[Show abstract][Hide abstract] ABSTRACT: Ursodeoxycholic acid (UDCA) can prevent chemical and colitis-associated colon carcinogenesis by unknown mechanism(s). One of the processes underlying the chemopreventive action could be the inhibition of proliferation by UDCA. To clarify the antiproliferative mechanism of UDCA, we used p53 wt colon carcinoma cell lines HCT8 and HCT116. UDCA-induced inhibition of proliferation was reversible and was associated with a decrease of the S-phase and an increase of G1 phase population, but not with apoptosis or senescence. The treatment suppressed the expression of c-Myc protein and, as a consequence, of several cell cycle regulatory molecules, including CDK4 and CDK6. Using the HCT8 cell line as a model, we show that UDCA suppresses c-Myc at the protein level. The suppression of c-Myc alone or a simultaneous suppression of CDK4 and of CDK6 kinase is sufficient to inhibit cell proliferation. In sum, we identified c-Myc as a primary UDCA target in colon carcinoma cells. The degradation of c-Myc protein decreases the expression of the cell cycle regulators CDK4 and CDK6, which reversibly slows down the cell cycle. The suppression of these proproliferatory molecules is the likely initial mechanism of antiproliferatory action of UDCA on colon cancer cells.
European journal of cancer prevention: the official journal of the European Cancer Prevention Organisation (ECP) 03/2012; 21(5):413-22. DOI:10.1097/CEJ.0b013e32834ef16f · 3.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The role of the mismatch repair (MMR) system in correcting base-base mismatches is well established; its involvement in the response to DNA double strand breaks, however, is less clear. We investigated the influence of the essential component of MMR, the hMLH1 protein, on the cellular response to DNA-double strand breaks induced by treatment with SN-38, the active metabolite of topoisomerase I inhibitor irinotecan, in a strictly isogenic cell system (p53(wt), hMLH1(+)/p53(wt), hMLH1(-)). By using hMLH1 expressing clones or cells transduced with the hMLH1-expressing adenovirus as well as siRNA technology, we show that in response to SN-38-induced DNA damage the MMR proficient (MMR(+)) cells make: (i) a stronger G2/M arrest, (ii) a subsequent longer tetraploid G1 arrest, (iii) a stronger activation of Chk1 and Chk2 kinases than the MMR deficient (MMR(-)) counterparts. Both Cdk2 and Cdk4 kinases contribute to the basal tetraploid G1 arrest in MMR(+) and MMR(-) cells. Although the Chk1 kinase is involved in the G2/M arrest, neither Chk1 nor Chk2 are involved in the enhancement of the tetraploid G1 arrest. The long-lasting tetraploid G1 arrest of MMR(+) cells is associated with their lower clonogenic survival after SN-38 treatment, the abrogation of the tetraploid G1 arrest resulted in their better clonogenic survival. These data show that the stabilization of the tetraploid G1 arrest in response to double strand breaks is a novel function of the MMR system that contributes to the lesser survival of MMR(+) cells.
International Journal of Cancer 06/2010; 126(12):2813-25. DOI:10.1002/ijc.24893 · 5.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: DNA damage induced by the topoisomerase I inhibitor irinotecan (CPT-11) triggers in p53(WT) 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Introduction: SN-38 is the active metabolite of the camptothecin analogue, irinotecan (CPT-11), which is the component of the first line chemotherapy against colorectal cancer. SN-38 inhibits topoisomerase I, causes DNA double strand breaks and inhibits DNA replication, transcription, recombination and repair. The mechanism of the cellular response to SN-38 is unclear. However, the effect of SN-38 appears to be mediated via p53-dependent as well as p53-independent pathways. In this study, we investigated the cellular, morphological and transcriptional response of colon carcinoma cells to SN-38 treatment in relation to p53 function. Materials and Methods: We used an isogenic system consisting of HCT116 p53+/+ and HCT116 p53−/− cell lines to analyse the impact of p53 function on treatment. Cells were treated with 10 nM SN-38 for different time periods and the cellular morphology, cell cycle distribution, and apoptosis were analysed. Cells were stained with DAPI and the anti-PARP (poly-ADP ribose polymerase) fragment p85 antibody to identify apoptotic cells. The transcriptional response was analysed using Affymetrix Genechip oligonucleotide microarrays. Results: HCT116 p53−/− cells underwent a short-term G2/M arrest after 48 h of treatment. The arrest, however, could not be sustained and the cells underwent mitotic catastrophe and finally apoptosis, detected by the fragmented PARP protein. The p53-independent mitotic catastrophe was evident through appearance of aberrant mitoses, nuclear condensation and micronucleation. The aberrant mitoses and condensed nuclei, but not micronucleated cells showed the presence of frag\. mented PARP protein. SN-38 treatment followed by nocodazole did not protect the cells from mitotic catastrophe. In contrast, HCT116 p53+/+ cells maintained the G2/M arrest up to 96 h after start of treatment and did not undergo apoptosis. p53-independent mitotic catastrophe was associated with the up-regulation of 90 genes and down-regulation of 25 genes. Conclusions: 1. SN-38 induces a p53-independent trigerring of G2/M arrest in colon carcinoma cells. 2. The release of cells from G2/M arrest leads to mitotic catastrophe, resulting in 2 morphologically and biologically different forms of cell deaths: (a) Apoptosis: associated with aberrant mitoses and condensed nuclei. (b) Micronucleation. 3. SN-38-induced mitotic catastrophe occurs before the nocodazole-induced prometaphase arrest. 4. Analysis of the transcriptional profile will allow the delineation of pathways which are causal to the DNA damage-induced p53-independent mitotic catastrophe in colon carcinoma cells.
Proceedings of the American Association of Cancer Research, Orlando FL, USA; 01/2004
[Show abstract][Hide abstract] 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
International Journal of Cancer 10/2002; 101(1):23-31. DOI:10.1002/ijc.10565 · 5.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Two factors potentially determining the consistent over-expression of sialyl-Lex antigen in colon carcinoma and metastases were investigated: (i) the expression of the mucins MUC1 and MUC2, known to carry sialyl-Lex, by Northern blotting; (ii) the extent of sialic acid O-acetylation, by Western blotting and HPLC. RNA and sialyl-Lex-positive mucins were purified from normal colonic mucosa (N), primary carcinomas (T) and their liver metastases (M). Northern blots showed that mRNA expression both of MUC1 and of MUC2 decreases during the progression of the disease, and is lowest in metastatic tissue. The expression of mucin-bound sialyl-Lex increased strongly from N to T and, to a lesser extent, to M. After alkali treatment of the mucins these differences disappeared, indicating that the total amount of mucin-bound sialyl-Lex is the same in the 3 types of tissues. The O-acetylation of mucin-bound sialyl-Lex gradually decreased from N to M. HPLC analysis showed that in N about 70%, in T 45% and in M only 20% of mucin-bound sialic acids are O-acetylated. Thus, the increase of sialyl-Lex detectable during colon-carcinoma progression is due to diminished O-acetylation and not to increased expression of mucin protein cores. The decrease of O-acetylation is therefore the primary chemical alteration contributing to colon carcinoma-associated overexpression of sialyl-Lex. Int. J. Cancer 72:258–264, 1997.
International Journal of Cancer 07/1997; 72(2):258-264. DOI:10.1002/(SICI)1097-0215(19970717)72:2<258::AID-IJC10>3.0.CO;2-C · 5.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 non-mucinous tumors the development of the majority of colonic carcinomas with the mucinous phenotype may be independent from p53 mutations.
Cancer Letters 07/1996; 103(2-103):163-170. DOI:10.1016/0304-3835(96)04208-5 · 5.62 Impact Factor