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Defective DNA double-strand break repair underlies enhanced tumorigenesis and chromosomal instability in p27-deficient mice with growth factor-induced oligodendrogliomas.

ABSTRACT: The tumor suppressive activities of the Kip-family of cyclin-dependent kinase (cdk) inhibitors often go beyond their role directly regulating the cell cycle. In this study, we show that p27 enhances Rad51 accumulation during repair of double-strand DNA breaks. Progression of platelet-derived growth factor (PDGF)-induced oligodendrogliomas was accelerated in mice lacking the cyclin-cdk binding activities of p27(kip1). To understand how p27 deficiency contributes, cell lines were developed from RCAS-PDGF infection of nestin-tv-a brain progenitor cells in culture. p27 deficiency did not affect cell proliferation in early passage cell lines; however, the absence of p27 affected chromosomal stability. In p27-deficient cells, the activation of Atm and Chk2 and the accumulation of gamma-H2AX was unaffected when compared with wild-type cells, and the number of phospho-histone H3 staining mitotic cells was decreased, consistent with G2/M checkpoint activation. However, the percentage of Rad51 foci-positive cells was decreased, and the kinase activity that targets the C-terminus of BRCA2, regulating BRCA2/Rad51 interactions, was increased in lysates derived from p27-deficient cells. Increased numbers of chromatid breaks in p27-deficient cells that adapted to the checkpoint were also observed. These findings suggest that Rad51-dependent repair of double-stranded breaks was hindered in p27-deficient cells, leading to chromosomal instability, a hallmark of cancers with poor prognosis.

SUBMITTER: See WL

PROVIDER: S-EPMC2845739 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Defective DNA double-strand break repair underlies enhanced tumorigenesis and chromosomal instability in p27-deficient mice with growth factor-induced oligodendrogliomas.

See W L WL Miller J P JP Squatrito M M Holland E E Resh M D MD Koff A A

Oncogene 20100111 12

The tumor suppressive activities of the Kip-family of cyclin-dependent kinase (cdk) inhibitors often go beyond their role directly regulating the cell cycle. In this study, we show that p27 enhances Rad51 accumulation during repair of double-strand DNA breaks. Progression of platelet-derived growth factor (PDGF)-induced oligodendrogliomas was accelerated in mice lacking the cyclin-cdk binding activities of p27(kip1). To understand how p27 deficiency contributes, cell lines were developed from RC ...[more]

PMID: 20062078

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Project description:BACKGROUND: Global hypomethylation and genomic instability are cardinal features of cancers. Recently, we established a method for the detection of DNA methylation levels at sites close to endogenous DNA double strand breaks (EDSBs), and found that those sites have a higher level of methylation than the rest of the genome. Interestingly, the most significant differences between EDSBs and genomes were observed when cells were cultured in the absence of serum. DNA methylation levels on each genomic location are different. Therefore, there are more replication-independent EDSBs (RIND-EDSBs) located in methylated genomic regions. Moreover, methylated and unmethylated RIND-EDSBs are differentially processed. Euchromatins respond rapidly to DSBs induced by irradiation with the phosphorylation of H2AX, gamma-H2AX, and these initiate the DSB repair process. During G0, most DSBs are repaired by non-homologous end-joining repair (NHEJ), mediated by at least two distinct pathways; the Ku-mediated and the ataxia telangiectasia-mutated (ATM)-mediated. The ATM-mediated pathway is more precise. Here we explored how cells process methylated RIND-EDSBs and if RIND-EDSBs play a role in global hypomethylation-induced genomic instability. RESULTS: We observed a significant number of methylated RIND-EDSBs that are retained within deacetylated chromatin and free from an immediate cellular response to DSBs, the gamma-H2AX. When cells were treated with tricostatin A (TSA) and the histones became hyperacetylated, the amount of gamma-H2AX-bound DNA increased and the retained RIND-EDSBs were rapidly repaired. When NHEJ was simultaneously inhibited in TSA-treated cells, more EDSBs were detected. Without TSA, a sporadic increase in unmethylated RIND-EDSBs could be observed when Ku-mediated NHEJ was inhibited. Finally, a remarkable increase in RIND-EDSB methylation levels was observed when cells were depleted of ATM, but not of Ku86 and RAD51. CONCLUSIONS: Methylated RIND-EDSBs are retained in non-acetylated heterochromatin because there is a prolonged time lag between RIND-EDSB production and repair. The rapid cellular responses to DSBs may be blocked by compact heterochromatin structure which then allows these breaks to be repaired by a more precise ATM-dependent pathway. In contrast, Ku-mediated NHEJ can repair euchromatin-associated EDSBs. Consequently, spontaneous mutations in hypomethylated genome are produced at faster rates because unmethylated EDSBs are unable to avoid the more error-prone NHEJ mechanisms.

Dataset Information

Defective DNA double-strand break repair underlies enhanced tumorigenesis and chromosomal instability in p27-deficient mice with growth factor-induced oligodendrogliomas.

Publications

Defective DNA double-strand break repair underlies enhanced tumorigenesis and chromosomal instability in p27-deficient mice with growth factor-induced oligodendrogliomas.

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