Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Trabectedin (Yondelis; ET-743) is a potent anticancer drug that binds to DNA by forming a covalent bond with a guanine in one strand and one or more hydrogen bonds with the opposite strand. Using a fluorescence-based melting assay, we show that one single trabectedin-DNA adduct increases the thermal stability of the double helix by >20 degrees C. As deduced from the analysis of phosphorylated H2AX and Rad51 foci, we observed that clinically relevant doses of trabectedin induce the formation of DNA double-strand breaks in human cells and activate homologous recombination repair in a manner similar to that evoked by the DNA interstrand cross-linking agent mitomycin C (MMC). Because one important characteristic of this drug is its marked cytotoxicity on cells lacking a functional Fanconi anemia (FA) pathway, we compared the response of different subtypes of FA cells to MMC and trabectedin. Our data clearly show that human cells with mutations in FANCA, FANCC, FANCF, FANCG, or FANCD1 genes are highly sensitive to both MMC and trabectedin. However, in marked contrast to MMC, trabectedin does not induce any significant accumulation of FA cells in G2-M. The critical relevance of FA proteins in the response of human cells to trabectedin reported herein, together with observations showing the role of the FA pathway in cancer suppression, strongly suggest that screening for mutations in FA genes may facilitate the identification of tumors displaying enhanced sensitivity to this novel anticancer drug.
Mol Cancer Ther 2008 May
PMID:Relevance of the Fanconi anemia pathway in the response of human cells to trabectedin. 1848 18

p53R2 is a p53-inducible ribonucleotide reductase that contributes to DNA repair by supplying deoxynucleotide triphosphate pools in response to DNA damage. In this study, we found that p53R2 was overexpressed in prostate tumor cell lines compared with immortalized prostatic epithelial cells and that the protein was induced upon DNA damage. We investigated the effects of p53R2 silencing on DNA damage in LNCaP cells (wild-type p53). Silencing p53R2 potentiated the apoptotic effects of ionizing radiation and doxorubicin treatment as shown by increased sub-G(1) content and decreased colony formation. This sensitizing effect was specific to DNA-damaging agents. Comet assay and gamma-H2AX phosphorylation status showed that the decreased p53R2 levels inhibited DNA repair. Silencing p53R2 also reduced the levels of p21(WAF1/CIP1) at the posttranscriptional level, suggesting links between the p53-dependent DNA repair and cell cycle arrest pathways. Using LNCaP sublines stably expressing dominant-negative mutant p53, we found that the sensitizing effect of p53R2 silencing is mediated by p53-dependent apoptosis pathways. In the LNCaP sublines (R273H, R248W, and G245S) that have defects in inducing p53-dependent apoptosis, p53R2 silencing did not potentiate DNA damage-induced apoptosis, whereas p53R2 silencing was effective in a LNCaP subline (P151S) which retains the ability to induce p53-dependent apoptosis. This study shows that p53R2 is a potential therapeutic target that could be used to enhance the effectiveness of ionizing radiation or DNA-damaging chemotherapy in a subset of patients with prostate cancer.
Mol Cancer Res 2008 May
PMID:Impairment of the DNA repair and growth arrest pathways by p53R2 silencing enhances DNA damage-induced apoptosis in a p53-dependent manner in prostate cancer cells. 1850 25

The differential diagnosis of metastatic renal cell carcinoma (RCC) includes, although is not limited to, hepatocellular carcinoma (HCC) and adrenocortical carcinoma (ACC) due to overlapping morphology. Immunohistochemical markers, including RCC marker (RCC-Ma) have been employed with varying success in the differential diagnosis of RCC. Our preliminary tissue microarray study demonstrated that gamma-H2AX, an antibody that specifically reacts with phosphorylated histone H2AX, stained many primary RCC strongly and did not stain HCC or ACC, prompting us to evaluate its utility in these tumors and to compare it with RCC-Ma. Seventy-one cases of metastatic RCC, 18 HCC, and 21 ACC were stained with gamma-H2AX and RCC-Ma and the sensitivity and specificity of each marker was compared. RCC-Ma demonstrated a membranous pattern of staining in 70% of RCC cases (50/71), and none of the ACC or HCC (100% specificity for RCC). Nuclear staining by gamma-H2AX had a similar sensitivity of 70% for RCC but a lower specificity of 77%, as it was seen in 1 of 18 HCC (5%) and 8 of 21 (38%)ACC. In metastatic RCC, 83% (39/47) of tumors with a higher nuclear grade stained with gamma-H2AX, compared with 46% (11/24) of low nuclear grade (equivalent of Fuhrman 2 and lower) tumors. RCC-Ma had a similar rate of staining in low and high-grade tumors, 75% (18/24) and 68% (32/47), respectively. More importantly, of RCCs that were negative for RCC-Ma, 14 of 21 (67%) were positive for gamma-H2AX. The results suggest gamma-H2AX is a useful adjunct in diagnosis of metastatic RCC when RCC-Ma is negative and in higher grade RCC, which are often a diagnostic challenge. A nuclear pattern of staining of gamma-H2AX has a comparable sensitivity with RCC-Ma, and the interpretation is easier and more reliable. RCC-Ma is 100% specific for RCC, but only when a membranous pattern of staining is interpreted as positive.
Appl Immunohistochem Mol Morphol 2008 Jul
PMID:Utility of antiphosphorylated H2AX antibody (gamma-H2AX) in diagnosing metastatic renal cell carcinoma. 1852 82

Mdm2, a regulator of the tumor suppressor p53, is frequently overexpressed in human malignancies. Mdm2 also has unresolved, p53-independent functions that contribute to tumorigenesis. Here, we show that increased Mdm2 expression induced chromosome/chromatid breaks and delayed DNA double-strand break repair in cells lacking p53 but not in cells with a mutant form of Nbs1, a component of the Mre11/Rad50/Nbs1 DNA repair complex. A 31-amino-acid region of Mdm2 was necessary for binding to Nbs1. Mutation of conserved amino acids in the Nbs1 binding domain of Mdm2 inhibited Mdm2-Nbs1 association and prevented Mdm2 from delaying phosphorylation of H2AX and ATM-S/TQ sites, repair of DNA breaks, and resolution of DNA damage foci. Similarly, the mutation of eight amino acids in the Mdm2 binding domain of Nbs1 inhibited Mdm2-Nbs1 interaction and blocked the ability of Mdm2 to delay DNA break repair. Both Nbs1 and ATM, but not the ubiquitin ligase activity of Mdm2, were necessary to inhibit DNA break repair. Only Mdm2 with an intact Nbs1 binding domain was able to increase the frequency of chromosome/chromatid breaks and the transformation efficiency of cells lacking p53. Therefore, the interaction of Mdm2 with Nbs1 inhibited DNA break repair, leading to chromosome instability and subsequent transformation that was independent of p53.
Mol Cell Biol 2008 Aug
PMID:Mdm2 promotes genetic instability and transformation independent of p53. 1854 70

Camptothecins (CPT) activate S or G(2)-M arrest and the homologous recombination (HR) repair pathway in tumor cells. In this process, both checkpoint kinases 1 and 2 (Chk1 and Chk2, respectively) are activated, but their differential roles, especially in the coordination of checkpoint and repair control, and potential clinic relevance remain to be fully elucidated. In this study, the repairable double-strand breaks were induced in human colon cancer HCT116 cells by 1-h exposure to 25 or 100 nmol/L CPT and its novel derivative chimmitecan. The cellular disposal of double-strand breaks was reflected as the progressive dispersal of gamma-H2AX foci, reduction of "comet" tails, dynamic activation of RAD51-mediated HR repair, and reversible G(2)-M arrest. In this model, the differential kinetics of Chk1 and Chk2 activation was characterized by the progressively increased phosphorylation of Chk2 until 72 h, the degradation of Chk1, and the disappearance of phosphorylated Chk1 48 h after drug removal. Using RNA interference, we further showed that Chk2 was essential to G(2)-M arrest, whereas Chk1 was mainly required for HR repair in CPT-treated HCT116 cells. Moreover, Chk2, rather than Chk1, predominated over the control of cell survival in this model. The differential roles of Chk1 and Chk2 in regulating HR repair and G(2)-M phase arrest were also confirmed in HT-29 colon cancer cells. Together, these findings systematically dissect the differential roles of Chk1 and Chk2 in a favorable model pursuing CPT-driven DNA damage responses, providing critical evidence to further explore checkpoint modulation, especially Chk2 inhibition as a therapeutic strategy in combination with CPT.
Mol Cancer Ther 2008 Jun
PMID:Chk1 and Chk2 are differentially involved in homologous recombination repair and cell cycle arrest in response to DNA double-strand breaks induced by camptothecins. 1856 16

Oncogenes can induce p53 through a signaling pathway involving p19/Arf. It was recently proposed that oncogenes can also induce DNA damage, and this can induce p53 through the Atm DNA damage pathway. To assess the relative roles of Atm, Arf, and p53 in the suppression of Ras-driven tumors, we examined susceptibility to skin carcinogenesis in 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate (TPA)-treated Atm- and p53-deficient mice and compared these results to previous studies on Arf-deficient mice. Mice with epidermal-specific deletion of p53 showed increased papilloma number and progression to malignant invasive carcinomas compared with wild-type littermates. In contrast, Atm-deficient mice showed no increase in papilloma number, growth, or malignant progression. gamma-H2AX and p53 levels were increased in both Atm(+/+) and Atm(-/-) papillomas, whereas Arf(-/-) papillomas showed much lower p53 expression. Thus, although there is evidence of DNA damage, signaling through Arf seems to regulate p53 in these Ras-driven tumors. In spontaneous and radiation-induced lymphoma models, tumor latency was accelerated in Atm(-/-)p53(-/-) compound mutant mice compared with the single mutant Atm(-/-) or p53(-/-) mice, indicating cooperation between loss of Atm and loss of p53. Although p53-mediated apoptosis was impaired in irradiated Atm(-/-) lymphocytes, p53 loss was still selected for during lymphomagenesis in Atm(-/-) mice. In conclusion, in these models of oncogene- or DNA damage-induced tumors, p53 retains tumor suppressor activity in the absence of Atm.
Mol Cancer Res 2008 Jul
PMID:Tumor suppression by p53 in the absence of Atm. 1858 27

Werner syndrome (WS) is a human genetic disorder characterized by extensive clinical features of premature aging. Ataxia-telengiectasia (A-T) is a multisystem human genomic instability syndrome that includes premature aging in some of the patients. WRN and ATM, the proteins defective in WS and A-T, respectively, play significant roles in the maintenance of genomic stability and are involved in several DNA metabolic pathways. A role for WRN in DNA repair has been proposed; however, this study provides evidence that WRN is also involved in ATM pathway activation and in a S-phase checkpoint in cells exposed to DNA interstrand cross-link-induced double-strand breaks. Depletion of WRN in such cells by RNA interference results in an intra-S checkpoint defect, and interferes with activation of ATM as well as downstream phosphorylation of ATM target proteins. Treatment of cells under replication stress with the ATM kinase inhibitor KU 55933 results in a S-phase checkpoint defect similar to that observed in WRN shRNA cells. Moreover, gamma H2AX levels are higher in WRN shRNA cells than in control cells 6 and 16 h after exposure to psoralen DNA cross-links. These results suggest that WRN and ATM participate in a replication checkpoint response, in which WRN facilitates ATM activation in cells with psoralen DNA cross-link-induced collapsed replication forks.
Mol Biol Cell 2008 Sep
PMID:WRN is required for ATM activation and the S-phase checkpoint in response to interstrand cross-link-induced DNA double-strand breaks. 1859 39

MAD2 (mitotic arrest deficient 2) is a key regulator of mitosis. Recently, it had been suggested that MAD2-induced mitotic arrest mediates DNA damage response and that upregulation of MAD2 confers sensitivity to DNA-damaging anticancer drug-induced apoptosis. In this study, we report a potential novel role of MAD2 in mediating DNA nucleotide excision repair through physical interactions with two DNA repair proteins, XPD (xeroderma pigmentosum complementation group D) and ERCC1. First, overexpression of MAD2 resulted in decreased nuclear accumulation of XPD, a crucial step in the initiation of DNA repair. Second, immunoprecipitation experiments showed that MAD2 was able to bind to XPD, which led to competitive suppression of binding activity between XPD and XPA, resulting in the prevention of physical interactions between DNA repair proteins. Third, unlike its role in mitosis, the N-terminus domain seemed to be more important in the binding activity between MAD2 and XPD. Fourth, phosphorylation of H2AX, a process that is important for recruitment of DNA repair factors to DNA double-strand breaks, was suppressed in MAD2-overexpressing cells in response to DNA damage. These results suggest a negative role of MAD2 in DNA damage response, which may be accounted for its previously reported role in promoting sensitivity to DNA-damaging agents in cancer cells. However, the interaction between MAD2 and ERCC1 did not show any effect on the binding activity between ERCC1 and XPA in the presence or absence of DNA damage. Our results suggest a novel function of MAD2 by interfering with DNA repair proteins.
J Mol Biol 2008 Aug 01
PMID:MAD2 interacts with DNA repair proteins and negatively regulates DNA damage repair. 1859 77

The histone H2A variant H2AX is rapidly phosphorylated in response to DNA double-stranded breaks to produce gamma-H2AX. gamma-H2AX stabilizes cell-cycle checkpoint proteins and DNA repair factors at the break site. We previously found that the protein phosphatase PP2A is required to resolve gamma-H2AX foci and complete DNA repair after exogenous DNA damage. Here we describe a three-protein PP4 phosphatase complex in mammalian cells, containing PP4C, PP4R2, and PP4R3beta, that specifically dephosphorylates ATR-mediated gamma-H2AX generated during DNA replication. PP4 efficiently dephosphorylates gamma-H2AX within mononucleosomes in vitro and does not directly alter ATR or checkpoint kinase activity, suggesting that PP4 acts directly on gamma-H2AX in cells. When the PP4 complex is silenced, repair of DNA replication-mediated breaks is inefficient, and cells are hypersensitive to DNA replication inhibitors, but not radiomimetic drugs. Therefore, gamma-H2AX elimination at DNA damage foci is required for DNA damage repair, but accomplishing this task involves distinct phosphatases with potentially overlapping roles.
Mol Cell 2008 Jul 11
PMID:A PP4-phosphatase complex dephosphorylates gamma-H2AX generated during DNA replication. 1861 45

The cellular activity of Yondelis (trabectedin, Ecteinascidin 743, Et743) is known to depend on transcription-coupled nucleotide excision repair (TCR). However, the subsequent cellular effects of Et743 are not fully understood. Here we show that Et743 induces both transcription- and replication-coupled DNA double-strand breaks (DSBs) that are detectible by neutral COMET assay and as gamma-H2AX foci that colocalize with 53BP1, Mre11, Ser(1981)-pATM, and Thr(68)-pChk2. The transcription coupled-DSBs (TC-DSBs) induced by Et743 depended both on TCR and Mre11-Rad50-Nbs1 (MRN) and were associated with DNA-PK-dependent gamma-H2AX foci. In contrast to DNA-PK, ATM phosphorylated H2AX both in NER-proficient and -deficient cells, but its full activation was dependent on H2AX as well as DNA-PK, suggesting a positive feedback loop: DNA-PK-gamma-H2AX-ATM. Knocking-out H2AX or inactivating DNA-PK reduced Et743's antiproliferative activity, whereas ATM and MRN tended to act as survival factors. Our results highlight the interplays between ATM and DNA-PK and their impacts on H2AX phosphorylation and cell survival. They also suggest that gamma-H2AX may serve as a biomarker in patients treated with Et743 and that molecular profiling of tumors for TCR, MRN, ATM, and DNA-PK might be useful to anticipate tumor response to Et743 treatment.
Mol Biol Cell 2008 Sep
PMID:Transcription-coupled DNA double-strand breaks are mediated via the nucleotide excision repair and the Mre11-Rad50-Nbs1 complex. 1863 84


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