Inhibition of poly(ADP-ribose) polymerase down-regulates BRCA1 and RAD51 in a pathway mediated by E2F4 and p130.

Inhibitors of poly(ADP-ribose) polymerase (PARP) are in clinical trials for cancer therapy, on the basis of the role of PARP in recruitment of base excision repair (BER) factors to sites of DNA damage. Here we show that PARP inhibition to block BER is toxic to hypoxic cancer cells, in which homology-dependent repair (HDR) is known to be down-regulated. However, we also report the unexpected finding that disruption of PARP, itself, either via chemical PARP inhibitors or siRNAs targeted to PARP-1, can inhibit HDR by suppressing expression of BRCA1 and RAD51, key factors in HDR of DNA breaks. Mechanistically, PARP inhibition was found to cause increased occupancy of the BRCA1 and RAD51 promoters by repressive E2F4/p130 complexes, a pathway prevented by expression of HPV E7, which disrupts p130 activity, or by siRNAs to knock down p130 expression. Functionally, disruption of p130 by E7 expression or by siRNA knockdown also reverses the cytotoxicity and radiosensitivity associated with PARP inhibition, suggesting that the down-regulation of BRCA1 and RAD51 is central to these effects. Direct measurement of HDR using a GFP-based assay demonstrates reduced HDR in cells treated with PARP inhibitors. This work identifies a mechanism by which PARP regulates DNA repair and suggests new strategies for combination cancer therapies.

Differing patterns of genetic instability in mice deficient in the mismatch repair genes Pms2, Mlh1, Msh2, Msh3 and Msh6.

Defects in genes associated with DNA mismatch repair (MMR) have been linked to hereditary colon cancer. Because the MMR pathway includes multiple factors with both overlapping and divergent functions, we sought to compare the impact of deficiencies in each of several MMR genes on genetic instability using a collection of knock-out mouse models. We investigated mutation frequencies and patterns in MMR-deficient mice using two transgenic reporter genes, supFG1 and cII, in the context of mice deficient for Pms2, Mlh1, Msh2, Msh3 or Msh6 or both Msh2 and Msh3 or both Msh3 and Msh6. We found that the mean mutation frequencies of all of the MMR-deficient mice were significantly higher than the mean mutation frequencies of wild-type mice. Mlh1-deficient mice and Msh2-deficient mice had the highest mutation frequencies in a comparison of the single nullizygous mice. Of all the mice studied, mice nullizygous for both Msh2 and Msh3 and those nullizygous for both Msh3 and Msh6 displayed the greatest overall increases in mutation frequencies compared with wild-type mice. Sequence analysis of the mutated reporter genes revealed significant differences between the individual groups of MMR-deficient mice. Taken together, our results further characterize the functions of the MMR factors in mutation avoidance and provide in vivo correlation to biochemical models of the MMR pathway.

Overexpression of the DNA mismatch repair factor, PMS2, confers hypermutability and DNA damage tolerance.

Inherited defects in genes associated with DNA mismatch repair (MMR) have been linked to familial colorectal cancer. Cells deficient in MMR are genetically unstable and demonstrate a tolerance phenotype in response to certain classes of DNA damage. Some sporadic human cancers also show abnormalities in MMR gene function, typically due to diminished expression of one of the MutL homologs, MLH1. Here, we report that overexpression of the MutL homolog, human PMS2, can also cause a disruption of the MMR pathway in mammalian cells, resulting in hypermutability and DNA damage tolerance. A mouse fibroblast cell line carrying a recoverable lambda phage shuttle vector for mutation detection was transfected with either a vector designed to express hPMS2 or with an empty vector control. Cells overexpressing hPMS2 were found to have elevated spontaneous mutation frequencies at the cII reporter gene locus. They also showed an increase in the level of mutations induced by the alkylating agent, methynitrosourea (MNU). Clonogenic survival assays demonstrated increased survival of the PMS2-overexpressing cells following exposure to MNU, consistent with the induction of a damage tolerance phenotype. Similar results were seen in cells expressing a mutant PMS2 gene, containing a premature stop codon at position 134 and representing a variant found in an individual with familial colon cancer. These results show that dysregulation of PMS2 gene expression can disrupt MMR function in mammalian cells and establish an additional carcinogenic mechanism by which cells can develop genetic instability and acquire resistance to cytotoxic cancer therapies.

Induction of aberrant crypt foci in DNA mismatch repair-deficient mice by the food-borne carcinogen 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP).

Disruption of the DNA mismatch repair (MMR) pathway results in elevated mutation rates, inappropriate survival of cells bearing DNA damage, and increased cancer risk. Relatively little is known about the impact of environmentally relevant carcinogens on cancer risk in individuals with MMR-deficiency. We evaluated the effect of MMR status (Mlh1(+/+) versus Mlh1(-/-)) on the carcinogenic potential of the cooked-meat mutagen, 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP) in mice. PhIP exposure did not obviously increase lymphoma or small intestinal tumorigenesis in either Mlh1-deficient or -proficient mice. In contrast, the frequency of aberrant crypt foci (ACF), a preneoplastic biomarker for colon tumorigenesis, was increased by PhIP, and the increase due to PhIP was significantly greater in Mlh1(-/-) versus wild-type littermates. This apparent heightened susceptibility to induction of ACF parallels the previously reported hypermutability of Mlh1-deficient mice to PhIP and is consistent with the hypothesis that MMR-deficiency would increase the likelihood of PhIP-induced carcinogenic mutations. Further evaluation of the risk that consumption of heterocyclic amines may impart to MMR-deficient individuals therefore is warranted.

Mlh1-dependent suppression of specific mutations induced in vivo by the food-borne carcinogen 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP).

Disruption of the DNA mismatch repair (MMR) pathway results in elevated mutation rates, inappropriate survival of cells bearing DNA damage, and increased cancer risk. Relatively little is known about the potential impact of environmentally relevant carcinogens on cancer risk in individuals with MMR-deficiency. We determined the effect of MMR status (Mlh1+/+ versus Mlh1-/-) on mutagenesis induced by the cooked-meat mutagen, 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP) within cII and supFG1 transgene reporters. Despite being a lymphomagen in mice, PhIP was not mutagenic in thymus. In colon, PhIP exposure induced 3-fold more mutations in Mlh1-deficient mice compared to their Mlh1+/+ littermates. Similar induction was seen in Mlh1-/- small intestine. Analysis of mutational spectra revealed that G/C to T/A transversions, the "signature PhIP mutation", were induced to similar levels regardless of Mlh1 status. In contrast, Mlh1-/- mice exhibited hypermutability to frameshifts, G/C to A/T transitions, and G/C to C/G transversions. Thus, both the level and types of mutation induced by PhIP are influenced by the activity of the MMR system. MMR may suppress PhIP-induced mutation through recognition and processing of specific mispairs (PhIP-G/T, PhIP-G/G, and PhIP-G/loop mispairs). In contrast, the PhIP-G/A mispair is unlikely to be a MMR substrate. In addition, the similar induction of both transversions and transitions in Mlh1-/- mice suggests that mutagenic bypass of PhIP-G is similarly efficient with dATP, dTTP, and dGTP, in contrast to previously published conclusions. Our data suggests that MMR-deficiency would increase the likelihood of PhIP-induced carcinogenic mutations. Further evaluation of the risk that consumption of heterocyclic amines may impart to MMR-deficient individuals therefore is warranted.