bluuebunny/crossref_metadata_2025 huggingface dataset:
N‐Methyl‐N‐nitrosourea Concentration‐dependent, Rather than Total Intake‐dependent, Induction of Adenocarcinomas in the Glandular Stomach of BALB/c Mice
Authors: Yamachika Takasuke , Nakanishi Hayao , Inada Ken‐ichi , Tsukamoto Tetsuya , Shimizu Nobuyuki , Kobayashi Kiyoshi , Fukushima Shoji , Tatematsu Masae
Published: August 2005

The influence of the level of carcinogen exposure on histopathological types and cellular differentiation of the induced tumors was examined in 100 male BALB/c mice given N‐methyl‐N‐nitrosourea (MNU) in their drinking water at 240 ppm on alternate weeks (total exposure: five weeks) (group 1), at 120 ppm similarly (total exposure: ten weeks) (group 2), at 60 ppm for 20 weeks continuously (group 3), or at 30 ppm for 40 weeks continuously (group 4). Forty‐three differentiated and 17 undifferentiated type adenocarcinomas were induced. Glandular stomach carcinomas and undifferentiated type lesions were more common in mice treated with a high concentration of MNU for a short period than with a low concentration of MNU for a long period, even though total measured intake of MNU was smaller (P<0.01). All the induced glandular stomach carcinomas, independent of the treatment schedule, consisted entirely of gastric phenotype cells. In conclusion, the induction of glandular stomach cancers and the proportion of undifferentiated type lesions depend not on the total quantity, but rather on the concentration of the carcinogen, while the phenotypic expression of tumor cells is not affected by the differences in the administration protocol.
Piroxicam and Acarbose as Chemopreventive Agents for Spontaneous Intestinal Adenomas in APC Gene 1309 Knockout Mice
Authors: Quesada Carlos F. , Kimata Hiroyuki , Mori Masayuki , Nishimura Masahiko , Tsuneyoshi Toshihiro , Baba Shozo
Published: August 2005

The use of nonsteroidal anti‐inflammatory drugs has been suggested to have a chemopreventive effect against colon carcinoma, through the inhibition of cyclooxygenases 1 and 2, in patients with familial adenomatous polyposis and in animal models. Acarbose, an alpha‐glycosidase inhibitor, may also be chemopreventive. In order to examine the effects of these drugs we employed APC gene knockout mice randomized into 3 groups, one for treatment with piroxicam (0.05% concentration in drinking water), one for acarbose (0.04% concentration in food) and another for the control. After 14 weeks of treatment, mice were killed for quantitation of gastric and intestinal adenomas. Tumor multiplicity in the whole gastrointestinal tract decreased from 33.89±13.07 tumors/mouse in the control group to 17.05±7 tumors/mouse in the piroxicam‐treated group (P<0.001). The decrease in the acarbose‐treated group (29.68±12.86 tumors/mouse) was not significant (P>0.05). The number of tumors ≥3 mm in diameter was also quantified in all gastrointestinal segments. The number of such tumors in the piroxicam group was decreased to 0.56±1.2 tumors/mouse from the control value of 3.78±1.17 tumors/mouse (P<0.001), while in the acarbose‐treated group the number decreased to 2.36±1.7 tumors/mouse (P<0.01). Thus, piroxicam decreases the size and number of gastrointestinal adenomas in APC 1309 knockout mice, while acarbose decreases only the size.
Heterogeneity of p53 Mutational Status in Esophageal Squamous Cell Carcinoma
Authors: Kuwabara Shirou , Ajioka Yoichi , Watanabe Hidenobu , Hitomi Jiro , Nishikura Ken , Hatakeyama Katsuyoshi
Published: August 2005

In esophageal squamous cell carcinoma, p53 gene mutations have been analyzed for inter‐ or intra‐patient heterogeneity but only a few studies have investigated intratumoral heterogeneity. We investigated this question within individual esophageal cancers, and also in their lymph‐node metastases in 8 cases. Analyzing the p53 gene sequence by direct sequencing of polymerase chain reaction products, we found heterogeneity for p53 mutations in the pre‐invasive area in 3 esophageal cancers. In all areas sampled in the invasive portion of each cancer, the p53 mutational status was identical in a given tumor. In heterogeneous tumors, the invasive area showed one of the p53 mutations found in the pre‐invasive area. In nodal metastases, the p53 mutation was identical to that in the invasive area of each primary tumor. These data suggest that the timing of p53 alteration is not as early as might have been expected, indicating that, in regard to p53 gene alteration, some esophageal cancers are composed of various subclones in the pre‐invasive stage with invasiveness developing in one of them, which becomes predominant through clonal selection.
Novel Point Mutations and Allele Loss at the RET Locus in Sporadic Medullary Thyroid Carcinomas
Authors: Uchino Shinya , Noguchi Shiro , Adachi Mitsuo , Sato Mari , Yamashita Hiroyuki , Watanabe Shin , Murakami Tsukasa , Toda Masakatsu , Murakami Nobuo , Yamashita Hiroto
Published: August 2005

Germline mutations in the RET proto‐oncogene have been shown to be the underlying cause of multiple endocrine neoplasia type 2 (MEN 2A and 2B) and familial medullary thyroid carcinoma (FMTC). Some cases of sporadic medullary thyroid carcinoma (sporadic MTC) are reported to have specific codon 918, 883 and 768 mutations of the RET gene in tumor tissues. We examined RET gene mutations in 40 Japanese cases who had previously undergone surgery for sporadic MTC. DNA extracted from formalin‐fixed tumor tissues and corresponding normal thyroid tissues or peripheral blood leukocytes was analyzed for mutations of exon 10, 11, 13, 14 and 16 of the RET gene by DNA sequencing and by mutation‐specific restriction enzyme analysis. Germline RET point mutations were found in six of 40 cases (15%), cysteine residues at codon 618 in two, codon 634 in three and valine residue at codon 804 in one, and were newly identified as heritable MTC. Of the remaining 34 sporadic MTC cases, four (12%) had tumor‐specific RET point mutations. Two were found in exon 16; one case showed an ATG to ACG (Met to Thr) mutation at codon 918, and the other showed two point mutations, ATG to ACG (Met to Thr) at codon 918 and GCA to GTA (Ala to Val) at codon 919 with loss of the wild‐type allele, suggesting that both alleles at the RET locus were altered. The other two were found in exon 13; one case showed a CCG to TCG (Pro to Ser) mutation at codon 766 and the other showed a silent mutation, GTC to GTT (Val) at codon 778 with loss of the wild‐type allele. There was no association of sporadic mutations with recurrence or prognosis in patients with sporadic MTCs. The low rate of somatic RET mutation at codon 918 in our sporadic MTC suggests that as yet unknown factors may be involved. Genetic alterations in both alleles may have an important role in a small fraction of sporadic MTCs.
Clinicopathological Criteria for Multicentricity of Hepatocellular Carcinoma and Risk Factors for Such Carcinogenesis
Authors: Kubo Shoji , Nishiguchi Shuhei , Hirohashi Kazuhiro , Shuto Taichi , Kuroki Tetsuo , Minamitani Shin , Ikebe Takashi , Yamamoto Takatsugu , Wakasa Kenichi , Kinoshita Hiroaki
Published: August 2005

Multicentric occurrence is an important characteristic of hepatocellular carcinoma. We evaluated clinicopathological criteria for multicentric hepatocellular carcinoma and identified risk factors for such carcinogenesis. Subjects were 251 consecutive patients undergoing liver resection for hepatocellular carcinoma. One kind of multicentric hepatocellular carcinoma had at least one tumor consisting of well‐differentiated hepatocellular carcinoma, together with moderately or poorly differentiated hepatocellular carcinoma located in a separate region. The other kind had an area of well‐differentiated component around hepatocellular carcinoma with less differentiation in all occurrences. The outcome of patients with tumors classified in this way was studied. Univariate and multivariate analyses were done to identify risk factors for multicentric hepatocellular carcinoma. The cumulative survival rate was significantly higher in patients with multicentric hepatocellular carcinoma than in patients with hepatocellular carcinoma associated with intrahepatic metastasis. Analysis by Cox's proportional hazard model showed that multicentricity was not a factor in the outcome. The risk of multicentric occurrence increases with progression of chronic liver disease. Univariate analysis showed hepatitis C virus marker and hepatitis B core antibody to be risk factors. By multivariate analysis, the odds ratio for multicentric occurrence in patients infected with hepatitis C virus and with serum hepatitis B virus core antibody compared with patients without either hepatitis C virus or hepatitis B virus was 10.86. This ratio in patients with hepatitis C virus alone was 4.30. These criteria for multicentric hepatocellular carcinoma seem to be clinically useful. Hepatitis C virus infection with or without former infection by hepatitis B virus is a strong risk factor for multicentric hepatocarcinogenesis.
Clotrimazole, an Imidazole Antimycotic, Is a Potent Inhibitor of Angiogenesis
Authors: Takahashi Hiroto , Abe Mayumi , Sugawara Toshiyuki , Tanaka Katsuhiro , Saito Yasuki , Fujimura Sigefumi , Shibuya Masabumi , Sato Yasufumi
Published: August 2005

Clotrimazole, an imidazole antimycotic, interferes with the rise in cytosolic Ca2+ and inhibits cell proliferation in a reversible manner. Here we describe the effect of clotrimazole on vascular endothelial cells (ECs). Clotrimazole inhibited the proliferation of ECs stimulated with typical angiogenic growth factors; vascular endothelial growth factor and basic fibroblast growth factor (bFGF). This inhibitory effect of clotrimazole was dose‐dependent and the maximal inhibition was observed at a concentration of 10 mM. We did not observe any increase in 51Cr release from ECs during treatment with 10 μM. clotrimazole. Moreover, clotrimazole inhibited the basal and bFGF‐stimulated migration of ECs. As clotrimazole inhibited two principle components of angiogenesis; the proliferation and migration of ECs, we examined whether clotrimazole inhibited angiogenesis. Tube formation by ECs in type 1 collagen gel was investigated, and clotrimazole was found to be significantly inhibitory. The inhibitory effect of clotrimazole on angiogenesis was further confirmed in an in vivo angiogenesis model of murine Matrigel plug assay. These results demonstrate that clotrimazole is a potent inhibitor of angiogenesis.
Intracellular Hyperthermia for Cancer Using Magnetite Cationic Liposomes: An in vivo Study
Authors: Yanase Mitsugu , Shinkai Masashige , Honda Hiroyuki , Wakabayashi Toshihiko , Yoshida Jun , Kobayashi Takeshi
Published: August 2005

The effect of hyperthermia on solid glioma tissue formed subcutaneously in the left femoral region of female F344 rats was investigated. Magnetite cationic liposomes (MCLs), which have a positive surface charge, were used as heating mediators for intracellular hyperthermia. MCLs were injected into the solid tumors, which were then subjected to irradiation by an alternating magnetic field (118 kHz, 384 Oe). The rats were divided into four groups, which received no irradiation (control: group I), or irradiation for 30 min given once (group II), twice (group III) or three times (group IV), and the hyperthermic effect on tumor growth was evaluated. Complete tumor regression was observed in 87.5% of the rats in group IV. In the other groups, tumors completely regressed in 20 and 60% of the rats in groups II and III, respectively. Histological observations showed that in group I tumors, MCLs were localized only around the point where they were injected, while in group II tumors they were a little more dispersed. In the cases of group III and IV tumors, however, the distribution of the MCLs was found to be widespread, and necrotic cells were observed throughout almost the entire tumor tissue. The high percentage of complete regression of group IV is considered to be due to this wide distribution of the MCLs. Furthermore, the treated rats showed no severe side‐effects. These results suggest that our magnetic particles are potentially effective tools for the treatment of solid tumors.
Mortality and Cancer Incidence in Misasa, Japan, a Spa Area with Elevated Radon Levels
Authors: Ye Weimin , Sobue Tomotaka , Lee Valerie S. , Tanooka Hiroshi , Mifune Masaaki , Suyama Akihiko , Koga Taeko , Morishima Hiroshige , Kondo Sohei
Published: August 2005

A historical cohort study was conducted in Misasa town, Tottori prefecture, Japan, where radon spas have been operating for a long time. Misasa town was divided into an elevated radon level area and a control area, with mean indoor radon levels of about 60 and 20 Bq/m3, respectively. In total, 3,083 subjects in the elevated radon level area and 1,248 in the control area, all aged 40 or older on January 1, 1976, were followed up until December 31, 1993, for a mean period of 14 years. The mortality rates from all causes exhibited no difference between the elevated radon level area and the control area for both sexes. No difference was observed in the incidence of all‐site cancers (age, period‐adjusted rate ratios by Poisson regression, RR=1.06, 95% confidence interval (CI) 0.79–1.42 for males, RR=0.90, 95% CI 0.65–1.24 for females), while stomach cancer incidence seemed to decrease for both sexes (RR=0.70, 95% CI 0.44–1.11 for male, RR=0.58, 95% CI 0.34–1.00 for female) and lung cancer incidence for males only seemed to increase (RR=1.65, 95% CI 0.83–3.30 for male, RR=1.07, 95% CI 0.28–4.14 for female) in the elevated radon level area. Caution is needed in the interpretation of these findings, however, since the individual exposure level was not measured and major confounding factors, such as smoking and diet, could not be controlled in this study.
Human T‐Lymphotropic Virus Type‐I Infection, Antibody Titers and Cause‐specific Mortality among Atomic‐bomb Survivors
Authors: Arisawa Kokichi , Soda Midori , Akahoshi Masazumi , Matsuo Tatsuki , Nakashima Eiji , Tomonaga Masao , Saito Hiroshi
Published: August 2005

There have been few longitudinal studies on the long‐term health effects of human T‐lymphotropic virus type‐I (HTLV‐I) infection. The authors performed a cohort study of HTLV‐I infection and cause‐specific mortality in 3,090 atomic‐bomb survivors in Nagasaki, Japan, who were followed from 1985–1987 to 1995. The prevalence of HTLV‐I seropositivity in men and women was 99/1,196 (8.3%) and 171/1,894 (9.0%), respectively. During a median follow‐up of 8.9 years, 448 deaths occurred. There was one nonfatal case of adult T‐cell leukemia/lymphoma (incidence rate=0.46 cases/1,000 person‐years; 95% confidence interval [CI] 0.01–2.6). After adjustment for sex, age and other potential confounders, significantly increased risk among HTLV‐I carriers was observed for deaths from all causes (rate ratio [RR]=1.41), all cancers (RR=1.64), liver cancer (RR=3.04), and heart diseases (RR=2.22). The association of anti‐HTLV‐I seropositivity with mortality from all non‐neoplastic diseases (RR=1.40) and chronic liver diseases (RR=5.03) was of borderline significance. Possible confounding by blood transfusions and hepatitis C/B (HCV/HBV) viral infections could not be precluded in this study. However, even after liver cancer and chronic liver diseases were excluded, mortality rate was still increased among HTLV‐I carriers (RR=1.32, 95% CI 0.99–1.78), especially among those with high antibody titers (RR=1.56, 95% CI 0.99–2.46, P for trend=0.04). These findings may support the idea that HTLV‐I infection exerts adverse effects on mortality from causes other than adult T‐cell leukemia/lymphoma. Further studies on confounding by HCV/HBV infections and the interaction between HCV/HBV and HTLV‐I may be required to analyze the increased mortality from liver cancer and chronic liver diseases.
Somatic Mutations of the PTEN/MMAC1 Gene in Fifteen Japanese Endometrial Cancers: Evidence for Inactivation of Both Alleles
Authors: Kurose Keisuke , Bando Koichi , Fukino Koichi , Sugisaki Yuichi , Araki Tsutomu , Emi Mitsuru
Published: August 2005

Loss of heterozygosity (LOH) of chromosome 10q is observed in approximately 40% of endometrial cancers. Mutations in PTEN/MMAC1, a gene recently isolated from the 10q23 region, are responsible for two dominantly inherited neoplastic syndromes, Cowden disease and Bannayan‐Zonana syndrome. Somatic mutations of this gene have also been detected in sporadic cancers of the brain, prostate and breast. To investigate the potential role of this putative tumor suppressor gene in endometrial carcinogenesis as well, we examined 46 primary endometrial cancers for LOH at the 10q23 region, and for mutations in the entire coding region and exon‐intron boundaries of the PTEN/MMAC1 gene. LOH was identified in half of the 38 informative cases, and subtle somatic mutations were detected in 15 tumors (33%). Our results suggest that of the genes studied so far in endometrial carcinomas, PTEN/MMAC1 is the most commonly mutated one, and that inactivation of both copies by allelic loss and/or mutation, a pattern that defines genes as “tumor suppressors,'’contributes to tumorigenesis in endometrial cancers.
Sequence Analysis of Genes Encoding Rodent Homologues of the Human Tumor‐rejection Antigen SART‐1
Authors: Gotoh Masashi , Shichijo Shigeki , Hoshino Tomoaki , Imai Yasuhisa , Imaizumi Toshihiro , Inoue Yoshiko , Takasu Hideo , Yamaoka Takashi , Itoh Kyogo
Published: August 2005

Human SART‐1 (hSART‐1) gene encodes a 125 kD protein with a leucine‐zipper motif expressed in the nucleus of all proliferating cells, and a 43 kD protein expressed in the cytosol of most epithelial cancers. In this study, two rodent genes (rSART‐1 and mSART‐1) homologous to hSART‐1 were cloned from cDNA libraries of murine brain and a rat tumor cell line, respectively. mSART‐1 and rSART‐1 were highly homologous to hSART‐1 with 86% and 84% identity at the nucleotide level, and 95% and 91% at the protein level, respectively. The leucine zipper domain and two basic amino acid portions that bind DNA, as well as peptide sequences recognized by human cyto‐toxic T lymphocytes (CTLs), were all conserved in these rodent genes. Nuclear protein homologous to the 125 kD hSART‐1800 protein, but not to the 43 kD cytosol SART‐1259 protein, was detectable with specific antibody in the nuclear fractions of rodent tumor cell lines, and normal rodent fetal liver and testis. These rodent genes should be a novel tool for studies on the biological roles of the SART‐1 gene, and also in the construction of animal models of specific immuno‐therapy using SART‐1 gene products.
The TdT‐mediated dUTP Nick End Labeling Assay Precisely Assesses the DNA Damage in Human Tumor Xenografts
Authors: Kikuchi Hiroaki , Ujiie Shigeki , Kanamaru Ryunosuke
Published: August 2005

Cultured HL‐60, HeLa S3 and WiDr cells grown in male BALB/c nu/nu mice were studied by conventional and field‐inversion DNA gel electrophoresis (FIGE), as well as by means of cytomorphological approaches, including TdT‐mediated dUTP nick end labeling (TUNEL) assay. Chemosensitivity tests revealed HL‐60 to be sensitive to vindesine (VDS), and HeLa S3 and WiDr to mitomycin C (MMC). Although VDS‐treated HL‐60 exhibited condensation of chromatin and a DNA ladder, MMC‐exposed HL‐60 cells showed apoptotic figures without typical DNA ladders. With MMC‐treated WiDr cells, neither DNA ladders nor apoptotic figures were observed. Cells characterized by chromatin condensation were TUNEL‐positive in both treated and untreated cases with the exception of the MMC‐treated WiDr case, in which many TUNEL‐positive cells were observed without cytomorphological changes. On FIGE, DNA fragments of approximately 50, 300 and 400 kbp were detected in groups treated with both effective and ineffective drugs, as well as in untreated controls. Furthermore, change of the time parameters in FIGE resulted in different sizes (550 and 850 kbp) of DNA fragments. These findings indicate that i) cell death is not always detectable in terms of apoptotic figures or DNA oligonucleosomal fragmentation, ii) only the TUNEL assay is a reliable tool to detect DNA damage and, iii) FIGE does not provide accurate size profiles of macromolecular DNA fragments.
Rhenium‐186‐mercaptoacetyltriglycine‐labeled Monoclonal Antibody for Radioimmunotherapy: In vitro Assessment, in vivo Kinetics and Dosimetry in Tumor‐bearing Nude Mice
Authors: Kinuya Seigo , Yokoyama Kunihiko , Tega Harunobu , Hiramatsu Takashi , Konishi Shota , Yamamoto Wakako , Shuke Noriyuki , Aburano Tamio , Watanabe Naoto , Takayama Terahiko , Michigishi Takatoshi , Tonami Norihisa
Published: August 2005

Stability and immunoreactivity of 186Re‐labeled monoclonal antibody were examined, and its in vivo kinetics was investigated in tumor‐bearing Balb/c nu/nu female mice to assess the feasibility of using it in radioimmunotherapy (RIT). A murine IgG1, A7, against a 45 kD glycoprotein in human colon cancer was radiolabeled with 186Re by using a chelating method with a mercaptoacetyltriglycine (MAG3). 186Re‐MAG3 complex was conjugated to A7 after esterification of 186Re‐MAG3 with tetrafluorophenol (TFP). The efficiency of 186Re‐MAG3‐TFP production and the labeling efficiency of A7 were 51–59% and 57–60%, respectively. Immunoreactivity of purified 186Re‐MAG3‐A7 was 68.2% at infinite antigen excess. In 0.9% NaCl at 4°C, the radioactivity (12.7 MBq/mg, 3.55 MBq/ml) dissociated with time from 186Re‐MAG3‐A7 as a small molecular weight moiety because of autoradiolysis. The addition of ascorbic acid, 5 mg/ml, as a radioprotectant or storage at –80°C could effectively prevent the radiolysis of 186Re‐MAG3‐A7 for 7 days. Immunoreactivity of 186Re‐MAG3‐A7, 6.70 MBq/mg (6.66 MBq/ml), stored in the presence of ascorbic acid was well retained up to 8 days after the preparation. In colon cancer xenografted mice, 31.0% of the injected dose/g of 186Re‐MAG3‐A7 had accumulated in the tumors at 24 h postinjection. Estimated radiation dose to tumors was 14.9 cGy/37 kBq up to 8 days postinjection which was 12‐fold greater than the whole‐body radiation dose. These in vivo characteristics were superior to those of A7 labeled with radioiodine, affording greater therapeutic ratios than 131I‐A7. Because of the better image quality of 186Re‐MAG3‐A7 as well as more favorable dosimetry, 186Re‐MAG3‐A7 would be a better choice for RIT of colon cancer than 131I‐A7. These results indicated the feasibility of RIT with 186Re‐MAG3‐A7, though the prevention of radiolysis of the labeled antibody should be considered.
Expression of Uroplakin Ib and Uroplakin III Genes in Tissues and Peripheral Blood of Patients with Transitional Cell Carcinoma
Authors: Yuasa Takeshi , Yoshiki Tatsuhiro , Tanaka Tsutomu , Kim Chol Jang , Isono Takahiro , Okada Yusaku
Published: August 2005

Uroplakins (UPs), urothelium‐specific transmembrane proteins, are present only in urothelial cells. We have determined the nucleotide sequences of human UP‐Ib and UP‐III and synthesized specific primer pairs. The two UP genes were expressed in both cancerous and noncancerous urothelia taken from all patients examined by reverse transcription‐polymerase chain reaction (RT‐PCR). These genes were also detected in the peripheral blood of 3 patients with metastatic transitional cell carcinoma (TCC), but not in that from 9 patients with non‐metastatic TCC or 3 healthy volunteers. The sensitivity of our assay was sufficient to detect one cancer cell in 5 ml of peripheral blood. Detection of UP gene‐expression in blood by RT‐PCR may provide helpful information for the diagnosis and management of TCC.
Somatic Mutations in the RET Protooncogene in Japanese and Chinese Sporadic Medullary Thyroid Carcinomas
Authors: Shan Liang , Nakamura Misa , Nakamura Yasushi , Utsunomiya Hirotoshi , Shou Nanhai , Jiang Xihong , Jing Xuefeng , Yokoi Toyoharu , Kakudo Kennichi
Published: August 2005

Despite advances in the understanding of the genotype‐phenotype correlation in multiple endocrine neoplasia type 2A and 2B (multiple endocrine neoplasia (MEN) 2A, MEN 2B), and familial medullary thyroid carcinoma (FMTC), the frequency and prognostic relevance of RET protooncogene mutations in sporadic medullary thyroid carcinomas (MTCs) remain controversial. To study somatic mutations in the RET protooncogene in Japanese and Chinese sporadic MTCs and to analyze comparatively the correlation between RET mutation and tumor differentiation, we investigated somatic mutations in the RET protooncogene in 20 Japanese and 20 Chinese sporadic MTCs by the polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) method. Of the 40 sporadic MTCs, 13 had a point mutation in codon 918 of exon 16, a frequency of 32.5%. There was no significant difference in the frequency between Japanese and Chinese sporadic MTCs, as 30% of the Japanese and 35% of the Chinese sporadic MTCs contained this mutation. We did not observe any correlation between the presence or absence of codon 918 mutation and tumor differentiation in either Japanese or Chinese sporadic MTCs. Our findings indicate that the frequency of RET somatic mutations is similar in Japanese and Chinese sporadic MTCs, and the presence or absence of RET mutation does not correlate with the differentiation of sporadic MTCs.
Abnormalities of the FHIT Transcripts in Osteosarcoma and Ewing Sarcoma
Authors: Hinohara Shin‐ichi , Satake Noriko , Sekine Ki‐ichi , Kaneko Yasuhiko
Published: August 2005

In a study of FHIT gene abnormalities by reverse transcription‐polymerase chain reaction (RT‐PCR) and sequence analysis of the PCR products, we found normal and abnormal PCR products in 11 osteosarcomas, one osteosarcoma cell line and 3 Ewing sarcomas, and a normal PCR product only in 5 osteosarcomas and 8 Ewing sarcomas. Sequence analysis of the abnormal PCR products revealed 7 osteosarcomas lacking exons 4 to 6, 7, 8 or 9, two lacking exons 5 to 7 or 10, and two lacking exons 6 to 8 or 10. In the aberrant transcripts of the 11 osteosarcomas, fusion had occurred in the exon/intron junctions in 2 tumors, between a segment within an exon and a complete exon in 3, and between segments within exons in 6. The 3 Ewing sarcomas had lost exon 4 or 5 to exon 6 or 10, and fusion had occurred in the exon/intron junction in one, and between segments within exons in 2. These findings suggest that both abnormal or variant splicing and other mechanisms such as genomic instabilities in the FHIT locus may have resulted in the expression of aberrant transcripts. One osteosarcoma and one cell line established from this osteosarcoma showed different abnormal FHIT transcripts, indicating that the tumor cells with the initial aberrant transcripts may not have had a selective advantage for proliferation. The FHIT abnormalities did not seem to be correlated with lung metastasis or a poor clinical outcome in our patients with osteosarcoma or Ewing sarcoma.
Association of Replication Error Positive Phenotype with Lymphocyte Infiltration in Endometrial Cancers
Authors: Kihana Toshimasa , Fujioka Toru , Hamada Katsuyuki , Kito Katsumi , Takahashi Akira , Tsukayama Choutatsu , Ito Masaharu
Published: August 2005

Microsatellite instability (MI) has been detected in certain sporadic cancers as well as in hereditary non‐polyposis colorectal cancer (HNPCC). In order to determine the precise clinicopathological characteristics of MI in endometrial cancer, we examined 90 sporadic endometrial cancers (83 endometrioid adenocarcinomas, 3 adenosquamous carcinomas, 3 papillary serous carcinomas, and 1 clear cell carcinoma) and eight lesions of endometrial hyperplasia for replication error (RER) using polymerase chain reaction amplification of CA repeated microsatellite sequences at 15 loci. RER was observed in 23 (28%) of the 83 endometrioid adenocarcinomas at at least one locus and in 19 (23%) at two or more loci (RER+ phenotype) in the seven most commonly observed loci, but not in carcinomas of other histological types or in endometrial hyperplasia. Lymphocyte infiltration around carcinoma cells, which is one of the histological features seen in tumors from HNPCC, was severer in RER+ phenotype tumors (79%, 11/14) than in the RER– tumors (25%, 11/44) (marked/moderate infiltration versus slight, P<0.001, χ2 test), when 58 tumors with muscular invasion were examined. The RER+ phenotype was associated with a higher parity and gravidity (P<0.05, Wilcoxon test). However, RER+ phenotype was not associated with tumor stage, histological grade, muscular invasion, lymph node metastasis or patient survival. In conclusion, MI occurs in a subset of endometrial cancers, which often show marked infiltration of lymphocytes around the tumor.
Immunological Quantitation of DT‐Diaphorase in Carcinoma Cell Lines and Clinical Colon Cancers: Advanced Tumors Express Greater Levels of DT‐Diaphorase
Authors: Mikami Koji , Naito Mikihiko , Ishiguro Tatsuaki , Yano Hideaki , Tomida Akihiro , Yamada Takeshi , Tanaka Noritake , Shirakusa Takayuki , Tsuruo Takashi
Published: August 2005

NAD(P)H:quinone oxidoreductase (DT‐diaphorase; DTD) plays a major role in activating mitomycin C (MMC) in human colon and gastric carcinoma cell lines. Thus, measurement of DTD in clinical tumor samples could be beneficial in designing adjuvant chemotherapy. We explored immunological quantitation of DTD protein using a monoclonal antibody against DTD, demonstrating a close correlation between protein expression and enzyme activity of DTD in colon and gastric carcinoma cell lines and in colorectal tumor samples. This indicates that such immunoblot analysis is a simple alternative method for quantitating DTD in clinically excised samples. In most colorectal tumor samples, the tumors expressed larger amounts of DTD than did the peripheral normal tissues, suggesting a selective toxicity of MMC toward tumor cells. Also tumors with nodal metastases showed significantly higher DTD levels than did tumors without metastasis. These results raise the possibility that DTD expression is related to tumorigenesis and malignant progression of colorectal tumors. Measurement of DTD by the immunological method described here could be beneficial in designing a rational adjuvant chemotherapy with MMC.
Induction of Apoptosis in Human Pancreatic Carcinoma Cells by a Synthetic Bleomycin‐like Ligand
Authors: Suginaka Rei , Izui Reiko , Inoue Jun‐ichiro , Muraoka Yasuhiko , Yamaguchi Ken , Otsuka Masami , Umezawa Kazuo
Published: August 2005

Histidine‐pyridine‐histidine‐3 (HPH‐3) is an oxygen‐activating ligand based on the structure of bleomycin. HPH‐3 induced the death of human pancreatic adenocarcinoma AsPC‐1 cells in 24 h, causing apoptotic morphology and internucleosomal degradation of DNA. HPH‐3‐induced cell death was not inhibited by antioxidants such as reduced glutathione and N‐acetylcysteine, whereas hydrogen peroxide‐induced cell death was inhibited by them, indicating that hydrogen peroxide is not involved in the induction of apoptosis by HPH‐3. Induction of apoptosis by HPH‐3 was inhibited by zinc and copper ions, indicating that chelation with ferrous ion is responsible for induction of apoptosis, as is the case in chelation by bleomycin to cleave DNA. Bleomycin A2 and its fragment having no DNA‐binding region, glycopeptide‐3, did not induce apoptosis in AsPC‐1 cells. Bleomycin A2 induced G2/M block in flow‐cytometric analysis, but HPH‐3 did not and instead induced an apoptotic pre‐G1 peak. Thus, HPH‐3 induced apoptosis in human pancreatic carcinoma cells, which is a unique characteristic among bleomycin‐related compounds.
Preferential Binding of E7010 to Murine β3‐Tubulin and Decreased β3‐Tubulin in E7010‐resistant Cell Lines
Authors: Iwamoto Yasuo , Nishio Kazuto , Fukumoto Hisao , Yoshimatsu Kentaro , Yamakido Michio , Saijo Nagahiro
Published: August 2005

N‐[2‐[(4‐Hydroxyphenyl)amino]‐3‐pyridyl]‐4‐methoxybenzenesulfonamide (E7010) is a novel sulfonamide antimitotic agent, which is active against mouse and human tumors. E7010 binds to β‐tubulin and inhibits polymerization of microtubules. In order to clarify the mechanisms of E7010‐resistance, two murine leukemic P388 subclones resistant to E7010, 0.5r‐D and 4.0r‐M, were characterized. The two clones showed approximately 10‐ and 100‐fold resistance to E7010‐induced growth‐inhibitory effects, respectively, compared with the parental cells in 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. These cell lines showed no cross‐resistance to other anticancer agents such as taxanes, vinca alkaloids, mitomycin C, cisplatin and irinotecan hydrochloride (CPT‐11). Increased α‐ and β‐tubulin protein and mRNA levels were observed in 0.5r‐D and 4.0r‐M cells as compared with the parental cells. We examined the isotype‐specific expression of β‐tubulin in these E7010‐resistant cells by a competitive reverse transcription‐polymerase chain reaction method. Although a 50% increase in β5 isotype mRNA levels was observed in 4.0r‐M cells, the levels of β3 isotype message in the two resistant clones were approximately 50% less than the parental cells. To elucidate the binding properties of E7010 with β‐tubulin isotypes, we prepared isotype‐specific fusion proteins of β‐tubulins. Direct photoaffinity labelling of the isotype‐specific fusion proteins with [14C]E7010 revealed that E7010 preferentially binds to the β3 isotype rather than β2, β4, and β5 isotype proteins. Therefore, altered expression of β‐tubulin isotypes, especially β3 isotype, to which E7010 binds with high affinity, may account for the decreased sensitivity of these resistant clones to E7010.
Association of Helicobacter pylori Infection with Gastric Adenocarcinoma
Authors: Lee Byung Mu , Jang Ja‐June , Kim Joung‐Soon , You Young Chan , Chun Sun Ah , Kim Hyung Sik , Han Hyung Mee , Ahn Mi Young , Byun Soo Hyun
Published: August 2005

Gastric adenocarcinoma is the most prevalent cancer in South Korea, and Helicobacter pylori (H. pylori) infection is also common. This study was performed to examine the association between H. pylori infection and gastric cancer, taking into account various other factors. To investigate the association between gastric adenocarcinoma and H. pylori infection, determined by urease‐positive reaction in the CLO test, a total of 175 paired specimens (175 tumor and 175 tissues adjacent to tumor) of stomach cancer patients and a total of 113 control specimens were obtained. The positive H. pylori infection rates were 78.9% (138/175) among the patients in specimens of tumor or tissues adjacent to the tumor and 41.6% (47/113) among controls in the CLO test. A positive correlation between H. pylori infection and gastric cancer was observed (age‐adjusted odds ratio, 7.0; MH χ2=34.5 with P<0.0005). These data suggest that stomach cancer patients in Korea have high infection rates of H. pylori regardless of site specificity, and this infection might be causally associated with stomach cancer.
Provirus Load in Patients with Human T‐Cell Leukemia Virus Type 1 Uveitis Correlates with Precedent Graves' Disease and Disease Activities
Authors: Ono Ayako , Ikeda Eiko , Mochizuki Manabu , Matsuoka Masao , Yamaguchi Kazunari , Sawada Takashi , Yamane Sumitaka , Tokudome Shinkan , Watanabe Toshiki
Published: August 2005

We previously demonstrated the increased provirus load in the peripheral blood of patients with human T‐cell leukemia virus type 1 (HTLV‐1) uveitis (HU). To delineate the relevance of the increased provirus load to clinical and immunologic parameters, we studied the correlation between them. Seventy‐nine HU patients (24 male and 55 female) were included in the study, with their informed consent. Plasma samples and genomic DNA of the peripheral blood mononuclear cells were isolated and the provirus load was estimated by semi‐quantitative polymerase chain reaction of the gag region sequence. Serum levels of anti‐HTLV‐1 antibodies and soluble IL‐2R were determined by electrochemiluminescence immuno assay and by ELISA, respectively. Disease activities were assessed and graded 0 to 4 according to the evaluation system. Recurrence of the disease during the follow‐up period was diagnosed ophthalmologically. The provirus load was significantly higher in the HU patients after Graves' disease (GD) than in those without GD (P<0.05). It correlated with disease activities assessed in terms of vitreous inflammation and interval to recurrence (both P<0.05). In the HU patients without GD, it correlated with the serum levels of soluble IL‐2 receptor (P<0.01), and nearly with those of HTLV‐1 antibody (P=0.063). These correlations were not found in the HU patients after GD under methimazole treatment. The results suggested a direct involvement of HTLV‐1‐infected cells in the pathogenesis of uveitis, and raise the possibility that hyperthyroidism may contribute to the clonal expansion of HTLV‐1‐infected cells.
Cell Cycle‐dependent Modulation of Promoter Activities of RB and WAF1/Cip1 Genes
Authors: Matsumoto Takuji , Ohtani‐Fujita Naoko , Sowa Yoshihiro , Bai Fulu , Nikaido Toshio , Tamaki Tetsuya , Sakai Toshiyuki
Published: August 2005

A universal inhibitor of cyclin‐dependent kinases, WAF1/Cip1 can dephosphorylate the RB gene product to arrest the cell cycle at the G1 phase. Here we show that the mRNA level and the promoter activities of the RB and WAF1/Cip1 genes exhibit cell cycle‐dependent change when cells are released from either serum‐starvation or the confluent cell state with serum. RB expression and promoter activity are elevated at middle to late G1. In contrast, the mRNA and promoter activity of the WAF1/Cip1 gene increase at early G1. These results suggest that the RB and WAF1/Cip1 expression and promoter activities depend not only on serum, but also on the cell cycle progression itself. Moreover, we identified the responsive region for serum‐released cell cycle progression in the RB promoter and mapped it to the region between –4 and –182 relative to the initiating codon of the RB gene. The region in the WAF1/Cip1 promoter responsible for the serum‐released cell cycle progression mapped not to the p53 binding site, but to the 374 base‐pair region between –1770 and –1396 from the transcription start site.
Alterations of RET, Oncogene in Human Adrenal Tumors
Authors: Lin Shiu‐Ru , Yang Yuan‐Chieh , Tsai Juei‐Hsiung , Hsu Chin‐Hsun
Published: August 2005

Previous studies have revealed specific activations of the RET oncogene in multiple endocrine neoplasia type 2 (MEN 2) and thyroid tumors. To understand the role of the RET proto‐oncogene activation in sporadic adrenal tumors, we analyzed the alterations of the RET proto‐oncogene in the cysteine‐rich extracellular domain (exons 6 and 10), the terminal region of the extracellular domain and transmembrane domain (exon 11) and the tyrosine kinase domain (exons 12–17) in 35 cases of adrenal tumors (including 18 Conn's syndrome, 3 Cushing's syndrome, 2 non‐functional adrenocortical tumor and 12 pheochromocytomas by polymerase chain reaction‐single strand conformational polymorphism and sequencing methods. One case with pheochromocytoma and one with Conn's syndrome had point mutation. We also detected the rearrangement of the RET gene by reverse transcription‐polymerase chain reaction and Southern hybridization. One case with Conn's syndrome and one with Cushing's syndrome were found to harbor RET/PTC1 (RET tyrosine kinase domain rearranged with H4 gene). The above results indicate that RET protooncogene mutations and RET/PTC1 are involved in the pathogenesis of sporadic adrenal tumors. Mutations at codon 634 of the RET gene were also found in adrenal tumors. This suggests that the RET oncogene may also play a role in the tumorigenesis of adrenal tumors, and this possibility requires further investigation.
Identification of Glutathione S‐Transferase p‐1 as the Class Pi Form Dominantly Expressed in Mouse Hepatic Adenomas
Authors: Ookawa Keizou , Nakano Hajime , Kakizaki Ikuko , Hatayama Ichiro , Kajihara‐Kano Hiroko , Kimura Junya , Hayakari Makoto , Takahata Takenori , Satoh Kimihiko , Tsuchida Shigeki
Published: August 2005

To clarify which of the two genes for pi class glutathione S‐transferases (GSTs) (p‐1 and p‐2) is dominantly expressed in mouse hepatic adenomas, the relative mRNA levels were examined by means of the reverse transcription‐polymerase chain reaction (RT‐PCR). Hepatic adenomas were induced in male and female B6C3F1 mice by diethylnitrosamine treatment. Northern blot analysis revealed that pi class mRNA levels were decreased in adenomas of male mice, but increased in those of females, with reference to the respective surrounding non‐adenoma tissues. In contrast to the marked sex difference in surrounding tissues, pi class GST mRNA levels in adenomas were almost the same in both males and females. To evaluate p‐1 and p‐2 mRNA levels separately, the products of RT‐PCR employing primers common for both cDNAs were digested with the endonuclease BanI (specific for p‐2) and then resolved by electrophoresis. The p‐1 mRNA was thus found to be dominant in adenomas of both female and male mice. The p‐2 mRNA levels were increased in the lesions as compared with those in the surrounding non‐adenoma tissues. Recombinant p‐1 and p‐2 proteins were expressed in Escherichia coli. Unlike p‐1, the p‐2 protein did not show any significant activity towards 1‐chloro‐2,4‐dinitrobenzene and did not bind to S‐hexylglutathione‐Sepharose despite immunological cross‐reactivity. The dominant pi class form in adenomas could also be identified as p‐1 by its binding to S‐hexylglutathione‐Sepharose. Single radial immunodiffusion analyses confirmed that the p‐1 protein levels were in line with the mRNA findings, i.e., 1.9±0.3 mg/g adenoma as compared to 6.5±1.2 mg/g non‐adenoma tissue for males and 2.2±0.6 mg/g as compared to 0.7±0.2 mg/g for females. The results thus indicated that the change of pi class forms in adenomas is caused mainly by alteration in the p‐1 level and the contribution of p‐2 is minimal.
Significant Correlation of Nitric Oxide Synthase Activity and p53 Gene Mutation in Stage I Lung Adenocarcinoma
Authors: Fujimoto Hisao , Sasaki Ji‐ichiro , Matsumoto Mitsuhiro , Suga Moritaka , Ando Yukio , Iggo Richard , Tada Mitsuhiro , Saya Hideyuki , Ando Masayuki
Published: August 2005

Nitric oxide (NO) and its derivatives can directly cause DNA damage and mutation in vitro and may play a role in the multistage carcinogenic process. It has been reported that NO induces mutation in the p53 tumor suppressor gene; we therefore analyzed the relationship between NO synthase 9 activity and p53 gene status in early‐stage lung adenocarcinoma. Surgical samples were classified into two categories: 14 lung adenocarcinomas with high NOS activity (>25 pmol/min/g tissue, category A), and 16 with low NOS activity (<25 pmol/min/g tissue, category B). A yeast functional assay for p53 mutations disclosed a red colony that corresponded to a mutation in the p53 gene in 8 cases (57.1%) in category A and 3 cases (18.8%) in category B, the frequency being significantly higher in the former (P<0.05). A p53 DNA sequence analysis revealed that 5 of the 8 p53 mutation‐positive samples in category A had a G:C‐to‐T:A transversion, which is reported to be a major target of NO. The mechanism of carcinogenesis of adenocarcinoma is not fully understood, but these results suggest that an excess of endogenously formed NO may induce a p53 gene mutation containing mainly G:C‐to‐T:A transversion in the early stage of lung adenocarcinoma. Our results suggest that NO has potential mutagenic and carcinogenic activity, and may play important roles in human lung adenocarcinoma.
Inhibitory Effects of Ginsenoside Rh2 on Tumor Growth in Nude Mice Bearing Human Ovarian Cancer Cells
Authors: Nakata Hideyuki , Kikuchi Yoshihiro , Tode Takehiko , Hirata Junko , Kita Tsunekazu , Ishii Kenji , Kudoh Kazuya , Nagata Ichiro , Shinomiya Nariyoshi
Published: August 2005

Ginsenoside Rh2 (Rh2), isolated from an ethanol extract of the processed root of Panax ginseng CA Meyer, inhibits the growth of B16 melanoma cells. This study was designed to evaluate the ability of Rh2 to inhibit growth of human ovarian cancer cells (HRA) in vitro and in nude mouse. Rh2 inhibited proliferations of various established human ovarian cancer cell lines in a dose‐dependent manner between 10 and 60 μM in vitro and induced apoptosis at around the IC50 dose. When HRA cells were inoculated s.c. into the right flank of nude mice, all mice formed a palpable tumor within 14 days. Although i.p. administration of Rh2 alone hardly inhibited the tumor growth, when Rh2 was combined with cis‐diamminedichloroplatinum(II) (CDDP) the tumor growth was significantly inhibited, compared to treatment with CDDP alone. When mice were treated p.o. with Rh2 daily (but not weekly), the tumor growth was significantly (P<0.01) inhibited, compared to CDDP treatment alone. When Rh2 was combined with CDDP, the degree of tumor growth retardation was not potentiated. The survival time was significantly (P<, we examined whether p.o. administration of Rh2 has a dose‐dependent inhibitory effect on the tumor growth. I.p. and weekly administration of CDDP had more potent antitumor activity in the order of 1 mg/kg, 2 mg/kg and 4 mg/kg, whereas p.o. and daily administration of Rh2 (0.4 to 1.6 mg/kg) not only had antitumor activity comparable to that of 4 mg/kg CDDP, but also resulted in a significant increase of the survival. Doses of Rh2 used in this study did not result in any adverse side‐effects as confirmed by monitoring hematocrit values and body weight, unlike 4 mg/kg CDDP, which had severe side‐effects. It is noteworthy that p.o. but not i.p. treatment with Rh2 resulted in induction of apoptotic cells in the tumor in addition to augmentation of the natural killer activity in spleen cells from tumor‐bearing nude mice. Thus, particularly in view of the toxicity of CDDP, Rh2 alone would seem to warrant further evaluation for treatment of recurrent or refractory ovarian tumor.
Growth Inhibition of CD20‐positive B Lymphoma Cell Lines by IDEC‐C2B8 Anti‐CD20 Monoclonal Antibody
Authors: Taji Hirohumi , Kagami Yoshitoyo , Okada Yasutaka , Andou Manabu , Nishi Yoshimi , Saito Hidehiko , Seto Masao , Morishima Yasuo
Published: August 2005

Treatment with IDEC‐C2B8 (C2B8), the chimeric anti‐CD20 antibody, was shown in a phase I‐II study to be very effective for the treatment of low‐grade B‐cell lymphoma, in contrast to the results of most previous immunotherapies with monoclonal antibodies. In a study designed to elucidate the reason for this efficacy, two cell lines derived from lymphomas with BCL2 gene rearrangement (SU‐DHL‐4 and SU‐DHL‐6) showed remarkable growth inhibition and cell‐death, and two other cell lines derived from a diffuse lymphoma (RC‐K8) and a mantle cell lymphoma (SP‐49) showed moderate growth inhibition, but neither a CD20 weakly positive cell line (NALL‐1) nor a negative cell line (MOLT‐4) showed any growth inhibition. An examination of the intensity of cell‐surface CD20 expression showed no correlation between intensity and degree of growth inhibition among the four cell lines showing growth inhibition. Morphological examination revealed condensed and fragmented nuclei and budding of the plasma membrane, both characteristic of apoptosis, with some cells in these cell lines showing growth inhibition by C2B8. Such apoptosis was also confirmed by flow cytometric analysis, suggesting that, at least in part, apoptosis plays a role in this growth inhibition. This growth‐inhibitory mechanism may thus account for the effectiveness of C2B8 antibody therapy.