Hypoxic and/or anoxic tumor cells can have increased rates of mutagenesis and altered DNA repair protein expression. Yet very little is known regarding the functional consequences of any hypoxia-induced changes in the expression of proteins involved in DNA double-strand break repair. We have developed a unique hypoxic model system using H1299 cells expressing an integrated direct repeat green fluorescent protein (DR-GFP) homologous recombination (HR) reporter system to study HR under prolonged chronic hypoxia (up to 72 h under 0.2% O 2 ) without bias from altered proliferation, cell cycle checkpoint activation, or severe cell toxicity. We observed decreased expression of HR proteins due to a novel mechanism involving decreased HR protein synthesis. Error-free HR was suppressed 3-fold under 0.2% O 2 as measured by the DR-GFP reporter system. This decrease in functional HR resulted in increased sensitivity to the DNA cross-linking agents mitomycin C and cisplatin but not to the microtubule-interfering agent, paclitaxel. Chronically hypoxic H1299 cells that had decreased functional HR were relatively radiosensitive [oxygen enhancement ratio (OER), 1.37] when compared with acutely hypoxic or anoxic cells (OER, 1.96-2.61). Using CAPAN1 cells isogenic for BRCA2 and siRNA to RAD51, we confirmed that the hypoxia-induced radiosensitivity was due to decreased HR capacity. Persistent down-regulation of HR function by the tumor microenvironment could result in low-fidelity DNA repair and have significant implications for response to therapy and genetic instability in human cancers. [Cancer Res 2008;68(2):605-14]

The breast/ovarian cancer susceptibility proteins BRCA1 and BRCA2 maintain genome stability, at least in part, through a functional role in DNA damage repair. They both colocalize with RAD51 at sites of DNA damage/replication and activate RAD51-mediated homologous recombination repair of DNA double-strand breaks (DSB). Whereas BRCA2 interacts directly with and regulates RAD51, the role of BRCA1 in this process is unclear. However, BRCA1 may regulate RAD51 in response to DNA damage or through its ability to interact with and regulate MRE11/RAD50/NBS1 (MRN) during the processing of DSBs into single-strand DNA (ssDNA) ends, prerequisite substrates for RAD51, or both. To test these hypotheses, we measured the effect of BRCA1 on the competition between RAD51-mediated homologous recombination (gene conversion and crossover) versus RAD51-independent homologous recombination [single-strand annealing (SSA)] for ssDNA at a site-specific chromosomal DSB within a DNA repeat, a substrate for both homologous recombination pathways. Expression of wild-type BRCA1 in BRCA1-deficient human recombination reporter cell lines promoted both gene conversion and SSA but greatly enhanced gene conversion. In addition, BRCA1 also suppressed both spontaneous gene conversion and deletion events, which can arise from either crossover or sister chromatid replication slippage (SCRS), a RAD51-independent process. BRCA1 does not seem to block crossover. From these results, we conclude that (a) BRCA1 regulates RAD51 function in response to the type of DNA damage and (b) BRCA1 suppresses SCRS, suggesting a role for this protein in sister chromatid cohesion/alignment. Loss of such control in response to estrogen-induced DNA damage after BRCA1 inactivation may be a key initial event that triggers genome instability and carcinogenesis. (Cancer Res 2005; 65(24): 11384-91)

BRCA2 has been implicated in the maintenance of genome stability and RAD51-mediated homologous recombination repair of chromosomal double-strand breaks (DSBs), but its role in these processes is unclear. To gain more insight into its role in homologous recombination, we expressed wild-type BRCA2 in the well-characterized BRCA2-deficient human cell line CAPAN-1 containing, as homologous recombination substrates, either direct or inverted repeats of two inactive marker genes. Whereas direct repeats monitor a mixture of RAD51-dependent and RAD51-independent homologous recombination events, inverted repeats distinguish between these events by reporting RAD51-dependent homologous recombination, gene conversion, and crossover events only. At either repeats, BRCA2 decreases the rate and frequency of spontaneous homologous recombination, but following chromosomal DSBs, BRCA2 increases the frequency of homologous recombination. At direct repeats, BRCA2 suppresses both spontaneous gene conversion and deletions, which can arise either from crossover or RAD51-independent sister chromatid replication slippage (SCRS), but following chromosomal DSBs, BRCA2 highly promotes gene conversion with little effect on deletions. At inverted repeats, spontaneous or DSB-induced crossover events were scarce and BRCA2 does not suppress their formation. From these results, we conclude that (i) BRCA2 regulates RAD51 recombination in response to the type of DNA damage and (ii) BRCA2 suppresses SCRS, suggesting a role for BRCA2 in sister chromatids cohesion and/or alignment. Loss of such control in response to estrogeninduced DNA damage after BRCA2 inactivation may be a key initial event triggering genome instability and carcinogenesis. (Cancer Res 2005; 65(10): 4117-25)