INHIBITION OF RADIATION-INDUCED G(2) DELAY POTENTIATES CELL-DEATH BY APOPTOSIS AND OR THE INDUCTION OF GIANT-CELLS IN COLORECTAL TUMOR-CELLS WITH DISRUPTED P53 FUNCTION/

We have previously identified a p53-independent apoptotic response tha t is delayed until 48-72 h after irradiation of colorectal adenoma and carcinoma cells, Because the delay appears to be in Dart due to a tra nsient G(2) cell cycle arrest, the importance of this checkpoint in th e mechanism of ionizing radiation (IR)-induced death of colorectal tum or cells was investigated, An adenoma cell line with (282Arg-->Trp) mu tant p53 (S/RG/C2) and a carcinoma cell line (PC/JW/FI) lacking p53 pr otein treated with 5 Gy IR in the presence of 1.5 mM caffeine (CAF) re duced IR-induced G, arrest and increased the level of apoptosis (1.5-1 .6-fold) 24 h after treatment, Increased IR apoptotic cell death with CAF significantly reduced IR cell survival over a 7-day period in S/RG /C2 and PC/JW/FI, To investigate whether CAF radiosensitization correl ated with lack of wild-type (mt) p53, we studied transfected derivativ es of an adenoma-derived cell line (PC/AA/C1), in which the endogenous wt p53 activity was disrupted by the expression of a dominant negativ e (273Arg-->His) p53 mutant protein (designated AA/273p53/B), This p53 -defective cell line was also radiosensitized by CAF, whereas the vect or control (AA/PCMV/D), which retained wt p53 activity, was not, In ad dition, as with the S/RG/C2 and PC/JW/FI cell lines, the 7-day IR cell survival was reduced significantly in AA/273p53/B compared with the v ector control cell line. This suggests that radiosensitization by CAF and increased cell death is dependent on loss of wt p53 function, Inte restingly, radiosensitization of the AA/273p53/B cell line was not ass ociated with accelerated apoptosis but correlated with increased polyp loid giant cells, which have been associated with disruption of cell c ycle checkpoints and genomic instability, These results demonstrate th at G(2) checkpoint inhibition with CAF leads to preferential IR cell k illing in cell lines in which wt p53 is inactivated and that this incr eased cell killing is not necessarily dependent on increased IR-induce d apoptosis.