One of the early events of apoptosis is proteolysis. The enzyme(s) involved in this event appear to be the serine protease(s), inasmuch as the reversible or irreversible inhibitors of serine proteases prevent DNA degradation and abrogate other signs of apoptosis in several cell systems (Gorczyca et al, Int J Oncol 1: 639-648, 1992). In the present studies, using multiparameter flow cytometry, we attempted to characterize the rate of disappearance of the nuclear and nucleolar proteins [Proliferating Cells Nuclear Antigen (PCNA), Ki-67, p120 and another nucleolar 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 topoisomerase inhibitors m-AMSA and teniposide, respectively, and was specific to cells in S phase. The loss of the protein reactive with Ki-67 antibody was the most rapid: two hours after administration of the drugs few S phase cells that expressed this protein remained in the cultures. Nearly all cells which were more advanced in apoptosis, and, due to extensive DNA degradation, had an already diminished DNA content, were negative with respect to expression of Ki-67. The nucleolar proteins, especially the one detected by the antibody distributed by Chemicon, appeared to be more stable compared to Ki-67. The most stable was PCNA: expression of this protein was high even after 6 h of treatment with each of the drugs, even in cells which had reduced DNA content. The data indicate that the proteolytic step is not entirely random and may involve different proteases having different substrate preferences or specific targeting of individual proteins for the degradation. Because spontaneous apoptosis appears to be common in tumors, the phenomenon of rapid degradation of some of the proliferation-associated antigens during apoptosis should be taken into account in the analysis of expression of these proteins in tumors (e.g. for estimation of the tumor growth fraction).

Related Articles