DNA-DAMAGE IN HUMAN B-CELLS CAN INDUCE APOPTOSIS, PROCEEDING FROM G(1) S WHEN P53 IS TRANSACTIVATION COMPETENT AND G(2)/M WHEN IT IS TRANSACTIVATION DEFECTIVE/

Cisplatin treatment of Epstein-Barr virus-immortalized human B lymphob lastoid cell lines (LCLs) results in p53-mediated apoptosis which occu rs largely in a population of cells at the G(1)/S boundary of the cell cycle. Cell cycle progression appears to be required for this apoptos is because arresting cells earlier in G(1) inhibited apoptosis despite the accumulation of p53, Overexpression of wild-type p53 also induces apoptosis in an LCL. Therefore six mutant genes derived from Burkitt' s lymphoma (BL) cells were assayed for their ability to induce apoptos is when similarly overexpressed. The same genes were analysed in trans ient transfection assays for their ability to transactivate appropriat e reporter plasmids. A correlation between the ability of p53 to trans activate and induce apoptosis was revealed. The only mutant capable of transactivation also induced apoptosis. Further analysis of the BL li nes in which p53 had been characterized showed that whereas some lines were essentially resistant to cisplatin, three were rapidly induced t o undergo apoptosis. All three have a single p53 allele encoding a mut ant which is incapable of transactivation or (for two tested) mediatin g apoptosis when expressed in an LCL. Cell cycle analysis revealed tha t this apparently p53-independent apoptosis did not follow G(1) arrest but in fact occurred largely in cells distributed in the G(2)/M phase of the cell cycle. These data suggest the existence of a second check point in the G(2) or M phase which, in the absence of a functional p53 , is the primary point of entry into the apoptosis programme following DNA damage.