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

The broad-range cyclin-dependent kinase inhibitor UCN-01 induces apoptosis in colon carcinoma cells through transcriptional suppression of the Bcl-xL 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-x(L) on mRNA and protein level. In contrast, in the apoptosis-resistant cell lines, Bcl-x(L) expression was not affected by UCN-01. Stable overexpression of the Bcl-x(L) 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-x(L) expression in colon carcinoma cells. Moreover, we show for the first time that UCN-01 induces apoptosis by suppression of Bcl-x(L) expression. The inhibition of this pathway is a new aspect of cytotoxic and modulatory potential of UCN-01

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 p53 MUT 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 p53 MUT cells activates the checkpoint and contributes to apoptosis. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc