ACTIVATION OF POLY(ADP-RIBOSE)POLYMERASE IN APOPTOTIC HUMAN-CELLS

We have studied the effect of the chemotherapeutic drug VP-16 (etoposi de) on the metabolism of HeLa cells by analysing different cellular pa rameters considered as markers of apoptosis. Typical features such as chromatin condensation and internucleosomal DNA cleavage are visible i n HeLa cells exposed to VP-16. We investigated whether the appearance of small-sized DNA fragments could regulate the ADP-ribosylation proce ss. To this purpose, we have analysed, by means of the activity gel te chnique, the structural and catalytical properties of poly(ADP-ribose) polymerase. In extracts from cells where etoposide-induced DNA fragmen tation occurred, we have shown that the label of the autoribosylated f orm of the enzyme is greatly increased even if the amount of the prote in remains constant. This phenomenon is completely abolished in cells preincubated with poly(ADP-ribose)polymerase inhibitor, 3-aminobenzami de. After VP-16 administration, we have observed that the level of NAD is not heavily decreased. It is widely agreed that zinc exerts an inh ibitory effect on the endonuclease(s) responsible for the fragmentatio n of DNA during apoptosis. After incubation of cells with zinc/VP-16 w e have found the occurrence of apoptotic parameters even in the absenc e of internucleosomal DNA cleavage. The inhibition of DNA fragmentatio n prevents the activation of poly(ADP-ribose)polymerase activity. Thes e results indicate that the activation of the enzyme towards the autom odification reaction is strictly dependent on the appearence of DNA in ternucleosomal fragments and could represent a way to control enzyme a ctivity.