While investigating sensitivity of hypoxic cells to various therapeutic agents we observed that HIF1-alpha mRNA expression is increased during hypoxia. We hypothesized that NF-kB may transcriptionally upregulate HIF1-alpha during hypoxia. We performed ChIP assays and found that hypoxia induces recruitment of NF-kB p65 to the HIF1-alpha promoter. Following exposure to hypoxia, there was increased p65 localization in the nucleus. Using real-time PCR we found that Jurkat mutant IkB-expressing cells that overexpress p65 in the cytoplasm have decreased levels of HIF1-alpha as compared to wild-type Jurkat cells, either during normoxia or hypoxia. We tested the influence of p65 knockdown on cell survival after treatment of wild-type p53-expressing or p53-null HCT116 colon cancer cells by CPT11 or TRAIL and exposure to hypoxia. While both p53+/+ and p53-/- HCT116 cells showed sensitivity to TRAIL, the cells were relatively resistant to CPT11 under normoxic conditions. Under hypoxia, as we previously observed (Mayes et al., Cancer Biology and Therapy 4:1068-1074, 2005), p53-null HCT116 (and not p53+/+ cells) exhibit greatly reduced TRAIL-induced apoptosis. p65 knockdown did not enhance TRAIL sensitivity under normoxic or hypoxic conditions. Strikingly, inhibition of p65 in CPT11-resistant p53+/+ HCT116 cells significantly increased their sensitivity to CPT11 during hypoxia. This effect was diminished in p53-null HCT116 cells. Our results indicate that wild-type p53-expressing HCT116 cells are resistant to CPT11 in normoxia as well as hypoxia and that p65 knockdown results in increased CPT11-sensitivity in hypoxic HCT116 cells. From these findings we postulate that one of the mechanisms by which hypoxic p53-wild-type HCT116 cells become resistant to CPT11 may involve the expression of HIF1-alpha via transcriptional upregulation by NF-kB, and that this may lead to chemoresistance to CPT11. Further studies are ongoing to establish the potentially important role of NF-kB-mediated regulation of HIF1-alpha in CPT11-resistance under hypoxia.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA