Cellular effects of CPT-11 on colon carcinoma cells: dependence on p53 and hMLH1 status.
Irinotecan (CPT-11), a recently introduced component of a standard chemotherapy for colorectal cancer, induces in colon cancer cell lines in vitro cell cycle arrest and apoptosis. Since sporadic colon carcinomas exhibit in 50-60% mutations in the p53 gene and in 10-15% an MSI phenotype due in the great majority of the cases to hMLH1 inactivation, we investigated how these lesions influence the cellular effects of CPT-11 by using colorectal carcinoma cell line HCT116 (which has the genotype p53(+/+),hMLH1(-)) and 2 derivative cell lines with the genotypes p53(+/+),hMLH1(+) and p53(-/-),hMLH1(-). CPT-11 treatment induced G2/M arrest in all 3 cell lines within 48 hr. In the p53(+/+),hMLH1(+) cell line, G2/M arrest was maintained for at least 12 days. There was little concomitant apoptosis, but this was enhanced when the hMLH1 protein was absent. This enhanced apoptosis was accompanied by a shorter duration of the G2/M arrest than in the hMLH1(+) cell line. Partial abrogation of G2/M arrest by caffeine enhanced apoptosis in both hMLH1(+) and hMLH1(-) cells. By contrast, in the p53(-/-) cell line, the G2/M arrest was terminated within 4 days. Termination of the G2/M arrest was accompanied by a high level of apoptosis detectable through poly(ADP-ribose)polymerase (PARP) cleavage, DNA fragmentation and by the appearance of cells with a DNA content <2N. The triggering of G2/M arrest was accompanied in the 3 cell lines by a transient phosphorylation of cdc-2, while the maintenance of the arrest in the p53(+/+) cell lines was accompanied by the overexpression of p53 and p21 proteins and, consequently, by the inhibition of cdc-2 kinase activity. These data indicate that: (i) CPT-11 induces long-term arrest in p53(+/+) cells and a short-term arrest followed by apoptosis in p53(-/-) cells; (ii) triggering of the arrest is p53 independent and is associated with a brief increase of phosphorylation of cdc-2, while the p53-dependent maintenance of G2/M arrest is associated with the inhibition of cdc-2 kinase activity by p21; and (iii) lack of hMLH1 protein enhances CPT-11-induced apoptosis. These results may be useful for designing rational therapies dependent on the p53 and mismatch-repair status in the tumor.
Magrini
Roberta
R
Department of Gastroenterology, University Clinic Benjamin Franklin, Berlin, Germany.
Bhonde
Mandar R
MR
Hanski
Marie-Luise
ML
Notter
Michael
M
Scherübl
Hans
H
Boland
C Richard
CR
Zeitz
Martin
M
Hanski
Christoph
C
D048868:Adaptor Proteins, Signal Transducing; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002110:Caffeine / Q000494:pharmacology; D002166:Camptothecin / Q000031:analogs & derivatives / Q000494:pharmacology*; D002352:Carrier Proteins; D002453:Cell Cycle / Q000187:drug effects; D003110:Colonic Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D019008:Drug Resistance, Neoplasm; D005434:Flow Cytometry; D017353:Gene Deletion; D006801:Humans; D000077146:Irinotecan; D000070957:MutL Protein Homolog 1; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins; D010766:Phosphorylation / Q000187:drug effects; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C501486:MLH1 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; D002110:Caffeine; D000077146:Irinotecan; D016203:CDC2 Protein Kinase; D000070957:MutL Protein Homolog 1; D002166:Camptothecin