Tadahiko Kubozoe

Gender differences in the dihydropyrimidine dehydrogenase expression of colorectal cancers

Dihydropyrimidine dehydrogenase (DPD) is the initial, rate-limiting enzyme in the catabolism of 5-fluorouracil (5-FU). DPD expression levels are believed to correlate with the 5-FU sensitivity of malignant tumors. In colorectal cancer (CRC), a few previous studies demonstrated that females could benefit more from adjuvant chemotherapy. However, it is still unknown why the effectiveness of postoperative chemotherapy is affected by gender. The objective of this study was to clarify the beneficial differences in 5-FU chemotherapy between genders in patients with the CRC based on DPD expression. Ninety-seven tumor specimens and 92 adjacent normal tissue specimens from 97 patients with the CRC and no prior therapy were obtained. The DPD expression in the tissues was quantified and analyzed based on clinicopathological factors. In the tumor tissue, the DPD expression in females was significantly lower than that in males. In the normal tissues, however, there were no significant differences in DPD expression between genders. In the treatment of CRC, cases who will benefit most because of 5-FU sensitivity; i.e. cases with lower DPD expression, must be given priority. Based on DPD expression, female gender seems to be a predictive factor for a better response to chemotherapy with 5-FU.

Alterations in the Fhit gene in pancreatic duct adenocarcinomas induced by N-nitrosobis(2-oxopropyl)amine in hamsters.

Alteration of the Fhit gene was investigated in pancreatic duct adenocarcinomas induced by N‐nitrosobis(2‐oxopropyl)amine (BOP) in Syrian golden hamsters. The animals received 70 mg/kg BOP, followed by repeated exposure to an augmentation pressure regimen consisting of a choline‐deficient diet combined with DL‐ethionine and then L‐methionine and administration of 20 mg/kg BOP. A total of 15 pancreatic duct adenocarcinomas were obtained 10 wk after the beginning of the experiment, and total RNAs were extracted from each for assessment of aberrant transcription of the Fhit gene by reverse transcription–polymerase chain reaction analysis. Aberrant transcripts lacking nucleotides in the regions of nt −75 to 348, nt −15 to 348, or nt −75 to 178 were detected in 11 adenocarcinomas (73.3%). Southern blot analysis of eight tumors did not show any evidence of gross rearrangement or deletion. These results indicated that changes in the Fhit gene occurred frequently and thus may have played a role in the development of pancreatic duct adenocarcinomas induced by BOP in hamsters.

Alterations of the Fhit gene in hepatocellular carcinomas induced by N-nitrosodiethylamine in rats.

The present study was conducted to assess whether Fhit gene alterations are a feature of hepatocellular carcinomas (HCCs) induced by N‐nitrosodiethylamine (DEN) in male Fischer 344 rats. Animals, 6 wk old, received a single intraperitoneal injection of DEN at a dose of 10 mg/kg body weight, followed by combined treatment with partial hepatectomy and colchicine to induce cell‐cycle disturbance and a selection procedure, consisting of 2‐acetylaminofluorene and carbon tetrachloride. Fourteen HCCs were obtained 42 wk after the beginning of the experiment; total RNA was extracted for the assessment of aberrant transcription of the Fhit gene by reverse transcriptase–polymerase chain reaction analysis. Aberrant transcripts were detected in nine of the 14 HCCs (64.3%). Sequence analysis showed that these resulted from the absence of nt −9 to 279, nt −9 to 348, nt −98 to 279, nt −26 to 365, or nt −98 to 348. Western blot analysis demonstrated reduced expression of Fhit protein in six of 10 HCCs (60.0%), with a perfect correlation with Fhit gene alterations. These results indicated that changes in the Fhit gene occur frequently and may thus play some role in the development of HCCs induced by DEN in rats. ©2002 Wiley‐Liss, Inc.

Overexpression of Midkine in Pancreatic Duct Adenocarcinomas Induced by N-Nitrosobis(2-oxopropyl)amine in Hamsters and Their Cell Lines

The expression of midkine (MK) was investigated in pancreatic ductal hyperplasias, atypical hyperplasias and adenocarcinomas induced by N‐nitrosobis(2‐oxopropyl)amine (BOP) in hamsters, and in hamster ductal adenocarcinoma cell lines (HPD‐1NR, ‐2NR and ‐3NR). MK mRNA was clearly overexpressed in invasive pancreatic duct adenocarcinomas (PCs) and the three cell lines as assessed by northern blot analysis, and MK protein expression increased from ductal hyperplasia through atypical hyperplasias, intraductal carcinomas and invasive PCs by immunohistochemistry. The extent of overexpression of MK mRNA in PCs was almost the same as in hamster whole embryonic tissue. MK is reported to be a retinoid‐responsive gene, but MK mRNA expression was not affected by treatment with all‐trans retinoic acid (tRA) or N‐(4‐hydroxyphenyl)retinamide (4‐ HPR) in HPD‐1NR cells. The results thus suggest that MK expression is involved in the development and progression of pancreatic ductal adenocarcinomas induced by BOP in hamsters, with loss of upregulation by retinoic acid.

Absence of p16, p21 and p53 gene alterations in hepatocellular carcinomas induced by N-nitrosodiethylamine or a choline-deficient l-amino acid-defined diet in rats

To clarify the involvement of tumor suppressor genes in exogenous and endogenous liver carcinogenesis, alterations of p16, p21 and p53 in hepatocellular carcinomas (HCCs) induced by N-nitrosodiethylamine (DEN) and a choline deficient L-amino acid-defined (CDAA) diet in rats were investigated. Male Fischer 344 rats received DEN at 6-week of age followed by partial hepatectomy (PH), with colchicine to induce cell cycle disturbance, and a selection pressure regimen. Sacrifice was after 42 weeks. Other animals continuously received a CDAA diet for 75 weeks and were then killed. Eleven and 15 HCCs were obtained, respectively. Total RNA was extracted from and cDNA was synthesized with reverse transcriptase to allow investigation of mutations in p16, p21 and p53 by polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) analysis. Expression of p16 and p21 mRNA was also analyzed by reverse transcription (RT)-PCR. The results showed no mutations or deletions of p16, p21 and p53 in any of the HCCs induced by DEN or CDAA. Loss or decrease of p16 and p21 expression were also not found, suggesting that p16, p21 and p53 alteration may not be necessary for either exogenous or endogenous liver carcinogenesis in rats.

Absence of β-catenin gene mutations in pancreatic duct lesions induced by N-nitrosobis(2-oxopropyl)amine in hamsters

The involvement of beta-catenin gene alterations in pancreatic duct carcinogenesis induced by N-nitrosobis(2-oxopropyl)amine (BOP) in hamsters was examined by polymerase chain reaction-single strand conformation polymorphism analysis and the expression of beta-catenin protein was examined by immunohistochemistry. No mutations of the beta-catenin gene were detected in 20 pancreatic duct adenocarcinomas (PDAs). Immunohistochemical staining showed the beta-catenin protein to be ubiquitously localized in the cell membranes. beta-Catenin accumulation was not identified in the cytoplasm and/or nucleus in any of 102 hyperplasias, 35 atypical hyperplasias, and 73 PDAs, as well as normal pancreatic duct cells. These results suggest that the Wnt/beta-catenin signaling pathway may not play an important role in pancreatic duct carcinogenesis induced by BOP in hamsters.

In vitro detection of cross-resistant and non-cross-resistant agents with fluorouracil for patients with colorectal cancer.

BackgroundFluorouracil-based chemotherapy, such as that with 5-fluorouracil (5-FU)/leucovorin, is standard as first-line chemotherapy for advanced colorectal cancer (CRC) in Japan. However, the best agent for second-line chemotherapy after fluorouracil failure is yet to be determined. This study was undertaken to find an appropriate agent for second-line chemotherapy.MethodsSeventy-five tumor specimens from CRC patients with no prior chemotherapy were obtained operatively and their chemosensitivity to five anticancer agents; i.e., 5-FU, mitomycin C (MMC), cisplatin, docetaxel, and an active metabolite of irinotecan (SN-38), was analyzed in an in vitro chemosensitivity test. In this method, the degree of chemosensitivity was expressed as the percent T/C ratio, where T was the total volume of the tumor colonies in the treated group and C was that of the control group. Pearsons correlation coefficients were used to assess the relationship between two agents.ResultsFifty-eight specimens (colon, 28; rectum, 30) were successfully analyzed. Positive correlations with 5-FU chemosensitivity were verified for the chemosensitivity of MMC, cisplatin, and docetaxel. No correlation with 5-FU chemosensitivity was verified for SN-38 chemosensitivity. Although the functional mechanism of each of the agents differs from that of 5-FU, with the exception of irinotecan, they all had a spectrum closely similar to the 5-FU spectrum.ConclusionOnly irinotecan exhibited a spectrum independent of that of 5-FU, thus indicating that it could be an appropriate agent for second-line chemotherapy after fluorouracil failure.