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Background: Microsatellite instability (MSI) is a biological characteristic of most tumours, being involved in 85% of hereditary non-polyposis colorectal cancer (HNPCC). It also occurs in 10-15% of sporadic colorectal cancers (CRC). HNPCC appears to be caused by germline mutations in mismatch repair (MMR) genes, which are responsible for repairing single base-pair mismatches. MSI is also associated with a better response of CRC to adjuvant chemotherapy with fluoropyrimidines. We investigated any relationship between the MSI status and the TSmRNA expression, the polymorphisms of 5-Fluorouracil (5-FU cellular target, the enzyme thymidylate synthase (TS) and TS expression evaluated by means of immunohistochemistry., Materials and Methods: A series of 80 colorectal cancers was evaluated for MSI and polymorphisms in the 3'UTR and the 5'UTR of the TS gene by a PCR assay. TSmRNA was quantified by real-time PCR and the TS expression by immunohistochemical assay., Results: There was no significant association between the polymorphisms in the TS gene and the MSI or between the TSmRNA expression and the MSI status. CRC with a 3R/3R or 2R/3R genotype showed a significantly higher TSmRNA expression than those with the 2R/2R genotype (p = 0.001 and p = 0.028, respectively). Another significant association was found between the TSmRNA expression and the TS immunohistochemical determination (p = < 0.05). No association was found between the polymorphism of the 3'UTR and the TSmRNA expression., Conclusion: Our data show that there is no association between MSI status and the polymorphisms in the 3' and 5' UTRs and the TS expression. Tumour samples displaying the 3R/3R or 2R/3R genotype of TS have higher TSmRNA levels than the 2R/2R genotype. Polymorphic variant of the 3'UTR does not influence the TSmRNA level. We found a relationship between the TSmRNA expression, evaluated by real-time PCR, and with the TS level determined by immunohistochemical assay. Thus, genotyping of the 5'UTR and quantification of the TSmRNA expression in human CRC could be considered as predictors for response to SFU-based chemotherapy. The evaluation of the TS expression by means of immunohistochemistry assay remains a safe and reliable assay in CRC.

Background: The T-cell lymphoma cell line HuT78B1, selected for resistance to Fas-mediated apoptosis, resulted unexpectedly resistant to the apoptotic and cytotoxic effects of gemcitabine (dFdC). We investigated whether this resistance was due to the impairment of the Fas/Fas-ligand (FasL) system., Materials and Methods: dFdC effects were studied in HuT78B1 and in the parental Fas-sensitive HuT78 cells exposed to inhibitors of the Fas/FasL system., Results: FasL- and Fas-blocking antibodies did not interfere with dFdC-induced apoptosis in HuT78 cells, whereas inhibitors of caspase-8, -9, -1 or -3 had partial inhibitory effects. Notably, in HuT78B1 cells there was a markedly reduced dFdC accumulation notwithstanding a high activity of the activating enzyme deoxycytidine kinase. dFdC accumulation in HuT78 cells was unaffected by a Fas-blocking antibody., Conclusion: This is the first time that the selection of a Fas-resistant cell line led to the isolation of a cell clone unable to accumulate the deoxycytidine analog dFdC. Our results show that this alteration is independent from the impairment of the Fas/FasL system.

Many anticancer drugs are able to induce apoptosis in tumor cells but the mechanisms underlying this phenomenon are poorly understood. Some authors reported that the p53 tumor suppressor gene may be responsible for drug-induced apoptosis; however, chemotherapy-induced apoptosis can also be observed in p53 negative cells. Recently, doxorubicin (DXR) was reported to induce CD95L expression to mediate apoptosis through the CD95/CD95L system. Thus, an impairment of such a system may be involved in drug resistance. We evaluated the in vitro antitumor activity of several cytotoxic drugs on two human p53-negative T-cell lymphoma cell lines, the HUT78-B1 CD95L-resistant cell line and the HUT78 parental CD95L-sensitive cell line. We demostrated by Western blotting assay that DXR and etoposide (VP-16) were able to induce CD95L expression after 4 h of treatment. In contrast, they were unable to induce the expression of p53. DXR, at concentrations ranging from 0.001 - 1 microg/ml, and VP16, at concentrations ranging from 0.05 - 1 microg/ml, were equally cytotoxic and induced apoptosis in both cell lines as assessed by fluorescence microscopy and flow cytometry analyses. Although we observed a slightly reduced percentage of apoptotic cells in HUT78B1 when compared with the parental HUT78 cells after few hours of drug exposure, this difference was no longer evident at 48 or 72 h. Similarly, the exposure of HUT78 cells to a CD95-blocking antibody partially reduced early apoptosis (24 h) without affecting the long-term effects of the drugs including cytotoxicity. Furthermore, as observed with DXR and VP-16, both the CD95L-sensitive and the CD95L-resistant cell lines resulted equally sensitive to the cytotoxic effects of a number of different cytotoxic drugs (vincristine, camptothecin, 5-fluorouracil and methotrexate). The treatment with the Caspase-3 tetrapeptide aldehyde inhibitor, Ac-DEVD-CHO, did not affect the DXR-induced apoptosis whereas it only modestly inhibited apoptosis and cytotoxicity of VP-16, while Z-VAD.FMK, a Caspase inhibitor that prevents the processing of Caspase-3 to its active form, was able to block DXR-induced apoptosis at 24 h but not at 48 h. Thus, our results do not confirm a crucial role for the CD95/CD95L system in drug-induced apoptosis and suggest the involvement of alternative p53-independent pathways at least in this experimental model system.

A group of 60 patients with advanced head/neck cancer were treated with high-dose folinic acid (500 mg/m-2/week-1) plus 5-fluorouracil (400 mg/m-2/week-1 on day 1, and cisplatin (20 mg/m-2/week-1) 24 h after folinic acid infusion was completed. Out of 55 evaluable patients, 10 patients (18%) experienced a complete response with a mean duration of 11.4+ months, 25 patients had a partial response (45%) of 6.7+ months, 6 patients (11%) showed a stabilization of 4.8+ months, and 14 (25%) progressed. The overall response rate was 63.6% (95% confidence limits 56.5%-69.5%). Patients pretreated with radiotherapy had a 67% overall response rate, while those pretreated with chemotherapy showed a 54% overall response rate. All patients with cancer of the oropharynx had a major response, while patients with cancer of the oral cavity had the lowest response rate. The mean survival of patients who attained a complete response was 14.5+ months. Partial responders had a mean survival of 10.6+ months, while patients who progresses survived a mean of 3.6+ months. The treatment has been very well tolerated with few cases of grade 3 gastrointestinal toxicity. Grade 1-2 leukopenia was recorded in 64% of cases, grade 1-2 nausea/vomiting in 85%. In one case therapy was stopped because of persistent diarrhoea.

A combination of mitomycin C, vindesine and melphalan was administered to 33 patients with heavily pretreated refractory breast cancer. The overall response rate was 27% with a mean duration of more than 10.2 months. A stabilization with a mean duration of 5.1 months was seen in 56% of cases, while 20% of patients progressed. Gastrointestinal toxicity, mostly grade 1 or 2 nausea/vomiting was seen in 85% of cases, grade 1 or 2 leukopenia in 60% of patients, and grade 1 or 2 thrombocytopenia in 42%. Considering the good compliance of this regimen and the poor prognosis of patients with refractory advanced breast cancer, this combination can be useful as a palliative treatment of breast carcinoma.

A group of 23 consecutive patients with biopsy-proven advanced or metastatic head and neck cancer were treated with cisplatinum, 100 mg/m2 i.v., on day 1 plus 5-fluorouracil, 1000 mg/m2, in continuous infusion for 5 days. Most patients (87%) had recurrent or metastatic cancer and were previously treated (78%). Out of 21 evaluable patients we obtained a 42% overall response rate (complete + partial responses) with a mean duration of more than 8 months and a 14% minimal response rate. A stabilization of disease was achieved in 28% of cases, while 14% of patients progressed. This response rate, as well as the duration of response, seems to be similar to those obtained in other series comprising previously treated patients with advanced or metastatic head and neck carcinoma. The toxicity was generally acceptable, with few cases of grade 3 (WHO criteria) toxicity. However most patients required hospitalization because of the length of treatment. In conclusion the response rate and the duration of responses obtained with cisplatinum plus a 5-day infusion of 5-FU in advanced or metastatic pretreated patients is, at present, unsatisfactory, even if the impact on survival is still not entirely clear.