Transformation mediated by RhoA requires activity of ROCK kinases.
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BACKGROUND: The Ras-related GTPase RhoA controls signalling processes required for cytoskeletal reorganisation, transcriptional regulation, and transformation. The ability of RhoA mutants to transform cells correlates not with transcription but with their ability to bind ROCK-I, an effector kinase involved in cytoskeletal reorganisation. We used a recently developed specific ROCK inhibitor, Y-27632, and ROCK truncation mutants to investigate the role of ROCK kinases in transcriptional activation and transformation. RESULTS: In NIH3T3 cells, Y-27632 did not prevent the activation of serum response factor, transcription of c-fos or cell cycle re-entry following serum stimulation. Repeated treatment of NIH3T3 cells with Y-27632, however, substantially disrupted their actin fibre network but did not affect their growth rate. Y-27632 blocked focus formation by RhoA and its guanine-nucleotide exchange factors Dbl and mNET1. It did not affect the growth rate of cells transformed by Dbl and mNET1, but restored normal growth control at confluence and prevented their growth in soft agar. Y-27632 also significantly inhibited focus formation by Ras, but had no effect on the establishment or maintenance of transformation by Src. Furthermore, it significantly inhibited anchorage-independent growth of two out of four colorectal tumour cell lines. Consistent with these data, a truncated ROCK derivative exhibited weak ability to cooperate with activated Raf in focus formation assays. CONCLUSIONS: ROCK signalling is required for both the establishment and maintenance of transformation by constitutive activation of RhoA, and contributes to the Ras-transformed phenotype. These observations provide a potential explanation for the requirement for Rho in Ras-mediated transformation. Moreover, the inhibition of ROCK kinases may be of therapeutic use. (+info)
Expression of Bcl-2 protein is decreased in colorectal adenocarcinomas with microsatellite instability.
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Bcl-2 is known to inhibit apoptosis and is thought to play a role in colorectal tumour development. Studies of the promoter region of bcl-2 have indicated the presence of a p53 responsive element which downregulates bcl-2 expression. Since p53 is commonly mutated in colorectal cancers, but rarely in those tumours showing microsatellite instability (MSI), the aim of this study was to examine the relationship of bcl-2 protein expression to MSI, as well as to other clinicopathological and molecular variables, in colorectal adenocarcinomas. Expression of bcl-2 was analysed by immunohistochemistry in 71 colorectal cancers which had been previously assigned to three classes depending upon their levels of MSI. MSI-high tumours demonstrated instability in three or more of six microsatellite markers tested, MSI-low tumours in one or two of six, and MSI-null in none of six. Bcl-2 expression in tumours was quantified independently by two pathologists and assigned to one of five categories, with respect to the number of cells which showed positive staining: 0, up to 5%; 1, 6-25%; 2, 26-50%; 3, 51-75%; and 4, > or =76%. Bcl-2 negative tumours were defined as those with a score of 0. Bcl-2 protein expression was tested for association with clinicopathological stage, differentiation level, tumour site, age, sex, survival, evidence of p53 inactivation and MSI level. A significant association was found between bcl-2 expression and patient survival (P = 0.012, Gehan Wilcoxon test). Further, a significant reciprocal relationship was found between bcl-2 expression and the presence of MSI (P = 0.012, Wilcoxon rank sum test). We conclude that bcl-2 expressing colorectal cancers are more likely to be MSI-null, and to be associated with improved patient survival. (+info)
Vasoactive intestinal peptide (VIP) is a naturally occurring 28-amino acid peptide with a wide range of biological activities. Recent reports suggest that VIP receptors are expressed on a variety of malignant tumor cells and that the receptor density is higher than for somatostatin. Our aims were to label VIP with 99mTc--a generator-produced, inexpensive radionuclide that possesses ideal characteristics for scintigraphic imaging--and to evaluate 99mTc-VIP for bioactivity and its ability to detect experimental tumors. METHODS: VIP28 was modified at the carboxy terminus by the addition of four amino acids that provided an N4 configuration for a strong chelation of 99mTc. To eliminate steric hindrance, 4-aminobutyric acid (Aba) was used as a spacer. VIP28 was labeled with 1251, which served as a control. Biological activity of the modified VIP28 agonist (TP3654) was examined in vitro using a cell-binding assay and an opossum internal anal sphincter (IAS) smooth muscle relaxivity assay. Tissue distribution studies were performed at 4 and 24 h after injection, and receptor-blocking assays were also performed in nude mice bearing human colorectal cancer LS174T. Blood clearance was examined in normal Sprague-Dawley rats. RESULTS: The yield of 99mTc-TP3654 was quantitative, and the yields of 125I-VIP and 1251-TP3654 were >90%. All in vitro data strongly suggested that the biological activity of 99mTc-TP3654 agonist was equivalent to that of VIP28. As the time after injection increased, radioactivity in all tissues decreased, except in the receptor-enriched tumor (P = 0.84) and in the lungs (P = 0.78). The tumor uptake (0.23 percentage injected dose per gram of tissue [%ID/g]) was several-fold higher than 125I-VIP (0.06 %ID/g) at 24 h after injection in the similar system. In mice treated with unlabeled VIP or TP3654, the uptake of 99mTc-TP3654 decreased in all VIP receptor-rich tissues except the kidneys. The blood clearance was biphasic; the alpha half-time was 5 min and the beta half-time was approximately 120 min. CONCLUSION: VIP28 was modified and successfully labeled with 99mTc. The results of all in vitro examinations indicated that the biological activity of TP3654 was equivalent to that of native VIP28 and tumor binding was receptor specific. (+info)
Genomic structure and alterations of homeobox gene CDX2 in colorectal carcinomas.
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Expression of CDX2, a caudal-related homeobox gene, was found to be decreased in colorectal carcinomas. Heterozygous null mutant mice as to Cdx2 develop multiple intestinal adenomatous polyps. To clarify the role of CDX2 in colorectal carcinogenesis, we determined its genomic structure, and searched for mutations of CDX2 in 49 sporadic colorectal carcinomas and ten hereditary non-polyposis colorectal cancers (HNPCC) without microsatellite instability. None of them exhibited a mutation. We further examined 19 HNPCC carcinomas with microsatellite instability for mutations in a (G)7 repeat site within CDX2. One of them (5.3%) exhibited one G insertion. Loss of heterozygosity was observed in 2 of the 20 (10%) informative sporadic carcinomas, and in one of the three (33.3%) informative HNPCC cancers. These data indicate that CDX2 may play only a minor role in colorectal carcinogenesis. (+info)
Marimastat in recurrent colorectal cancer: exploratory evaluation of biological activity by measurement of carcinoembryonic antigen.
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Marimastat is a specific inhibitor of matrix metalloproteinases that has been shown to be effective in cancer models. A pilot, escalating-dose study of oral marimastat was performed in patients with recurrent colorectal cancer, in whom evaluation of serological response was made by measurement of carcinoembryonic antigen (CEA) levels. The study assessed the safety and tolerability of 4 weeks administration of marimastat, and determined a dose range producing detectable serological effects. Patients were recruited with a serum CEA level greater than 5 ng ml(-1), and rising by more than 25% over a 4-week screening period. Patients were treated for 28 days and entered into a continuation protocol if a serological response or clinical benefit was observed. Pharmacokinetic and safety data determined that groups of patients were recruited sequentially at 25 mg and 50 mg twice daily, and, thereafter, 10 mg twice daily, 10 mg once daily, 5 mg once daily and 20 mg once daily. A biological effect (BE) was defined as a CEA value on day 28 no greater than on day 0; a partial biological effect (PBE) was defined as a rise in CEA over the 28-day treatment period of less than 25%. Of 70 patients recruited, 63 completed the 28-day treatment period, and 55 were eligible for cancer antigen analysis. Examination of the dose-effect relationships provides evidence for a causal relationship between marimastat and biological effects: the proportion of patients with BE or PBE was higher with twice daily dosing (16 out of 25, 64%) than with once daily dosing (11 out of 30, 37%) (P = 0.043, chi2 test). Furthermore, the median rates of rise of CEA fell markedly during treatment compared with the screening period for patients receiving twice daily marimastat (P<0.0001), but not for patients receiving marimastat once daily (P = 0.25). Musculoskeletal adverse events emerged as the principal drug-related toxicity of marimastat, occurring in a dose- and time-dependent fashion. It was concluded that marimastat was associated with dose-dependent biological effects in cancer patients. The occurrence of musculoskeletal side-effects define 25 mg twice daily as the upper limit of the dose range for continuous use in further studies. Therefore, a dose range of 20 mg once daily to 25 mg twice daily seems appropriate for further studies, which should aim to demonstrate the efficacy of the drug in terms of conventional clinical end points and describe the long-term tolerability of this novel agent. (+info)
Low tumour cell proliferation at the invasive margin is associated with a poor prognosis in Dukes' stage B colorectal cancers.
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The conflicting results about the prognostic impact of tumour cell proliferation in colorectal cancer might be explained by the heterogeneity observed within these tumours. We have investigated whether a systematic spatial heterogeneity exists between different compartments, and whether the presence of such a systematic heterogeneity has any impact on survival. Fifty-six Dukes' stage B colorectal cancers were carefully morphometrically quantified with respect to the immunohistochemical expression of the proliferative marker Ki-67 at both the luminal border and the invasive margin. The proliferative activity was significantly higher at the luminal border compared with the invasive margin (P<0.001), although the two compartments were also significantly correlated with each other. Tumours with low proliferation at the invasive margin had a significantly poorer prognosis both in univariate (P = 0.014) and in multivariate survival analyses (P = 0.042). We conclude that Dukes' B colorectal cancers exhibit a systematic spatial heterogeneity with respect to proliferation, and tumours with low proliferation at the invasive margin had a poor prognosis. The present data independently confirm recent results from the authors, and provide new insights into the understanding of tumour cell proliferation in colorectal cancer. (+info)
Profound variation in dihydropyrimidine dehydrogenase activity in human blood cells: major implications for the detection of partly deficient patients.
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Dihydropyrimidine dehydrogenase (DPD) is responsible for the breakdown of the widely used antineoplastic agent 5-fluorouracil (5FU), thereby limiting the efficacy of the therapy. To identify patients suffering from a complete or partial DPD deficiency, the activity of DPD is usually determined in peripheral blood mononuclear cells (PBM cells). In this study, we demonstrated that the highest activity of DPD was found in monocytes followed by that of lymphocytes, granulocytes and platelets, whereas no significant activity of DPD could be detected in erythrocytes. The activity of DPD in PBM cells proved to be intermediate compared with the DPD activity observed in monocytes and lymphocytes. The mean percentage of monocytes in the PBM cells obtained from cancer patients proved to be significantly higher than that observed in PBM cells obtained from healthy volunteers. Moreover, a profound positive correlation was observed between the DPD activity of PBM cells and the percentage of monocytes, thus introducing a large inter- and intrapatient variability in the activity of DPD and hindering the detection of patients with a partial DPD deficiency. (+info)
Faecal occult blood screening and reduction of colorectal cancer mortality: a case-control study.
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To estimate the efficacy of screening on colorectal cancer mortality, a population-based case-control study was conducted in well-defined areas of Burgundy (France). Screening by faecal occult blood test prior to diagnosis in cases born between 1914 and 1943 and who died of colorectal cancer diagnosed in 1988-94 was compared with screening in controls matched with the case for age, sex and place of residence. Cases were less likely to have been screened than controls, with an odds ratio (OR) of 0.67 [95% confidence interval (CI) 0.48-0.94]. The negative overall association did not differ by gender or by anatomical location. The odds ratio of death from colorectal cancer was 0.64 (95% CI 0.46-0.91) for those screened within 3 years of case diagnosis compared with those not screened. It was 1.14 (95% CI 0.50-2.63) for those screened more than 3 years before case diagnosis. There was a negative association between the risk of death from colorectal cancer and the number of participations in the screening campaigns. The inverse association between screening for faecal occult blood and fatal colorectal cancer suggests that screening can reduce colorectal cancer mortality. This report further supports recommendations for population-based mass screening with faecal occult blood test. (+info)