Chronic Hypoxia Decreases Synthesis of Homologous Recombination Proteins to Offset Chemoresistance and Radioresistance

Abstract

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]

Hypoxia Provokes Base Excision Repair Changes and a Repair-Deficient, Mutator Phenotype in Colorectal Cancer Cells

“…The residual 8-oxo-dG accumulated under hypoxic conditions is therefore probably a result of impaired BER and MMR pathways. It has previously been documented that hypoxiapretreated cells have increased radiosensitivity and at the time this was attributed to a hypoxia-mediated defect in HR (23). However, in light of these findings, decreased BER may also play a role.…”

“…Logarithmically growing cells were exposed to 0.2% O 2 with 5% CO 2 and balance N 2 using an Invivo2 400 Hypoxic Workstation (Ruskinn). The chronic hypoxic gassing condition (72 hours with 0.2% O 2 ) was based on previous studies in which the translation of DNA repair genes was downregulated by hypoxia (23,45). This gassing condition for RKO and HCT116 cells was optimal for (i) minimizing toxicity (e.g., 35%-50% cell kill based on clonogenic survival assays); (ii) no effect on S-phase fraction or cell proliferation; and (iii) maintaining the inhibitory effect of hypoxia on protein translation (see Supplementary Fig.…”

“…Protein expression was determined by Western blot analysis based on a previously described protocol (23 RNA isolation, reverse transcription, and qRT-PCR Total RNA was isolated using the RNeasy isolation kit (Qiagen). Sample RNA or human reference RNA (Stratagene) was treated with DNase I (Roche Diagnostics).…”

“…Hypoxic tumor cells exist in colorectal cancer, and the level of hypoxia is prognostic for clinical outcome (10)(11)(12)(13)(14)(15)(16)(17)(18)(19). Hypoxia may give rise to genetic instability by downregulating multiple DNA repair pathways, including the DNA mismatch repair (MMR) and homologous recombination (HR) pathways (20)(21)(22)(23)(24)(25)(26)(27). For example, Chan and colleagues demonstrated an alternate mechanism of translational inhibition of the HR proteins RAD51 and BRCA2 (23).…”

“…Hypoxia may give rise to genetic instability by downregulating multiple DNA repair pathways, including the DNA mismatch repair (MMR) and homologous recombination (HR) pathways (20)(21)(22)(23)(24)(25)(26)(27). For example, Chan and colleagues demonstrated an alternate mechanism of translational inhibition of the HR proteins RAD51 and BRCA2 (23). Suppression of mRNA translation under hypoxia is controlled through at least two distinct pathways; first by PERK-mediated phosphorylation of eIF2a which is required for the recruitment of aminoacylated tRNA, and second by disruption of the mRNA capbinding complex, eIF4F (28).…”