Cellular effects of CPT‐11 on colon carcinoma cells: Dependence on p53 and hMLH1 status

Abstract

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.

Microsatellite instability due to hMLH1 deficiency is associated with increased cytotoxicity to irinotecan in human colorectal cancer cell lines

“…These results are in agreement with the 'fork collision model' and the S-phase specificity of CPT-11. Therefore, this work reinforces previous reports of higher sensitivity of MMR-deficient cell lines to CPT-11 and offers a potential explanation for it (Jacob et al, 2001;Magrini et al, 2002). Several authors have demonstrated that MSI is a prognostic marker in patients with CRC (Gryfe et al, 2000;Popat et al, 2005).…”

“…In accordance with this fact, emerging clinical data suggest that MSI-H CRC patients may obtain more benefit from CPT-11-based chemotherapy than patients bearing MSS tumours (Fallik et al, 2003;Bertagnolli et al, 2006). Still, preclinical evidence suggesting a higher sensitivity of MMR-deficient tumours to irinotecan (CPT-11) is controversial due to discrepant results coming from different studies (Hausner et al, 1999;Jacob et al, 2001;Magrini et al, 2002;Fedier and Fink, 2004).…”

“…Two independent research groups have reported resistance to camptothecins in MMR-deficient cell lines (Hausner et al, 1999;Fedier et al, 2001). In contrast with these reports, we and others (Jacob et al, 2001;Magrini et al, 2002) have demonstrated higher sensitivity to CPT-11 in MMR-deficient human CRC cell lines than in MMR-proficient cells and G2/M arrest after exposure to CPT-11. Likewise, the body of evidence supporting the sensitivity to CPT-11 in MSI-H CRC at the clinical level is limited.…”

Mucinous histology predicts for poor response rate and overall survival of patients with colorectal cancer and treated with first-line oxaliplatin- and/or irinotecan-based chemotherapy

“…It is recognised that defective DNA mismatch repair (MMR) leads to MSI and results in resistance to many antineoplastic drugs, such as antimetabolites, alkylating, and platinum agents, and inhibitors of topoisomerases. There are some evidences in vitro that suggest a correlation between response to 5-FU, OXA, and IRI and MSI (Magrini et al, 2002;Arnold et al, 2003;Warusavitarne and Schnitzler, 2007). Our analysis plan did not include an MSI analysis.…”

Phenotypic and genotypic heterogeneity in the Lynch syndrome: diagnostic, surveillance and management implications

“…Interestingly, Magrini et al 42 showed that the absence of MLH1 protein actually enhances CPT-11-induced apoptosis, and that triggering of cell cycle arrest was p53 independent. Thus, a theoretical basis was formed for the development of chemotherapeutic strategies specifically targeted to MSI þ cells.…”