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 apoptosis as revealed by DNA laddering and cleavage of poly(ADP-ribose) polymerase (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 performed a time-course analysis of (i) poly(ADP-ribose) synthesis and degradation as well as (ii) the subnuclear localization of PARP and its fragments by using confocal laser scanning immunofluorescence microscopy. PARP was activated within 15 min post-treatment, as revealed by nuclear immunostaining with 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 antibodies 10H and C-II-10 (recognizing the 85-kDa PARP fragment) was lost by 15 min post-treatment, whereas F-I-23 signals (recognizing the 29-kDa fragment) persisted. We hypothesize that the 85-kDa PARP fragment is translocated, along with covalently bound poly(ADP-ribose), from nucleoli to the nucleoplasm, whereas the 29-kDa fragment is retained, because it binds to DNA strand breaks. Our data (i) provide a link between the known time-dependent bifunctional role of PARP in apoptosis and the subcellular localization of PARP fragments and also (ii) add to the evidence for early proteolytic changes in nucleoli during apoptosis.