubR1(+/-)Apc(Min)(/+) compound mutant mice developed 10 times more colonic tumors than Apc(Min)(/+) mice. The colonic tumors in BubR1(+/-)Apc(Min)(/+) mice were in higher grades than those observed in Apc(Min)(/+) mice. Consistently, BubR1(+/-)Apc(Min)(/+) murine embryonic fibroblasts (MEFs) contained more beta-catenin and proliferated at a faster rate than WT or BubR1(+/-) MEFs. Moreover, BubR1(+/-)Apc(Min)(/+) MEFs slipped through mitosis in the presence of nocodazole and exhibited a higher rate of genomic instability than that of WT or BubR1(+/-) or Apc(Min)(/+) MEFs, accompanied by premature separation of sister chromatids. Together, our studies suggest that BubR1 and Apc functionally interact in regulating metaphase-anaphase transition, deregulation of which may play a key role in genomic instability and development and progression of colorectal cancer
— id: 70082, year: 2005, vol: 102, page: 4365, stat: Journal Article,

Ultraviolet irradiation-induced K(+) channel activity involving p53 activation in corneal epithelial cells
Wang, Ling; Dai, Wei; Lu, Luo
2005 Apr 21;24(18):3020-3027, Oncogene
Recent studies from our lab found that ultraviolet (UV) irradiation induces a voltage-gated potassium (Kv) channel activation and subsequently activates JNK signaling pathway resulting in apoptosis. The present study in rabbit corneal epithelial (RCE) cells is to investigate mechanisms of UV irradiation-induced Kv channel activity involving p53 activation in parallel to DNA damage-induced signaling pathway. UV irradiation-induced signaling events were characterized by measurements of JNK activation and further downstream p53 phosphorylation. UV irradiation elicited an early response in the cell membrane through activation of Kv channels to activate the JNK signaling pathway and p53 phosphorylation. Exposure of RCE cells to UV irradiation within a few min resulted in JNK and p53 activations that were markedly inhibited by suppression of Kv channel activity. However, suppression of Kv channel activity failed to prevent p53 activation induced by extended DNA damages through prolonging UV exposure time (more than 15 min). In addition, caffeine inhibited UV-induced activation of SEK, an upstream MAPK kinase of JNK, resulting in suppression of both Kv channel-involved and DNA damage-induced p53 activation. Our results indicate in these cells that UV irradiation induces earlier and later intracellular events that link to activation of JNK and p53. The early event in response to UV irradiation is initiated by activating Kv channels in the cell membrane, and the later event is predominated by UV irradiation-caused DNA damage
— id: 76362, year: 2005, vol: 24, page: 3020, stat: Journal Article,

Regulation of cell cycle checkpoints by polo-like kinases
Xie, Suqing; Xie, Bin; Lee, Marietta Y; Dai, Wei
2005 Jan 10;24(2):277-286, Oncogene
Protein kinases play a pivotal role in execution of cell division. Polo and Polo-like kinases have emerged as major regulators for various cell cycle checkpoints. Early genetic studies have demonstrated that CDC5, a budding yeast counterpart of vertebrate Plks, is essential for successful mitotic progression. Mammalian Plks localize primarily to the centrosome during interphase and the mitotic apparatus during mitosis. Many key cell cycle regulators such as p53, Cdc25C, cyclin B, components of the anaphase-promoting complex, and mitotic motor proteins are directly targeted by Plks. Although the exact mechanism of action of these protein kinases in vivo remains to be elucidated, Plks are important mediators for various cell cycle checkpoints that monitor centrosome duplication, DNA replication, formation of bipolar mitotic spindle, segregation of chromosomes, and mitotic exit, thus protecting cells against genetic instability during cell division
— id: 70081, year: 2005, vol: 24, page: 277, stat: Journal Article,

Slippage of mitotic arrest and enhanced tumor development in mice with BubR1 haploinsufficiency
Dai, Wei; Wang, Qi; Liu, Tongyi; Swamy, Malisetty; Fang, Yuqiang; Xie, Suqing; Mahmood, Radma; Yang, Yang-Ming; Xu, Ming; Rao, Chinthalapally V
2004 Jan 15;64(2):440-445, Cancer research
A compromised spindle checkpoint is thought to play a key role in genetic instability that predisposes cells to malignant transformation. Loss of function mutations of BubR1, an important component of the spindle checkpoint, have been detected in human cancers. Here we show that BubR1(+/-) mouse embryonic fibroblasts are defective in spindle checkpoint activation, contain a significantly reduced amount of securin and Cdc20, and exhibit a greater level of micronuclei than do wild-type cells. RNA interference-mediated down-regulation of BubR1 also greatly reduced securin level. Moreover, compared with wild-type littermates, BubR1(+/-) mice rapidly develop lung as well as intestinal adenocarcinomas in response to challenge with carcinogen. BubR1 is thus essential for spindle checkpoint activation and tumor suppression
— id: 70074, year: 2004, vol: 64, page: 440, stat: Journal Article,

BUBR1 deficiency results in abnormal megakaryopoiesis
Wang, Qi; Liu, Tongyi; Fang, Yuqiang; Xie, Suqing; Huang, Xuan; Mahmood, Radma; Ramaswamy, Gita; Sakamoto, Kathleen M; Darzynkiewicz, Zbigniew; Xu, Ming; Dai, Wei
2004 Feb 15;103(4):1278-1285, Blood
The physiologic function of BUBR1, a key component of the spindle checkpoint, was examined by generating BUBR1-mutant mice. BUBR1(-/-) embryos failed to survive beyond day 8.5 in utero as a result of extensive apoptosis. Whereas BUBR1(+/-) blastocysts grew relatively normally in vitro, BUBR1(-/-) blastocysts exhibited impaired proliferation and atrophied. Adult BUBR1(+/-) mice manifested splenomegaly and abnormal megakaryopoiesis. BUBR1 haploinsufficiency resulted in an increase in the number of splenic megakaryocytes, which was correlated with an increase in megakaryocytic, but a decrease in erythroid, progenitors in bone marrow cells. RNA interference-mediated down-regulation of BUBR1 also caused an increase in polyploidy formation in murine embryonic fibroblast cells and enhanced megakaryopoiesis in bone marrow progenitor cells. However, enhanced megakaryopoiesis in BUBR1(+/-) mice was not correlated with a significant increase in platelets in peripheral blood, which was at least partly due to a defect in the formation of proplatelet-producing megakaryocytes. Together, these results indicate that BUBR1 is essential for early embryonic development and normal hematopoiesis
— id: 70075, year: 2004, vol: 103, page: 1278, stat: Journal Article,

MEK1-induced Golgi dynamics during cell cycle progression is partly mediated by Polo-like kinase-3
Xie, Suqing; Wang, Qi; Ruan, Qin; Liu, Tongyi; Jhanwar-Uniyal, Meena; Guan, Kunliang; Dai, Wei
2004 May 6;23(21):3822-3829, Oncogene
MEK1, a gene product that regulates cell growth and differentiation, also plays an important role in Golgi breakdown during the cell cycle. We have recently shown that polo-like kinase (Plk3) is Golgi localized and involved in Golgi dynamics during the cell cycle. To study the mode of action of Plk3 in the Golgi fragmentation cascade, we examined functional as well as physical interactions between Plk3 and MEK1/ERKs. In HeLa cells, although a significant amount of Plk3 signals dispersed in a manner similar to those of Golgi during mitosis concentrated Plk3 was detected at spindle poles, which colocalized with phospho-MEKs and phospho-ERKs. Pull-down assays showed that Plk3 physically interacted with MEK1 and ERK2. Nocodazole activated Plk3 and its activation was blocked by MEK-specific inhibitors (PD98059 or U0126). Moreover, transfection of activated MEK1 resulted in an enhanced kinase activity of Plk3; Plk3-induced fragmentation of Golgi stacks was significantly reduced after treatment with MEK inhibitors. Consistently, ectopic expression of activated MEK1, but not kinase-dead MEK1(K97R), stimulated Plk3 to induce Golgi breakdown and the stimulation was not observed in cells expressing Plk3(K52R). Furthermore, PLK3(-/-) murine embryonic fibroblast cells exhibited a significantly less fragmentation of the Golgi complex than that in wild-type cells after exposed to nocodazole. Thus, our studies strongly suggest that Plk3 may be a key protein kinase mediating MEK1 function in the Golgi fragmentation pathway during cell division
— id: 70072, year: 2004, vol: 23, page: 3822, stat: Journal Article,