The effect of DN (dominant-negative) Ku70 and reoxygenation on hypoxia cell-kill: evidence of hypoxia-induced potentially lethal damage.

Overview

PURPOSE: To study the effect of DN (dominant-negative) Ku70 and reoxygenation on the hypoxia-induced cell-kill. MATERIALS AND METHODS: Cell lines were human colorectal carcinoma HCT8 and HT29 cells and their respective derivatives, v-HCT8 and v-HT29 infected with DNKu70-containing adenovirus. Cells were plated in glass tubes and made hypoxic by flushing N(2) gas containing 0, 0.1 or 0.5% O(2). Cell survival was determined by colony formation assay immediately after 0-96 h hypoxia. To reoxygenate medium were replaced fresh following 48 or 72 h in hypoxia and cells were incubated in aerobic environment for 2-24 h before survival assay. RESULTS: When incubated in hypoxia, cells lost reproductive capability ∼ exponentially as a function of time in hypoxia, and depending on the O(2) concentration. DNKu70 rendered cells more prone to hypoxia-induced cell-kill. Following reoxygenation cell survival increased rapidly but without detectable cell proliferation during first 24 hours. This evinced hypoxia-induced potentially lethal damage (PLD) that was repairable upon reoxygenation. DNKu70 did not significantly inhibit this repair. CONCLUSION: Hypoxia-induced cell lethality was facilitated by DNKu70, but substantially repaired upon reoxygenation. This may have negative impact on the effect of reoxygenation in cancer therapy.