HYPOXIA-INDUCED APOPTOSIS IN HUMAN-CELLS WITH NORMAL P53 STATUS AND FUNCTION, WITHOUT ANY ALTERATION IN THE NUCLEAR-PROTEIN LEVEL

We have studied hypoxia-induced inactivation of cells from three estab lished human cell lines with different p53 status. Hypoxia was found t o induce apoptosis in cells expressing mild-type p53 (MCF-7 cells), bu t not in cells where p53 is either mutated (T-47D cells), or abrogated by expression of the HPV18 E6 oncoprotein (NHIK 3025 cells). Apoptosi s was demonstrated by DNA fragmentation, using agarose gel electrophor esis of DNA and DNA nick end labeling (TUNEL). We demonstrate that ext remely hypoxic conditions (<4 ppm O-2) do not cause any change of expr ession in the p53 protein level in these three cell lines. In addition , the localization of p53 in MCF-7 cells was found exclusively in the nucleus in only some of the cells both under aerobic and hypoxic condi tions. Furthermore, no correlation was found between the p53-expressio n level and whether or not a cell underwent apoptosis. Flow cytometric TUNEL analysis of MCF-7 cells revealed that initiation of apoptosis o ccurred in all phases of the cell cycle, although predominantly for ce lls in S phase. Apoptosis was observed only during a limited time wind ow (i.e., approximate to 10 to approximate to 24 h) after the onset of extreme hypoxia. While 66% of the MCF-7 cells lost their ability to f orm visible colonies following 15 h exposure to extreme hypoxia, only similar to 28% were induced to apoptosis, suggesting that similar to 3 8% were inactivated by other death processes. Commitment to apoptotic cell death was observed in MCF-7 cells even for oxygen concentrations as high as 5000 ppm. Our present results indicate that the p53 status in these three tumor cell lines does not have any major influence on c ell's survival following exposure to extremely hypoxic conditions, whe reas following moderate hypoxia, cells expressing functional p53 enhan ced their susceptibility to cell death. Taken together, although these results suggest that functional p53 might play a role in the inductio n of apoptosis during hypoxia, other factors seem to be equally import ant. (C) 1997 Academic Press.