Associations of various gene polymorphisms with toxicity in colorectal cancer patients receiving oral uracil and tegafur plus leucovorin: a prospective study.

BACKGROUND: To assess the predictive value of polymorphism in nine genes, primarily thymidylate synthase (TS) and orotate phosphoribosyltransferase (OPRT), which relates to 5-fluorouracil (5-FU) metabolism, for toxicity in patients treated with oral uracil/tegafur (UFT) plus leucovorin (LV). PATIENTS AND METHODS: We treated 99 patients with stage II or III colorectal carcinoma with oral UFT + LV. Germline DNA from patients was genotyped for 5-FU and folate metabolism-relating genes. CYP2A6, tegafur-activating enzyme, and uridine diphosphate-glucuronosyltransferase 1A1 genetic variation were also assessed. Toxicity was graded by the National Cancer Institute Common Toxicity Criteria, version 2.0. RESULTS: The multivariate logistic regression revealed that OPRT 638G>C polymorphism was associated with grade 3 diarrhea [odds ratio (OR) 19.84 for patients with the C/C homozygous type compared with patients with wild type, P = 0.014] and polymorphisms of UGT1A1 were associated with hyperbilirubinemia (OR 38.76 for homozygotes and double heterozygotes of *6 or *28 compared with wild type, P = 0.0008). No relationships were observed between TS polymorphisms and any toxicity. CONCLUSIONS: OPRT polymorphism predicts toxicity, especially grade 3 or greater diarrhea to oral UFT + LV adjuvant chemotherapy, whereas TS does not, in our study cohort. UGT1A1 polymorphism seems to be a risk factor for hyperbilirubinemia due to UFT+LV.

Low expression of gamma-glutamyl hydrolase mRNA in primary colorectal cancer with the CpG island methylator phenotype.

The CpG island methylator phenotype (CIMP+) in colorectal cancer (CRC) is defined as concomitant and frequent hypermethylation of CpG islands within gene promoter regions. We previously demonstrated that CIMP+ was associated with elevated concentrations of folate intermediates in tumour tissues. In the present study, we investigated whether CIMP+ was associated with a specific mRNA expression pattern for folate- and nucleotide-metabolising enzymes. An exploratory study was conducted on 114 CRC samples from Australia. mRNA levels for 17 genes involved in folate and nucleotide metabolism were measured by real-time RT-PCR. CIMP+ was determined by real-time methylation-specific PCR and compared to mRNA expression. Candidate genes showing association with CIMP+ were further investigated in a replication cohort of 150 CRC samples from Japan. In the exploratory study, low expression of gamma-glutamyl hydrolase (GGH) was strongly associated with CIMP+ and CIMP+-related clinicopathological and molecular features. Trends for inverse association between GGH expression and the concentration of folate intermediates were also observed. Analysis of the replication cohort confirmed that GGH expression was significantly lower in CIMP+ CRC. Promoter hypermethylation of GGH was observed in only 5.6% (1 out of 18) CIMP+ tumours and could not account for the low expression level of this gene. CIMP+ CRC is associated with low expression of GGH, suggesting involvement of the folate pathway in the development and/or progression of this phenotype. Further studies of folate metabolism in CIMP+ CRC may help to elucidate the aetiology of these tumours and to predict their response to anti-folates and 5-fluorouracil/leucovorin.

[Analysis of cell kinetics using BrdU monoclonal antibody on cultured tumor cells after irradiation and treatment with anti-cancer drugs].

Flow cytometry (FCM) permits instantaneous determination of the percentages of cells in various phases of cell cycle using BrdU-PI double staining method, and allowing rapid evaluation of the effects of irradiation and anti-cancer drugs (ACNU, ADR, BLM) on the cell kinetics. In this study, the growth inhibition and changes in the cell kinetics after irradiation and chemotherapy were examined according to the growth curve analysis and BrdU-PI method to evaluate the usefulness of BrdU-PI method for assessment of the effect of the treatments. By the conventional method based on the DNA histogram, accurate determination of S cell fraction was difficult due to overlapping of the DNA contents of G1 cells and early S cells and those of late S cells and G2 cells. BrdU-PI double staining allowed direct differentiation of G1, S, and G2 + M cells, especially between G1-S and S-G2 + M cells. The analysis of cell kinetics using BrdU is advantageous in comparison to the conventional autoradiographic methods because it allows more rapid assay with very high sensitivity. By the present BrdU method, rapid transition to the G1-S phase was observed within 4 hours after exposure to radiation and anti-cancer drugs. This initial G1 arrest induced by irradiation was confirmed for the first time by the present BrdU-PI double staining. The present method is considered to be indispensable for evaluation of the percentage of S cells in the tumor tissue and analysis of cell kinetics after irradiation and chemotherapy against cancer.

Improvement of IGCR technique using FRET.

The fluorescent detection system has been introduced into the study on denaturation/reassociation process of DNA fragments in gel, for improving In-Gel Competitive Reassociation technique, one of genome subtraction methods. The annealing behaviour of the mixture of 3'-Fluorescein-labelled and 5'-Cy5-labelled DNA fragments was analysed by Fluorescence Resonance Energy Transfer (FRET) technique from donor Fluorescein to acceptor Cy5. We showed that two fluorescent dyes labelled at 3' and 5' ends of DNA fragments caused FRET in both the solution and the gel. The characterisation of fluorescence-labelled fragments in gel and the changes of their fluorescence intensity will be reported.

Analysis of DNA hybridization in polyacrylamide gel.

We have made the first apparatus for fluorescent detection, monitoring the hybridization process of fluorescently labeled DNA fragments in a polyacrylamide gel. Using this, the analysis on the thermal denaturation/reassociation process of DNA fragments in the gel was employed, for improving the performance of In-Gel Competitive Reassociation (IGCR) technique, one of genome subtraction methods. We showed that Fluorescence Resonance Energy Transfer (FRET) in the gel occurred by positioning two fluorescent dyes at 3' and 5' ends of DNA fragments. The characterization of fluorescence-labelled fragments in gel and the changes of their fluorescence intensity will be reported.

[Biochemical characterization of phenytoin-induced hyperplastic human gingival fibroblasts. Non-collagenous proteins biosynthesis].

Phenytoin (PHT), administered as an anticonvulsant, has a side effect gingiva overgrowth in approximately 50% of patients. The present study was attempted to explore the biochemical mechanism on non-collagenous protein biosynthesis as affected by PHT. Responder cells (RES A3, RES C2) of a patient with gingival overgrowth were obtained by the method of Kawase et al. Normal human gingival fibroblasts (Gin-1), purchased from ATCC, were also used. All cells were inoculated at 1 x 10(4) cells/cm2 (12 multi-well plate or 60 mm tissue culture dish), and then cultured for 4, 8 and 12 days with or without PHT (5 micrograms/ml). Prior to harvesting at the indicated times, cells were incubated with 14C-amino acids (1.25 microCi/ml) for 24 hours. The 14C-labeled proteins were isolated from the cell layers including extracellular matrix, following Kurkinen et al. with a minor change. Each 14C-labeled fraction was dissolved in 3 ml of Aquasol-2 and the radioactivity by a liquid scintillation counter. The DNA content of cell layers affected by PHT was increased on Gin-1, RES A3 and RES C2 at the post-confluence, resulting also in an increase in cell number. Two morphologically different phenotypes of responder cells were observed, differing in nuclear and cell sizes. At 12 days culture, RES A3, were stimulated by PHT, showed increased synthesis of both total extractable proteins (EP) and binding proteins (BP) labeled with 14C-amino acids. Therefore, at least two distinct phenotypic responder cells are present in the PHT-induced overgrowth gingiva, alter the synthesis of non-collagenous proteins.