Selective loss of poly(ADP-ribose) and the 85-kDa fragment of poly(ADP-ribose) polymerase in nucleoli during alkylation-induced apoptosis of HeLa cells

Alkylation treatment of HeLa cells results in the rapid induction of apopto sis as revealed by DNA laddering and cleavage of poly(ADP-ribose) polymeras e (PARP) into the 29-and 85-kDa fragments (Kumari S. R., Mendoza-Alvarez, H . & Alvarez-Gonzalez, R, (1998) Cancer Res. 58, 5075-5078). Here, we perfor med a time-course analysis of (i) poly(ADP-ribose) synthesis and degradatio n as well as (ii) the subnuclear localization of PARP and its fragments by using confocal laser scanning immunofluorescence microscopy, PARP was activ ated within 15 min post-treatment, as revealed by nuclear immunostaining wi th antibody 10H (recognizing poly(ADP-ribose)), This was followed by a late , time-dependent, progressive decline of 10H signals that coincide with the time of PARP cleavage. Strikingly, nucleolar immunostaining with antibodie s 10H and C-LI-IO (recognizing the 85-kDa PARP fragment) was lost by 15 min post-treatment, whereas F-I-23 signals (recognizing the 29-kDa fragment) p ersisted. We hypothesize that the 85-kDa PARP fragment is translocated, alo ng with covalently bound poly(ADP-ribose), from nucleoli to the nucleoplasm , whereas the 29-kDa fragment is retained, because it binds to DNA strand b reaks. Our data (i) provide a Link between the known time-dependent bifunct ional role of PARP in apoptosis and the subcellular localization of PARP fr agments and also (ii) add to the evidence for early proteolytic changes in nucleoli during apoptosis.