title: Differences in induction of p53, p21WAF1 and apoptosis in relation to cell cycle phase of MCF-7 cells treated with camptothecin.
abstract: The DNA topoisomerase I (topI) inhibitor camptothecin (CPT), stabilizes so-called cleavable complexes which consist of topI covalently attached to 3' OH ends of DNA nicks. Collisions between the progressing DNA replication forks (occurring in S phase cells) or between the transcription driven RNA polymerase molecules (occurring in G1, S and G2 cells) and these complexes convert the latter into secondary DNA lesions which are unrepairable and lethal to the cell. Changes induced by CPT in the level of the tumor suppressor p53, cyclin-dependent kinase inhibitor p21WAF1 and proapoptotic protein Bax (all detected immunocytochemically), were measured separately in the nucleus and cytoplasm of individual human breast carcinoma MCF-7 cells by laser scanning cytometry (LSC) in relation to cell cycle position and induction of apoptosis. The initial transient cell arrest at the G1 checkpoint seen at 8-16 h of treatment with 0.15 microM CPT was accompanied by the rapid accumulation of p53 (preventable by cycloheximide) in the nucleus; the rise (>20-fold) in p53 was maximal for S phase cells. The magnitude of the nuclear p53 increase induced by CPT, at maximum, was 2-fold higher than that induced by the proteasome inhibitor N-acetyl-Leu-Leu-norleucinal (LLnL). While the accumulation of p53 was seen in all phases of the cycle, only G1 cells responded by induction ( approximately 60-fold increase) of p21WAF1. Inhibition of DNA replication by aphidicolin prevented the accumulation of p53 in S and G2/M but had no effect on its induction in G1 cells. Perturbation of cell progression through S phase was seen between 24-72 h of treatment, and it coincided with induction of Bax and apoptosis (both maximal in S phase cells). Thus, the changes observed in S phase cells (nuclear accumulation of p53 preventable by aphidicolin, induction of Bax, apoptosis), triggered by the collisions of DNA replication forks with the CPT-induced lesions, were distinct from the changes in G1 (nuclear p53 accumulation unaffected by aphidicolin, induction of p21WAF1) presumably triggered by collisions of RNA polymerase with the CPT-lesions. Great heterogeneity in expression of p53 and p21WAF1 of the G1 cell population in response to CPT was observed, which may reflect the intercellular variability in the rate of transcription (i.e., frequencies of collisions of RNA polymerase with the lesions). Thus, differences in the transcriptional activity of G1 cells may play a role in their sensitivity to CPT and similar topI inhibitors.
authors.affiliation: Brander Cancer Research Institute, New York Medical College, Valhalla, NY 10595, USA.
authors.forename: A
authors.initials: A
authors.lastname: Deptala
authors.forename: X
authors.initials: X
authors.lastname: Li
authors.forename: E
authors.initials: E
authors.lastname: Bedner
authors.forename: W
authors.initials: W
authors.lastname: Cheng
authors.forename: F
authors.initials: F
authors.lastname: Traganos
authors.forename: Z
authors.initials: Z
authors.lastname: Darzynkiewicz
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D001943:Breast Neoplasms; D002166:Camptothecin / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects*; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000096:biosynthesis*; D003513:Cycloheximide / Q000494:pharmacology; D004261:DNA Replication / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000032:analysis; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis; D019253:Proto-Oncogene Proteins c-bcl-2; D059004:Topoisomerase I Inhibitors; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis*; D051028:bcl-2-Associated X Protein
chemical_list: C494826:BAX protein, human; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004791:Enzyme Inhibitors; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D059004:Topoisomerase I Inhibitors; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D003513:Cycloheximide; D002166:Camptothecin