Christoph Hanski

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.

DNA Damage-induced Expression of p53 Suppresses Mitotic Checkpoint Kinase hMps1 THE LACK OF THIS SUPPRESSION IN p53MUT CELLS CONTRIBUTES TO APOPTOSIS

DNA damage induced by the topoisomerase I inhibitor irinotecan (CPT-11) triggers in p53WT colorectal carcinoma cells a long term cell cycle arrest and in p53MUT cells a transient arrest followed by apoptosis (Magrini, R., Bhonde, M. R., Hanski, M. L., Notter, M., Scherübl, H., Boland, C. R., Zeitz, M., and Hanski, C. (2002) Int. J. Cancer 101, 23-31; Bhonde, M. R., Hanski, M. L., Notter, M., Gillissen, B. F., Daniel, P. T., Zeitz, M., and Hanski, C. (2006) Oncogene 25, 165-175). The mechanism of the p53-independent apoptosis still remains largely unclear. Here we used five p53WT and five p53MUT established colon carcinoma cell lines to identify gene expression alterations associated with apoptosis in p53MUT cells after treatment with SN-38, the irinotecan metabolite. After treatment, 16 mitosis-related genes were found to be expressed at least 2-fold stronger in the apoptosis-executing p53MUT cells than in the cell cycle-arrested p53WT cells by oligonucleotide microarray analysis. One of the genes whose strong post-treatment expression was associated with apoptosis was the mitotic checkpoint kinase hMps1 (human ortholog of the yeast monopolar spindle 1 kinase). hMps1 mRNA and protein expression were suppressed by the treatment-induced and by the exogenous adenovirus-coded p53 protein. The direct suppression of hMps1 on RNA level or inhibition of its activity by a dominant-negative hMps1 partly suppressed apoptosis. Together, these data indicate that the high expression of mitotic genes in p53MUT cells after SN-38 treatment contributes to DNA damage-induced apoptosis, whereas their suppression in p53WT cells acts as a safeguard mechanism preventing mitosis initiation and the subsequent apoptosis. hMps1 kinase is one of the mitotic checkpoint proteins whose expression after DNA damage in p53MUT cells activates the checkpoint and contributes to apoptosis.

Equivalent effect of DNA damage-induced apoptotic cell death or long-term cell cycle arrest on colon carcinoma cell proliferation and tumour growth.

Knowledge of the type of biological reaction to chemotherapy is a prerequisite for its rational enhancement. We previously showed that irinotecan-induced DNA damage triggers in the HCT116p53wt colon carcinoma cell line a long-term cell cycle arrest and in HCT116p53−/− cells apoptosis (Magrini et al., 2002). To compare the contribution of long-term cell cycle arrest and that of apoptosis to inhibition of cell proliferation after irinotecan-induced DNA damage, we used this isogenic system as well as the cell lines LS174T (p53wt) and HT-29 (p53mut). Both p53wt cell lines responded to damage by undergoing a long-term tetraploid G1 arrest, whereas the p53mut cell lines underwent apoptosis. Cell cycle arrest as well as apoptosis caused a similar delay in cell proliferation. Irinotecan treatment also induced in mouse tumours derived from the p53wt cell lines a tetraploid G1 arrest and in those derived from the p53-deficient cell lines a transient G2/M arrest and apoptosis. The delay of tumour growth was in the same range in both groups, that is, arrest- and apoptosis-mediated tumour growth inhibition was comparable. In conclusion, cell cycle arrest as well as apoptosis may be equipotent mechanisms mediating the chemotherapeutic effects of irinotecan.

Low frequency of p53 gene mutation and protein expression in mucinous colorectal carcinomas

Immunohistochemical data indicate that the frequency of p53 protein overexpression is consistently lower in the mucinous than in the non-mucinous carcinomas of the breast, ovary, pancreas and colon. This peculiar immunohistochemical behavior of the mucinous phenotype could be due to the effect of large amounts of mucus on the staining or to an actual mutation frequency difference between mucinous and non-mucinous carcinomas. This question was investigated on a group of mucinous colorectal carcinomas. DNA was extracted from paraffin sections of 16 human mucinous colorectal carcinomas and the mutation frequency was determined by sequencing of p53 exons amplified in PCR. The expression of p53 protein was determined with the avidin-biotin complex-peroxidase staining procedure and CM-1 antiserum. Twenty-five percent of the tumors, exhibited p53 protein overexpression and in 31% a mutation was detected. Concordance between the two techniques was found in 69% of tumors. Overexpression without mutation was observed in 12% and mutation without overexpression in 19%. G:C --> A:T transitions represented the most frequent lesion (80%), as previously observed in non-mucinous colorectal carcinomas. These data indicate that the mutation pattern in the p53 gene is similar in mucinous and non-mucinous colorectal carcinomas. The low frequency of p53 overexpression in the mucinous phenotype is not due to a mucus effect on the staining but is related to the low mutation frequency of p53 gene. These results lead to the hypothesis that in contrast to the nonmucinous tumors the development of the majority of colonic carcinomas with the mucinous phenotype may be independent from p53 mutations.

Regulation of the intestinal mucin MUC2 gene expression in vivo: evidence for the role of promoter methylation

In the present work we investigated the in vivo regulation of the mucin gene MUC2, which is overexpressed in all mucinous colorectal carcinomas. The inhibition of methylation by 5-azadeoxycytidine induces de novo expression of MUC2 in the colon carcinoma cell line COLO 205. The expression is retained in xenograft tissue and the cells give rise to MUC2-expressing tumours in nude mice. The strong expression of MUC2 in the normal human goblet cells and in the tissue of human mucinous colorectal carcinomas is associated with the average methylation of about 50% at every investigated CpG site of the MUC2 promoter. In contrast, MUC2 promoter in the non-expressing normal columnar cells and in the non-mucinous carcinoma tissue is methylated to nearly 100%. These data show that (i) low methylation of MUC2 promoter is associated with MUC2 expression in vivo and (ii) the pattern of MUC2 promoter methylation in the normal goblet or columnar cells most closely resembles that in mucinous or non-mucinous colorectal carcinomas, respectively. They indicate that MUC2 expression in vivo is regulated by promoter methylation and support the hypothesis that cells with goblet-like differentiation give rise to mucinous colonic carcinomas.

Expression of p53 protein in invasive colorectal carcinomas of different histologic types

Background. This study was performed to determine whether morphologic differences of colonic cancer types can be related to different genotypes of these tumors.

Prevention of colitis-associated carcinogenesis in a mouse model by diet supplementation with ursodeoxycholic acid

Bile acids in the intestinal lumen contribute to the homeostatic regulation of proliferation and death of the colonic epithelial cells: Deoxycholic acid (DCA) appears to enhance and ursodeoxycholic acid (UDCA) to attenuate the process of chemically induced carcinogenesis. We studied the effects of UDCA on colitis‐related colorectal carcinogenesis. Three groups of 25 mice were given 0.7% dextran sulphate in drinking water for 7 days and pure water for 10 days and were fed a standard diet containing double iron concentration. In 2 groups, the diet was supplemented with 0.2% cholic acid (CA), the precursor of DCA, or with 0.4% UDCA. After 15 cycles, the histology, the expression of MUC2, β‐catenin, p27 and p16 and the fecal water concentration of DCA and UDCA were investigated. All animals showed colitis with similar severity and histologic as well as immunophenotypic alterations, resembling those of human colitis. Among the animals fed the nonsupplemented diet, 46% developed colorectal adenocarcinomas and 54% anal‐rectal squamous cell carcinomas. The prevalence of dysplasia and carcinomas did not change significantly in the animals given CA. Among the mice fed with UDCA, none developed adenocarcinomas and 20% squamous carcinomas. Dysplastic lesions were found in 88%, 67% and 40% of each group, respectively. The prevalence of dysplasia as well as of carcinoma showed an inverse relationship to the UDCA concentration in the fecal water. These data indicate that UDCA suppresses colitis‐associated carcinogenesis. This model is suitable for investigation of the mechanism of the anticarcinogenic effect of UDCA in vivo.

The broad-range cyclin-dependent kinase inhibitor UCN-01 induces apoptosis in colon carcinoma cells through transcriptional suppression of the Bcl-x L protein

The broad-range cyclin-dependent kinase inhibitor 7-hydroxystaurosporine (UCN-01) is known to induce both a G1 cell cycle arrest and apoptosis. The mechanism of UCN-01-induced apoptosis is largely unknown. We analysed the mechanism of cytotoxicity of UCN-01 in four established colon carcinoma cell lines. The cell lines SW48 and LS513 responded to UCN-01 treatment by undergoing apoptosis in a concentration-dependent manner while the cell lines HT-29 and WiDr were completely resistant. Apoptosis in LS513 and SW48 cell lines was concomitant with the suppression of Bcl-xL on mRNA and protein level. In contrast, in the apoptosis-resistant cell lines, Bcl-xL expression was not affected by UCN-01. Stable overexpression of the Bcl-xL protein abrogated UCN-01-triggered apoptosis, but only partially restored growth, indicating that both cell cycle arrest and apoptosis exert the anticancer effect in a coordinated manner. The inhibition of Akt phosphorylation did not correlate with the apoptotic phenotype. UCN-01 inhibited the activating STAT3 phosphorylations on Ser727 and, notably, on Tyr705, but STAT3 did not contribute to Bcl-xL expression in colon carcinoma cells. Moreover, we show for the first time that UCN-01 induces apoptosis by suppression of Bcl-xL expression. The inhibition of this pathway is a new aspect of cytotoxic and modulatory potential of UCN-01.

A modification of the JAM test is necessary for a correct determination of apoptosis induced by FasL+ adherent tumor cells

Tumor cells from several organs including colon have recently been shown to express Fas ligand (FasL) in vitro and in vivo. The expression, which in some tumours occurs de novo, was suggested to facilitate immune escape of malignant cells by killing tumor-infiltrating lymphocytes via Fas-FasL-induced apoptosis. An argument to support this hypothesis is the detection of tumor cell-induced apoptosis in Jurkat cells (as model T cells) by means of the widely used JAM test. In the present work the validity of this test for the analysis of colon carcinoma cell-mediated apoptosis in Jurkat cells was scrutinized in detail. The presented data show that the JAM test as described previously is prone to false-positive detection of apoptosis, when adherent epithelial cells are used as effectors. Furthermore, three lines of evidence indicated that several FasL+ colon carcinoma cell lines did not induce detectable apoptosis in Jurkat cells in vitro. We conclude that: (1) The JAM test must be modified for testing DNA fragmentation induced through adherent effector cells and (2) FasL+ colon carcinoma cells may be unable to induce apoptosis in vitro.

Prognostic value of multimarker analysis in stage III colorectal cancer: one step forward towards an individualized therapy decision.

Background: Recently, we have analyzed new prognostic markers in colorectal cancer including neuroendocrine differentiation, overexpression of the sialyl-Lex antigen, overexpression of the peripheral benzodiazepine receptor (PBR), BAX protein expression and p53 mutational status. The predictive power of all markers in combination has not yet been evaluated. Patients and Methods: Between 1989 and 1991, 48 consecutive patients underwent surgery for stage III colorectal cancer at our hospital. All patients received a complete 5-year follow-up. Paraffin-embedded tumor samples were analyzed for all 5 markers. Multivariate discriminant analysis was performed to determine the prognostic value of all markers in combination. Results: Based on these prognostic markers a mathematical discriminant function was obtained. This function allowed to correctly predict the further course of disease in 77% of the patients (specificity: 83.3%, sensitivity: 70.8%). The discriminant function was confirmed in another group of 19 patients. Single marker analysis allowed the prediction of the further course of disease only in 58-70%. Conclusion: Our study shows that in colorectal cancer, multimarker analysis is superior to unimarker analysis in predicting prognosis. The derived discriminant function allows patient stratification according to risk. Therefore, a multimarker analysis provides a rationale for future individualized risk-adapted therapies in stage III colorectal cancer.