ucn-1028-c and 3-aminobenzamide

The rapid accumulation of the p53 gene product is considered to be an important component of the cellular response to a variety of genotoxins. In order to gain insights on the biochemical pathways leading to p53 stabilization, the effect of (+/-) 7,8-dihydroxy-anti-9, 10-epoxy-7,8,9,10-tetrahydrobenzo(a)-pyrene [(+/-)-anti-BPDE] induced DNA damage on p53 protein levels was investigated in various repair-proficient and repair-deficient human cells. Brief exposure of normal human fibroblasts to 0.05-1 microM (+/-)-anti-BPDE resulted in elevated p53 protein levels as compared to the constitutive levels of control cells. The rapid induction response, detectable within a few hours, was sustained up to a period of at least 24 h. Repair-proficient and repair-deficient (XPA) human lymphoblastoid cells showed a similar response. The poly(ADP-ribose) polymerase inhibitor, 3-aminobenzamide (3-AB), diminished the p53 induction response by concomitantly decreasing the extent of (+/-)-anti-BPDE induced DNA damage in cells pretreated with the inhibitor. However, the direct involvement of poly ADP-ribosylation was also apparent as 3-AB was able to attenuate (approximately 50%) the p53 response by post-damage inhibitor treatment of the cells. Inhibition of cellular DNA replication by hydroxyurea and AraC, in the presence or absence of DNA damage, also resulted in rapid p53 accumulation in repair-deficient cells. On the contrary, inhibition of protein kinase C (PKC) by calphostin-C led to an abrogation of (+/-)-anti-BPDE mediated p53 induction. Analysis of the downstream effects of carcinogen treatment showed that the lymphoblastoid cells undergo DNA fragmentation indicative of apoptosis while fibroblasts exhibit cell cycle arrest at the G1-S boundary.

Topics: 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; Apoptosis; Benzamides; Carcinogens; Cell Cycle; Cells, Cultured; DNA Damage; DNA Replication; Dose-Response Relationship, Drug; Humans; Naphthalenes; Poly(ADP-ribose) Polymerase Inhibitors; Protein Kinase C; Tumor Suppressor Protein p53

Electron microscopy studies demonstrate unequivocally that the observed oligonucleosome-sized secondary DNA fragmentation in human promyelocytic HL-60 cells treated with the topoisomerase inhibitors camptothecin and teniposide is correlated with the morphological changes in cell structure typical of programmed cell death (apoptosis). Since apoptosis has been associated with potential involvement of intracellular signaling linked to the Ca2+/calmodulin and protein kinase C transduction pathways, we also investigated the effects of signaling modulators on camptothecin- and teniposide-induced secondary DNA fragmentation in HL-60 cells. Neither calcium chelators, calcium/calmodulin inhibitors (calmidazolium or cyclosporine A), protein kinase C stimulation by TPA, protein phosphatase inhibition by okadaic acid, protein kinase inhibition by staurosporine, calphostin C, genistein or H7, nor cell cycle alterations by caffeine had any detectable effect. Interestingly, most of these intracellular signaling modulators were able to induce DNA fragmentation in HL-60 cells by themselves. These results may suggest that even though modulation of these signaling pathways was unable to prevent topoisomerase inhibitor-induced apoptosis, their sole deregulations could induce apoptosis in HL-60 cells. In contrast, aphidicolin blocked camptothecin-induced secondary DNA fragmentation, indicating that replication-induced DNA damage is required for camptothecin- but not teniposide-induced secondary DNA fragmentation. Zinc, 3-aminobenzamide, and spermine also modulated both camptothecin- and teniposide-induced secondary DNA fragmentation without significant alteration of topoisomerase-mediated primary DNA strand breaks. Hence, poly(ADP-ribosyl)ation and chromatin structure may be important in modulating oligonucleosome-sized DNA fragmentation associated with apoptosis in HL-60 cells treated with topoisomerase inhibitors.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Aminoquinolines; Aphidicolin; Apoptosis; Benzamides; Calcium; Calmodulin; Camptothecin; Cell Cycle; Chromatin; Cyclosporine; DNA; Egtazic Acid; Ethers, Cyclic; Humans; Isoquinolines; Leukemia, Experimental; Leukemia, Promyelocytic, Acute; Microscopy, Electron; Naphthalenes; Okadaic Acid; Phosphoprotein Phosphatases; Piperazines; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerase Inhibitors; Polycyclic Compounds; Protein Kinase C; Spermine; Staurosporine; Teniposide; Tetradecanoylphorbol Acetate; Topoisomerase I Inhibitors; Topoisomerase II Inhibitors; Tumor Cells, Cultured; Zinc