DIFFERENT RATES OF DEGRADATION OF THE PROLIFERATION-ASSOCIATED NUCLEAR AND NUCLEOLAR ANTIGENS DURING APOPTOSIS OF HL-60 CELLS INDUCED BY DNA TOPOISOMERASE INHIBITORS

One of the early events of apoptosis is proteolysis. The enzyme(s) inv olved in this event appear to be the serine protease(s), inasmuch as t he reversible or irreversible inhibitors of serine proteases prevent D NA degradation and abrogate other signs of apoptosis in several cell s ystems (Gorczyca et al, Int J Oncol 1: 639-648, 1992). In the present studies, using multiparameter flow cytometry, we attempted to characte rize the rate of disappearance of the nuclear and nucleolar proteins [ Proliferating Cells Nuclear Antigen (PCNA), Ki-67, p120 and another nu cleolar protein] detected by mononuclear antibodies, during apoptosis of HL-60 cells. Apoptosis was induced by the topoisomerase I inhibitor camptothecin and by the intercalating and nonintercalating topoisomer ase inhibitors m-AMSA and teniposide, respectively, and was specific t o cells in S phase. The loss of the protein reactive with Ki-67 antibo dy was the most rapid: two hours after administration of the drugs few S phase cells that expressed this protein remained in the cultures. N early all cells which were more advanced in apoptosis, and, due to ext ensive DNA degradation, had an already diminished DNA content, were ne gative with respect to expression of Ki-67. The nucleolar proteins, es pecially the one detected by the antibody distributed by Chemicon, app eared to be more stable compared to Ki-67. The most stable was PCNA: e xpression of this protein was high even after 6 h of treatment with ea ch of the drugs, even in cells which had reduced DNA content. The data indicate that the proteolytic step is not entirely random and may inv olve different proteases having different substrate preferences or spe cific targeting of individual proteins for the degradation. Because sp ontaneous apoptosis appears to be common in tumors, the phenomenon of rapid degradation of some of the proliferation-associated antigens dur ing apoptosis should be taken into account in the analysis of expressi on of these proteins in tumors (e.g. for estimation of the tumor growt h fraction).