Background: Recent evidence indicates that programmed cell death or apoptosis may be an important mechanism for the lethality of cancer chemotherapy drugs in tumor cells. Apoptosis may be induced in tumor cells by a number of physical stresses, including radiation, UV light, heat shock, and cold. In preliminary studies, we have found that exposure of cells to hypoxia rapidly induces the expression of several immediate early genes, including c-jun, jun-D, and c-fos, and of a bifunctional redox protein/endonuclease, Ref-1.
Purpose: Our purpose was to determine if hypoxia-induced overexpression of the endonuclease was associated with altered DNA integrity and manifestations of apoptosis.
Methods: We examined cultured cells for the induction of apoptosis by electrophoresis and immunofluorescence techniques and related these findings to the induction of gene expression by hypoxia.
Results: We found that an 8-hour exposure of human adenocarcinoma HT29 cells to hypoxia (which results in only 14% loss of viability) induced internucleosomal DNA fragmentation beginning after 8 hours of hypoxia and before reoxygenation. Immunofluorescence of hypoxia-treated cells showed that apoptotic cells were detectable initially after 4 hours of hypoxia and reached a peak (31.3% of cells) at 12 hours after reoxygenation and that by 36 hours after reoxygenation all apoptotic cells had been eliminated from the population. Hypoxia-induced apoptosis was associated with a marked induction of c-myc messenger RNA (mRNA). We have previously shown that hypoxia is associated with ref-1 mRNA induction. While expression of c-myc declined with a time course similar to that of the disappearance of apoptotic cells, that of ref-1 remained elevated. Coincident with the decline of c-myc, we observed a late increase in the expression of bcl-2 beginning 24 hours after reoxygenation.
Conclusions and implications: These results suggest a possible relationship between Ref-1 induction and the occurrence of apoptosis following a hypoxic exposure.