bluuebunny/crossref_metadata_2025 huggingface dataset:
Mapping of Melanoma Modifier Loci in RET Transgenic Mice
Authors: Dragani Tommaso A. , Peissel Bernard , Zanesi Nicola , Aloisi Alessandra , Dai Yan , Kato Masashi , Suzuki Haruhiko , Nakashima Izumi
Published: August 2005

Transgenic mice carrying the RET oncogene under the control of the metallothionein promoter exhibit severe pigmentation of the whole skin and melanocytic tumors. The genetic background influences melanoma development in RET mice; founder mice crossed with BALB/c mice show decreased incidence and increased latency of melanocytic tumors, whereas progeny of C57BL/6 mice show the opposite effect. Using partially congenic RET mice on a C57BL/6 genetic background (N3/RET mice), we studied genetic linkage in (N3/RETxBALB/c)xN3/RET backcross mice. We mapped three melanoma modifier loci, on chromosome 1 (Melm1 and Melm2) and chromosome 11 (Melm3), that are linked with early melanoma incidence and latency. Mapping of Melm loci and of five additional regions on chromosomes 6, 8, 9, 12, and 13 indicated allelic imbalance in N3/RET mice, with a significant excess of BALB/c alleles, suggesting the presence of additional putative melanoma modifier loci on these chromosomes.
Induction of Apoptosis and Cell Cycle Arrest in Mouse Colon 26 Cells by Benastatin A
Authors: Kakizaki Ikuko , Ookawa Keizoh , Ishikawa Takashi , Hayakari Makoto , Aoyagi Takaaki , Tsuchida Shigeki
Published: August 2005

Benastatin A, isolated from Streptomyces bacteria, is reported to inhibit mammalian glutathione transferases (GSTs). Since GST inhibitors such as ethacrynic acid are suggested to induce apoptosis in some cell lines, the effect of benastatin A on the survival of mouse colon 26 adenocarcinoma cells was compared with that of ethacrynic acid. When cells in stationary phase were treated with benastatin A, viable cells were found to be dose‐dependently decreased after 3 days. In the case of ethacrynic acid, this became apparent within 24 h. Electrophoretic analysis revealed DNA fragmentation, indicating that cell loss was due to apoptosis in both cases. The dominant GST in colon 26 cells was identified as the class Pi‐form (GST‐II), and the activities in crude extracts as well as purified GST‐II were almost completely inhibited by 50 μM ethacrynic acid. Immunoblot and northern blot analyses revealed increased GST‐II protein and mRNA levels in cells treated with ethacrynic acid. Benastatin A did not significantly affect the activity in the crude extract even at 20 μM, a 10‐fold higher concentration than that which almost completely inhibited the activity of purified GST‐II. However, GST activity and GST‐II protein were decreased in colon 26 cells treated with benastatin A for 5 days, no significant activity being detected in the range of 16–20 μM. In addition, β‐actin and bax mRNAs were also decreased in a dose‐dependent manner. Furthermore, flow cytometric analysis of colon 26 cells revealed that benastatin A blocked the cell cycle at the G1/G0 phase. Thus, benastatin A also induces apoptosis of colon 26 cells, but this is unlikely to be due to inhibition of GST activity.
Expression of Tumor‐rejection Antigens in Gynecologic Cancers
Authors: Tanaka Shoko , Tsuda Naotake , Kawano Kouichirou , Sakamoto Masaru , Nishida Takashi , Hashimoto Takashi , Shichijo Shigeki , Kamura Toshiharu , Itoh Kyogo
Published: August 2005

We recently reported the four tumor‐rejection antigens (SART1259 SART2, SART3, and ART4) that possess tumor epitopes capable of inducing HLA‐A2402‐restricted cytotoxic T lymphocytes (CTLs) in cancer patients. This study investigated the expression of these tumor antigens in gynecologic cancers, including 33 ovarian cancers, 38 cervical cancers, and 40 endometrial cancers. SART1259 antigen was detected in 56%, 35%, and 30% of ovarian, cervical and endometrial cancers, while SART2 antigen was detected in 46%, 66%, and 30% of these cancers, respectively. Both SART3 and ART4 antigens were detectable in the majority of these gynecologic cancers tested. In contrast, none of these antigens was detectable in any of the normal ovarian and uterine tissues tested. Peripheral blood mononuclear cells (PBMCs) of HLA‐A24+ patients with gynecologic cancers were found to produce significant levels of interferon‐γ in response to HLA‐A24+ SART3+ gynecologic cancer cells after having been stimulated three times in vitro with either SART3109–118 or SART3315–323 peptide. These PBMCs lysed HLA‐A24+ SART3+ gynecologic cancer cells, but not HLA‐A24‐ SART3+ gynecologic cancer cells or HLA‐A24+ normal cells. Therefore, these four antigens and their peptides, including SART3 peptides, would be appropriate molecules for use in specific immunotherapy of HLA‐A24+ gynecologic cancer patients.
Inhibitory Effects of 1′‐Acetoxychavicol Acetate on N‐Nitrosobis(2‐oxopropyl)‐amine‐induced Initiation of Cholangiocarcinogenesis in Syrian Hamsters
Authors: Miyauchi Makoto , Nishikawa Akiyoshi , Furukawa Fumio , Nakamura Hideaki , Son Hwa‐ Young , Murakami Akira , Koshimizu Koichi , Ohigashi Hajime , Hirose Masao
Published: August 2005

The influence of 1′‐acetoxychavicol acetate (ACA) during the initiation stage was investigated in the N‐nitrosobis(2‐oxopropyl)amine (BOP)‐initiated hamster tumorigenesis model. Ninety male 5‐week‐old hamsters were divided into three groups, each consisting of 30 animals, and s.c. injected with 20 mg/kg of BOP twice with a one‐week interval. Groups 1 through 3 were fed diet supplemented with ACA at concentrations of 500, 100 and 0 ppm, respectively, for 3 weeks starting one week before the first carcinogen application. At the termination of experimental week 54, the total incidence and multiplicity of cholangiocellular adenomas and carcinomas in group 1 (17.9% and 0.3±0.9) were significantly (P< 0.05 and P< 0.01) decreased as compared to the group 3 values (50.0% and 0.7±0.8). The ACA treatments also showed a tendency to reduce the development of preneoplastic lesions in the pancreas, a main target organ of BOP, although this was not statistically significant. Our results thus indicate that ACA exerts an inhibitory effect on BOP‐induced cholangiocarcinogenesis in hamsters. Taken together with previous findings of inhibited colon, oral and skin carcinogenesis in rats and mice, they suggest that ACA is a candidate chemopreventive agent with a wide spectrum of activity.
MDM2 Overexpression with Alteration of the p53 Protein and Gene Status in Oral Carcinogenesis
Authors: Li Xiao‐Jun , Murai Mutsuhiko , Koyama Takashi , Li Xiao‐Jin , Wang Dong‐Yu , Hashimoto Kenji
Published: August 2005

In this study, to better understand the mechanism of oral squamous cell carcinoma (SCC) carcinogenesis, alterations of the p53 gene and overexpression of MDM2 and p53 were analyzed in 38 oral SCC samples. Twelve of the 38 specimens revealed mutant‐type p53. Moreover, coexpression of MDM2 and p53 was found most frequently in dysplastic lesions (P< 0.05). Expression of MDM2 and p53 was significantly increased in accordance with the histological progression of multistep carcinogenesis (P< 0.05). No significant correlation was found between the expression of MDM2 and the alteration of p53 protein or p53 gene status. MDM2 overexpression with mutant p53 was significantly associated with poorly differentiated SCCs (P< 0.05) and tumor stages III and IV of oral SCCs (P< 0.05). These results suggest that MDM2 overexpression is an early event in oral carcinogenesis through the functional inactivation of the wild‐type p53, and corresponding alterations of MDM2 and p53 contribute to the oral carcinogenesis. We propose that it would be clinically more instructive to evaluate MDM2 overexpression combined with p53 gene status, compared to the evaluation of either MDM2 or p53 alteration alone.
Promotion of Skin Carcinogenesis by Dimethylarsinic Acid in Keratin (K6)/ODC Transgenic Mice
Authors: Morikawa Takashi , Wanibuchi Hideki , Morimura Keiichirou , Ogawa Motome , Fukushima Shoji
Published: August 2005

Dimethylarsinic acid (DMA) is a major metabolite of inorganic arsenicals in mammals, and arsenic exposure is associated with tumor development in a wide variety of human tissues, particularly the skin. Transgenic mice with ornithine decarboxylase (ODC) targeted to hair follicle keratinocytes are much more sensitive than littermate controls to carcinogens. In this study we investigated the promoting effect of DMA on skin carcinogenesis in such K6/ODC transgenic mice. The back skin of female C57BL/6J K6/ODC transgenic mice, 10 to 14 weeks old, was initiated with topical application of 7,12‐dimethylbenz[α]anthracene (DMBA) at a dose of 50 μg or acetone alone on day 1 of the experiment, followed by treatment with 3.6 mg of DMA, 5 μg of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) or neutral vehicle (control) twice a week for 18 weeks. Mice were killed 1 week after the end of the treatment. Induction of skin tumors was significantly accelerated in the DMA‐treated group, as well as in the TPA‐treated group, indicating that DMA has a promoting effect on skin tumorigenesis in K6/ODC transgenic mice.
Chemopreventive Effects of Bovine Lactoferrin on N‐Butyl‐N‐(4‐ hydroxybutyl)nitrosamine‐induced Rat Bladder Carcinogenesis
Authors: Masuda Chikayoshi , Wanibuchi Hideki , Sekine Kazunori , Yano Yoshihisa , Otani Shuzo , Kishimoto Taketoshi , Tsuda Hiroyuki , Fukushima Shoji
Published: August 2005

Chemopreventive effects of bovine lactoferrin (bLF), which is found at high concentrations in colostrum, on rat bladder carcinogenesis were investigated using a rat bladder medium‐term bioassay. In experiment 1, a total of 80 F344 male rats, 6 weeks old, were divided into 5 groups. Groups 1 and 2 were treated with 0.05%N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine (BBN) in the drinking water for 8 weeks and after a 1‐week interval, received dietary supplementation with 2% and 0.2% bLF, respectively. Group 3 received 0.05% BBN for 8 weeks and then no treatment. Group 4 was administered 2% bLF alone from week 9, without prior carcinogen exposure. Group 5 was maintained without any treatment throughout the experiment. All rats were killed at the end of week 36. Group 1 demonstrated a significantly decreased multiplicity of the bladder tumors (carcinomas and papillomas) as compared with group 3, Maximum cut surface areas of bladder tumors were also significantly decreased in groups 1 and 2 compared with group 3. No bladder tumors were observed in groups 4 or 5. In experiment 2, a total of 60 rats were divided into two groups (30 rats each); both were treated with 0.05% BBN for 4 weeks and after a 1‐week interval, one received 2% bLF (group 1) and the other, basal diet (group 2) for 4 weeks. Group 1 demonstrated a tendency for decrease of the 5‐bromo‐2′‐deoxyuridine (BrdU) labeling index. bLF was detected in the urine of rats fed bLF by ELISA as well as western blot analysis. The findings indicate that 2% bLF can inhibit BBN‐induced rat bladder carcinogenesis, and that this may be due to bLF in the urine.
Three New Regions on Chromosome 17p13.3 Distal to p53 with Possible Tumor Suppressor Gene Involvement in Lung Cancer
Authors: Tsuchiya Eiju , Tanigami Akira , Ishikawa Yuichi , Nishida Kazunori , Hayashi Moriaki , Tokuchi Yoshio , Hashimoto Takehisa , Okumura Sakae , Tsuchiya Shigehiro , Nakagawa Ken
Published: August 2005

We investigated loss of heterozygosity (LOH) at the distal portion of the p53 gene on the short arm of chromosome 17 in lung cancers in order to search for new tumor suppressor genes. The roles of the putative genes were also studied in terms of pathological features. One hundred and forty‐five resected non‐small cell lung cancers were examined for LOH using 11 markers mapped on, and distal to the p53 locus, and deletion maps were constructed. Four commonly deleted regions were found: one from TP53 to ENO3, where the p53 gene resides, and three others from ENO3 to D17S1566, D17S379 to D17S1574 and distal to ABR, with LOH frequencies almost the same as, or higher than, at the TP53 locus. Examination of the relationship between LOH of the latter three regions and histopathological parameters of adenocarcinomas (genetically negative for p53 mutation) revealed allelic losses on D17S379 to be associated with advanced lesions, while D17S513 was more frequently deleted in poorly differentiated tumors. These results indicate that new tumor suppressor gene(s) may reside on these three distinctly deleted regions on chromosome 17p13.3 distal to the p53 gene in lung cancer, with possible roles in progression and differentiation of adenocarcinomas.
Spicamycin and KRN5500 Induce Apoptosis in Myeloid and Lymphoid Cell Lines with Down‐regulation of Bcl‐2 Expression and Modulation of Promyelocytic Leukemia Protein
Authors: Zhang Wen Jie , Ohnishi Kazunori , Yoshida Hitoshi , Pan Ling , Maksumova Lola , Muratkhodjaev Farkhad , Luo Jian Min , Shigeno Kazuyuki , Fujisawa Shinya , Naito Kensuke , Nakamura Satoki , Shinjo Kaori , Takeshita Akihiro , Ohno Ryuzo
Published: August 2005

Spicamycin is a potent inducer of differentiation of human myeloid leukemia cells (HL‐60) and murine myeloid leukemia cells (M1). One of the spicamycin derivatives, KRN5500, shows a broad spectrum of antitumor activity against human tumor xenografts in nude mice. In this study, we first investigated the differentiation efficacy of spicamycin and KRN5500 in HL‐60 and acute promyelocytic leukemia cell line, NB4, and found that low concentrations of both compounds induced differentiation to a small extent in both cell lines, but markedly induced apoptosis in NB4 cells. Further investigation in a myeloid leukemia cell line, NKM‐1, a lymphoma cell line, Daudi, and multiple myeloma cell line, NOP‐1, showed that high concentrations of both compounds also induced apoptosis in these cells. The 50% inhibitory concentration (IC50) determined by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay showed that myeloid cells were more sensitive to both compounds than lymphoid cells, and spicamycin was more potent than KRN5500. Western blot analysis of Bcl‐2, Bcl‐xL and Bax expression and immunofluorescence analysis of promyelocytic leukemia (PML) protein indicated that apoptosis induced by spicamycin and KRN5500 was associated with down‐regulation of Bcl‐2 expression and modulation of PML protein. Thus, spicamycin and KRN5500 may be useful for the treatment of myeloid and lymphoid neoplasms.
A Single Nucleotide Polymorphism in the Matrix Metalloproteinase‐1 Promoter in Endometrial Carcinomas
Authors: Nishioka Yoshihiro , Kobayashi Kanji , Sagae Satoru , Ishioka Shin‐ichi , Nishikawa Akira , Matsushima Mieko , Kanamori Yasunobu , Minaguchi Takeo , Nakamura Yusuke , Tokino Takashi , Kudo Ryuichi
Published: August 2005

Recent studies demonstrated that a single guanine insertion polymorphism in a matrix metalloprotease‐1 promoter created an Ets binding site and affected the elevation of the transcriptional level of matrix metalloproteinase‐1 (MMP‐1). Furthermore, in tumor cell lines derived from melanoma and breast cancer, the incidence of the 2G/2G genotype was significantly higher than that in the normal population. To evaluate the contribution of this polymorphism in endometrial carcinomas, we genotyped 100 endometrial carcinomas and then analyzed immunoexpression of MMP‐1 in these carcinomas. We found that endometrial carcinoma patients showed a significantly higher rate of 1G/2G or 2G/2G genotype than control individuals, and that tumors containing the 2G allele(a) expressed MMP‐1 protein more frequently than those with 1G/1G genotype. Therefore, the single nucleotide polymorphism at the MMP‐1 promoter affected the expression level of the MMP‐1 protein, which may result in the association with more aggressive character in endometrial carcinoma. Our result suggests that the presence of 2G polymorphism at the MMP‐1 promoter may be one of the risk factors for the development and/or progression of endometrial carcinoma.
Induction of Cytotoxic T Lymphocytes from Peripheral Blood of Human Histocompatibility Antigen (HLA)‐A31+ Gastric Cancer Patients by in vitro Stimulation with Antigenic Peptide of Signet Ring Cell Carcinoma
Authors: Nabeta Yuki , Sahara Hiroeki , Suzuki Kazuhiro , Kondo Hiroaki , Nagata Masami , Hirohashi Yoshihiko , Sato Yuriko , Wada Yoshimasa , Sato Takashi , Wada Takuro , Yamashita Toshihiko , Kikuchi Kokichi , Sato Noriyuki
Published: August 2005

Antigenic peptides have been used as a cancer vaccine in melanoma patients and have led to a drastic regression of metastatic tumors. However, few antigens have been identified in non‐melanoma tumors. We recently purified a new natural antigenic peptide, designated F4.2, by biochemical elution from a human gastric signet cell carcinoma cell line and showed that it is recognized by an autologous human histocompatibility antigen (HLA)‐A31‐restricted cytotoxic T lymphocyte (CTL) clone. Here we describe in vitro induction of F4.2‐specific CTLs from peripheral blood T lymphocytes of HLA‐A31+ gastric cancer patients. The T cells of seven HLA‐A31+ patients with gastric cancers were stimulated in vitro by F4.2‐pulsed autologous dendritic cells which had been induced from peripheral blood of each patient by incubation in the presence of granulocyte macrophage colony‐stimulating factor (GM‐CSF) and IL‐4. We tested the cytotoxicity of the T cells against F4.2‐loaded C1R‐A *31012 by a 6‐h 51Cr release assay after 3 stimulations with F4.2‐pulsed dendritic cells. F4.2‐specific cytotoxicity was detectable in the stimulated T cells from two of the seven HLA‐A31+ patients. Further, both F4.2‐specific CTLs also lysed the gastric cancer cell line, HST‐2, from which F4.2 was derived. These results suggest that F4.2 peptide may be useful as an HLA‐A31‐restricted peptide vaccine in certain patients with gastric cancer.
Suppression of Tumor Growth and Downregulation of Platelet‐derived Endothelial Cell Growth Factor/Thymidine Phosphorylase in Tumor Cells by Angiogenesis Inhibitor TNP‐470
Authors: Mori Jun , Haisa Minoru , Naomoto Yoshio , Takaoka Munenori , Kimura Masashi , Yamatsuji Tomoki , Notohara Kenji , Tanaka Noriaki
Published: August 2005

We investigated the effects of the angiogenesis inhibitor TNP‐470 on human lung squamous cell carcinoma cell lines H226B and H226Br both in vivo and in vitro. H226B was established from human lung squamous cell carcinoma and H226Br was established from a brain metastatic lesion of H226B in nude mice. Nude mice inoculated with these cells were treated with 30 mg/kg of TNP‐470 subcutaneously every other day. At this dose, TNP‐470 only significantly suppressed the growth of H226Br tumor, but not H226B tumor. Attempts to use a high dose of TNP‐470 (100 mg/kg) resulted in a severe loss of body weight. Immunohistochemical studies showed marked tumor vascularization in H226Br tumor, but the formation of new blood vessels was suppressed by 30 mg/kg of TNP‐470. Investigation of the mechanism of anti‐angiogenic effects of TNP‐470 in vivo showed that the expression and the activity of platelet‐derived endothelial cell growth factor/thymidine phosphorylase (PD‐ECGF/dThdPase) in H226Br tumor was significantly suppressed by 30 mg/kg of TNP‐470. Furthermore, TNP‐470 inhibited cell growth of cultured H226Br dosedependently at concentrations of ≥1 μg/ml. Immunoblot analysis revealed H226Br cells gave a stronger PD‐ECGF signal than H226B cells, and the expression of PD‐ECGF/dThdPase in H226Br was also suppressed by treatment with TNP‐470 at ≥0.1 μg/ml. No change in basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) was noted in these cell lines. Our results suggested that TNP‐470 acts, at least in part, by downregulation of PD‐ECGF/ dThdPase in this cell line.
Effects of 1‐Methyl‐3‐propyl‐7‐butylxanthine (MPBX) on Idarubicin‐induced Antitumor Activity and Bone Marrow Suppression
Authors: Sadzuka Yasuyuki , Egawa Youichi , Sugiyama Tomomi , Sawanishi Hiroyuki , Miyamoto Ken‐ichi , Sonobe Takashi
Published: August 2005

The effects of 1‐methyl‐3‐propyl‐7‐butylxanthine (MPBX), a xanthine derivative, on idarubicin (IDA)‐induced antitumor activity against P388 leukemia cells (P388) and bone marrow suppression were examined. In P388 tumor‐bearing mice, the combination of MPBX with IDA increased the antitumor activity of IDA. The IDA concentration in the tumors in the MPBX combination group increased by 2.0‐fold compared to the level in the IDA‐alone group. On the other hand, as regards IDA‐induced bone marrow suppression, the combination of MPBX with IDA reduced the decrease in the bone marrow cell number by 30% compared to that in the IDA‐alone group. In addition, the IDA concentration in the bone marrow cells was decreased by the combination of MPBX with IDA. An in vitro experiment showed that MPBX facilitated IDA influx and suppressed IDA efflux in P388 cells. In conclusion, the combination of MPBX with IDA increased the antitumor activity and decreased the bone marrow suppression. Therefore, we expect that the combination of MPBX with IDA will be useful for leukemia chemotherapy.
Mouse Strain Susceptibility to Diethylnitrosamine Induced Hepatocarcinogenesis Is Cell Autonomous Whereas Sex‐susceptibility Is Due to the Micro‐environment: Analysis with C3H ↔ BALB/c Sexually Chimeric Mice
Authors: Tsukamoto Tetsuya , Inada Ken‐ichi , Fukami Hiroko , Yamamoto Masami , Tanaka Harunari , Kusakabe Moriaki , Bishop Colin E. , Tatematsu Masae
Published: August 2005

In man, liver cancer is on the increase, especially in males. Sex differences also exist in rodent models. To elucidate the mechanisms, chimeric mice were produced by amalgamation of early embryos from high and low hepatocarcinogen‐susceptible strains, C3H and BALB/c. Tumor formation was initiated with 10 mg/kg of diethylnitrosamine at the ages of 7 and 14 days and mice were sacrificed at 30 and 45 weeks. The chimeras were classified into XY↔XY, XY↔XX, XX↔XY, and XX↔XX in terms of sex chromosomes by means of polymerase chain reaction‐simple sequence length polymorphism analysis (SSLP) using Y chromosome‐specific Sry primers in combination with the D3Mit21 marker. Liver lesions were analyzed histopathologically, by immunostaining using a C3H strain‐specific antibody and by DNA in situ hybridization with the Y chromosomespecific digoxigenin‐labeled Y353/B probe. Sex and strain genotyping by SSLP analysis matched histological observations, confirming the reliability of our system. The strain differences in liver tumor numbers of each strain type in XY↔XY and XX↔XX subtypes of C3H↔BALB/c chimeras were retained well (P< 0.0001 and P< 0.001, respectively), indicating a minimum influence of the C3H or BALB/c surrounding milieu on development of individual lesions. On the other hand, significant promotion of XX cell tumors was evident in phenotypically male sexually chimeric XY↔XX and XX↔XY chimeras for both C3H (P< 0.02) and BALB/c (P< 0.01) lesions compared to the XX↔XX case. The results suggest the presence of hormonal or micro‐environmental factors specific for males, which are not caused cell‐autonomously. Basic strain differences, however, are determined by intrinsic genetic factors rather than the strain‐dependent micro‐environment
Mutation Analysis of the PTEN/MMAC1 Gene in Japanese Patients with Cowden Disease
Authors: Sawada Takeshi , Hamano Naomichi , Satoh Hiro , Okada Toshihide , Takeda Yasuo , Mabuchi Hiroshi
Published: August 2005

Cowden disease (CD), also known as multiple hamartoma syndrome, is an autosomal dominant cancer syndrome associated with high risk of breast and thyroid cancer. Recently, germline mutations in PTEN/MMAC1, which has nine exons encoding a dual specificity phosphatase with homology to tensin and auxilin, have been identified on chromosome 10q23 in some 40 to 80% of CD patients. Our polymerase chain reaction amplification and sequence analysis of all coding regions identified five different mutations including four novel germline mutations among 5 of 12 unrelated Japanese CD patients. The novel findings included a missense mutation (G→T) at nucleotide 1004 in exon 8 resulting in an arginine‐to‐leucine change at codon 335 (R335L), two novel splice‐site mutations (209+1delGT and 209+1delGTAA) in intron 3, and insertion of G at nucleotide 632 in exon 6 (632insG). We also detected a nonsense mutation (C→T) at nucleotide 697 producing R233X in exon 7, which has been reported previously. From reported phenotypic data concerning CD patients from five different families who had the R233X mutation, it may be suggested that R233X mutation correlates with macrocephaly. Although previous reports have implicated exon 5 as a “hot spot,” we found no mutation in exon 5.
Combination Therapy with Vascular Endothelial Growth Factor Neutralizing Antibody and Mitomycin C on Human Gastric Cancer Xenograft
Authors: Matsumoto Keigo , Konno Hiroyuki , Tanaka Tatsuo , Baba Megumi , Kanai Toshikazu , Kamiya Kinji , Ohba Koji , Nakamura Satoshi
Published: August 2005

Antiangiogenic therapy has been proposed as a new strategy for the treatment of solid tumors. To enhance the therapeutic effect of antiangiogenic agents, combination with conventional anticancer therapy should be investigated. In the present study, we investigated the therapeutic effect of the combination of vascular endothelial growth factor neutralizing antibody (VEGF Ab) and mitomycin C (MMC) on MT‐2, a human gastric cancer xenograft. When small pieces of MT‐2 were transplanted orthotopically into 62 nude mice, liver metastasis developed 6 weeks after transplantation. The VEGF Ab (100 μg/mouse) was administered i.p. in the VEGF Ab group (n=14) and the combination group (n=16) twice a week from day 10 after transplantation. MMC (2 mg/kg) was administered in the MMC group (n=16) and the combination group (n=16) on days 10, 17 and 24 after transplantation. Compared with the control group, in which saline solution was administered i.p., all three treatments inhibited tumor growth significantly and the effects of MMC and combination therapy were potent. Liver metastases were also inhibited significantly by the administration of VEGF Ab alone, MMC alone or combination therapy. Liver metastasis developed in 9 mice of the control group, 3 of the VEGF Ab group, and 4 of the MMC group, but no mice had liver metastasis in the combination therapy group. However a significant body weight loss and a decrease in spleen weight were observed in the MMC and combination groups, with no significant difference between the two groups. These results suggest that combination therapy with VEGF Ab and MMC may be a potent therapy for human gastric cancer.
Diagnostic Usefulness of Telomerase Activity in Nasopharyngeal Carcinoma
Authors: Hwang Chung‐Feng , Su Chih‐Ying , Kou Shih‐Chang , Huang Shung‐Chen , Leung Stephen‐Wan , Huang Chao‐Min , Cho Chung‐Lung
Published: August 2005

Telomeres are specialized structures at the ends of eukaryotic chromosomes which are composed of simple repetitive G‐rich hexameric sequences. Activation of telomerase, a ribonucleoprotein that synthesizes telomeric DNA, is found in most malignant tumors. However, little data is available concerning the correlation between telomerase activity and NPC (nasopharyngeal carcinoma). In this study, telomerase activation was determined using the TRAP (telomerase repeat amplification protocol) assay in 62 nasopharyngeal biopsies (25 NPC, 25 non‐malignant nasopharyngeal lymphoid tissues, 12 post‐irradiated nasopharyngeal tissues). The results showed that strong telomerase activity was present in both NPC and non‐malignant nasopharyngeal biopsies. Post‐irradiated nasopharyngeal samples had a significantly lower telomerase activity than NPC and non‐malignant nasopharyngeal lymphoid tissues. It is well known that nasopharyngeal tissue is infiltrated by numerous lymphocytes, which might retain telomerase activity. Therefore, the finding that the telomerase activation was lowest in post‐irradiated nasopharyngeal tissues is reasonable because of the destruction of activated lymphocytes and NPC by radiation. NPC biopsies with positive lymph node involvement exhibited higher levels of telomerase compared to those without lymph node involvement. Our data indicate a positive association between telomerase activity and tumor potential for lymphatic spreading in limited local tumors. In addition, telomerase activity may be useful as a diagnostic marker in the detection of tumor cells in recurrent NPC, but not in primary NPC.
Inhibitory Effects of S‐Methylcysteine and Cysteine on the Promoting Potential of Sodium Phenobarbital on Rat Liver Carcinogenesis
Authors: Vijayaraghavan Meenakshi , Wanibuchi Hideki , Takada Nobuyasu , Yano Yoshihisa , Otani Shuzo , Yamamoto Shinji , Fukushima Shoji
Published: August 2005

The effects of S‐methylcysteine (SMC) and eysteine on the promotion stages of rodent hepatocar‐cinogenesis in a medium‐term bioassay previously developed by Ito were examined. Initiation was induced by a single dose of diethylnitrosamine (DEN), followed by dietary administration of the promoter sodium phenobarbital (NaPB) 2 weeks later, for 6 weeks. Partial hepatectomy was conducted on all the animals at week 3. Inhibitory potential was evaluated by analyzing two markers of carcinogenesis, namely numbers of glutathione S‐transferase placental form (GST‐P)‐positive foci, and proliferating cell nuclear antigen (PCNA). In addition, the level of ornithine decarboxylase (ODC), one of the rate‐limiting enzymes of polyamine metabolism induced by promoters, was analyzed. SMC and cysteine induced significant reduction in the areas of GST‐P‐positive foci. A significant reduction in the PCNA index was observed in the entire liver as well as in GST‐P‐positive areas. SMC also induced down‐regulation of the ODC enzyme activity. Thus, SMC and cysteine were found to inhibit the promotion stage of DEN‐induced hepatocarcinogenesis. No cocarcinogenic effects were evident on administration of either of these chemicals with NaPB.
More Frequent β‐Catenin Gene Mutations in Adenomas than in Aberrant Crypt Foci or Adenocarcinomas in the Large Intestines of 2‐Amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP)‐treated Rats
Authors: Tsukamoto Tetsuya , Tanaka Harunari , Fukami Hiroko , Inoue Manami , Takahashi Mami , Wakabayashi Keiji , Tatematsu Masae
Published: August 2005

Alteration of adenomatous polyposis coli (APC) is known to be an early event in neoplasia, causing activation of the β‐catenin/Tcf pathway. Although it is thought that alterations in APC and β‐catenin may complement one another, the contribution of β‐catenin mutations to colorectal carcino‐genesis remains unclear. We therefore performed PCR‐single strand conformation polymorphism analysis and direct sequencing of exon 3 of β‐catenin gene in adenomas, adenocarcinomas, and aberrant crypt foci (ACF), considered to be putative precursor lesions of colorectal neoplasias, in 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5b]pyridine (PhIP) treated F344 rats. β‐Catenin mutations were identified in all of 7 adenomas (100%) and 6 of 12 (50%) adenocarcinomas. All of the mutations were found in codons 32 through 34, the serine encoded by codon 33 being an important phosphorylation site by glycogen synthase kinase‐3β. Regarding ACF, 14 of 46 (30.4%) were found to be mutated, eleven (78%) in codon 34, and the others in codon 45 (frequently altered in human colon cancer), and codons 47 and 56 (which have not been previously reported). The frequency of β‐catenin mutations in adenomas was significantly higher than in ACF (P < 0.001) and adenocarcinomas (P < 0.05). Thus, β‐catenin mutations may have more importance in the genesis of adenomas than ACF or adenocarcinomas in rat colon carcinogens by PhIP.
Allelic Loss of the Region of Chromosome 1p35‐pter Is Associated with Progression of Human Gastric Carcinoma
Authors: Igarashi Jun , Nimura Yoshinori , Fujimori Minoru , Mihara Motohiro , Adachi Wataru , Kageyama Hajime , Nakagawara Akira
Published: August 2005

In order to identify the region on distal chromosome 1p that is thought to include one or more tumor suppressor genes for gastric carcinoma, 39 gastric carcinomas were examined for allelic loss using 11 polymorphic microsatellite markers and 1 marker of single strand conformation polymorphism. Loss of heterozygosity (LOH) was found in 18 (46%) of 39 informative patients. The regions with high frequency of loss of heterozygosity were the loci at D1S548 (6/17; 35.3%) and D1S2843 (7/20; 35%), and we found three commonly deleted regions on chromosome 1p35‐pter. The frequency of allelic loss in the region of chromosome 1p35‐pter was significantly associated with advanced‐stage gastric carcinoma, but not with early‐stage tumor or with the histology. These results suggest that allelic loss at chromosome 1p35‐pter may play a role in the progression of gastric carcinoma.
Preparation and Characterization of Antibodies against Human Ribosomal Proteins: Heterogeneous Expression of S11 and S30 in a Panel of Human Cancer Cell Lines
Authors: Nadano Daita , Ishihara Goshi , Aoki Chikage , Yoshinaka Toko , Irie Shinji , Sato Taka‐Aki
Published: August 2005

Mutants of model eukaryotic organisms have revealed that most ribosomal proteins are essential for cell viability. However, the precise functional role of each ribosomal protein is largely unknown. Recent reports on the involvement of ribosomal proteins in various genetic diseases and studies on the extraribosomal functions of these proteins have cast some light on their localization and functions. Here we prepared rabbit polyclonal antibodies against 26 human ribosomal proteins; each of these reagents recognized a single band in immunoblots of the purified ribosome. We used these antibodies to evaluate a panel of human cancer cell lines. Although no deficiency of ribosomal proteins was observed, the abundance of S11 and S30 varied substantially among the cell lines, but the difference did not affect the biogenesis or composition of the ribosome. Therefore, the heterogeneity may be related to extraribosomal functions of S11 and S30. The antibodies described here are powerful tools for research into the molecular mechanisms of protein translation, cell‐biological and medical studies on the ribosomal proteins, and ultimately a comprehensive understanding of all ribosomal proteins (“ribosomics”).
Y‐27632, an Inhibitor of Rho‐associated Protein Kinase, Suppresses Tumor Cell Invasion via Regulation of Focal Adhesion and Focal Adhesion Kinase
Authors: Imamura Fumio , Mukai Mutsuko , Ayaki Masako , Akedo and Hitoshi
Published: August 2005

Migration of rat ascites hepatoma (MM1) cells, invasion and phagokinetic movement were induced by the combination of lysophosphatidic acid (LPA) and fibronectin (FN). Induction of migratory activity was tightly correlated with morphological change of MM1 cells from spherical or polygonal‐shaped cells to fusiform‐shaped ones with pseudopodia. MM1 cells were mobile in a fusiform shape, whereas those of a spherical or polygonal shape were not. A small GTPase Rho and one of its downstream effectors ROCK (Rho‐associated coiled‐coil forming protein kinase), play essential roles in these processes, as evidenced by suppression of migration and morphological change of MM1 cells by Clostridium botulinum C3 exoenzyme, an inhibitor of Rho, or by Y‐27632, an inhibitor of ROCK. Y‐27632 also suppressed the formation of fusiform‐shaped pseudopodia‐carrying MM1 cells that was induced by stimulation with the combination of LPA and FN. LPA and FN also evoked the formation of focal adhesions and actin bundles, and tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin. The inhibitory effect of Y‐27632 on LPA‐induced migration and morphological change of MM1 cells was considered to be mediated, at least in part, by impaired formation of focal adhesions and actin bundles. Y‐27632 suppressed LPA‐induced tyrosine phosphorylation of FAK and paxillin, suggesting that ROCK regulates these molecules and Y‐27632 inhibits cellular migration and morphological change, at least in part, through this regulation.
Antibody‐dependent Cytotoxicity Mediated by Chimeric Monoclonal Antibody Nd2 and Experimental Immunotherapy for Pancreatic Cancer
Authors: Nishihara Tamahiro , Sawada Tetsuji , Yamamoto Atsushi , Yamashita Yoshito , Ho Jenny J. L. , Kim Young‐Sik , Chung Kosei Hirakawa‐YS
Published: August 2005

In a previous study, mouse monoclonal antibody (MoAb) Nd2 (m‐Nd2, mouse IgGl) labeled with 131 I exhibited efficacy in in vivo radioimmunotherapy against pancreatic cancer. In this study we prepared mouse/human chimeric antibody Nd2 (c‐Nd2, human IgG1) for clinical use and examined whether c‐Nd2 induced antibody‐dependent cell‐mediated cytotoxicity (ADCC). Cytotoxicity to pancreatic cancer (PC) cell lines, including Nd2 antigen‐positive (SW1990, RWP‐1, Capan‐1) and Nd2 antigen‐negative (Panc‐1, MiaPaca‐2, Capan‐2) lines, was evaluated by mixed human leukocyte and tumor cell culture (MLTC) at an effector cell to target cell (E/T) ratio of 50 with or without Nd2. Cytotoxicities to SW1990 with no antibody, m‐Nd2 and c‐Nd2 (1 μg/ml) were 26.7%, 38.0% and 55%, respectively; to RWP‐1, 28%, 41% and 70%; to Capan‐1, 26%, 30% and 52%; to Panc‐1, 24%, 28% and 30%; to MiaPaca‐2, 18%, 20% and 27% and to Capan‐2, 29.7%, 35.0% and 40.6%. Cytotoxic capacity during MLTC with c‐Nd2 was significantly higher than during MLTC with m‐Nd2 or with no antibody. These findings indicated that cytotoxicity to Nd2‐positive PC cells during MLTC is induced by ADCC. Intraperitoneal injection of c‐Nd2 inhibited the tumor growth of SW1990 xenografted subcutaneously in nude mice and prolonged the survival of nude mice in which SW1990 tumor was transplanted orthotopically at the tail of the pancreas. These findings suggested that, because of its ability to induce ADCC, c‐Nd2 may be clinically useful for the immunotherapeutic treatment of pancreatic cancer.
TZT‐1027, an Antimicrotubule Agent, Attacks Tumor Vasculature and Induces Tumor Cell Death
Authors: Otani Masashi , Natsume Tsugitaka , Watanabe Jun‐ichi , Kobayashi Motohiro , Murakoshi Masanori , Mikami Takashi , Nakayama Takaharu
Published: August 2005

TZT‐1027, a dolastatin 10 derivative, is an antimicrotubule agent with potent antitumor activity both in vitro and in vivo. In this study, we performed biochemical and histopathological examinations, and evaluated TZT‐1027‐induced tumoral vascular collapse and tumor cell death in an advanced tumor model, murine colon 26 adenocarcinoma. In addition, we studied the effects of TZT‐1027 on cultured human umbilical vein endothelial cells (HUVEC). Tolerable doses of TZT‐1027 induced tumor‐selective hemorrhage within 1 h. This hemorrhage occurred mainly in the peripheral area of the tumor mass. Measurements of tumoral hemoglobin content and dye permeation revealed that the hemorrhage occurred firstly and tumor blood flow stopped secondarily. The vascular damage was followed by continuous induction of apoptosis of the tumor cells, tumor tissue necrosis, and tumor regression. In cultured HUVEC, TZT‐1027 induced marked cell contraction with membrane blebbing in 30 min. These cell changes were completely inhibited by K252a, a broad‐spectrum inhibitor of protein kinases. These effects of TZT‐1027 on both tumor vasculature and HUVEC were greater than those of vincristine. In conclusion, TZT‐1027 quickly attacked the well‐developed vascular system of advanced tumors by a putative protein kinase‐dependent mechanism, and then blocked tumor blood flow. Therefore, TZT‐1027 has both a conventional antitumor activity and a unique anti‐tumoral vascular activity, making it a potentially powerful tool for clinical cancer therapy.
In vitro Plasma Protein Binding and Cellular Uptake of ATX‐S10(Na), a Hydrophilic Chlorin Photosensitizer
Authors: Mori Masahiko , Kuroda Toyoshi , Obana Akira , Sakata Isao , Hirano Toru , Nakajima Susumu , Hikida Muneo , Kumagai Toshio
Published: August 2005

ATX‐S10(Na), a hydrophilic chlorin photosensitizer having an absorption maximum at 670 nm, is a candidate second‐generation photosensitizer for photodynamic therapy (PDT) for cancer treatment. In this study, we examined plasma protein binding, cellular uptake and subcellular targets of ATX‐S10(Na) in vitro. Protein binding ratios of 50 μg/ml ATX‐S10(Na) in rat, dog and human plasma were 73.0%, 87.2% and 97.7%, respectively. Gel filtration chromatography revealed that 1 mg/ml ATX‐S10(Na) bound mainly to high‐density lipoprotein (HDL) and serum albumin at the protein concentration of 0.4%, with binding ratios of 46% and 36%, respectively. The free form of ATX‐S10(Na) was mostly incorporated into T.Tn cells, and its cellular uptake was partially but significantly inhibited by endocytosis inhibitors such as phenylarsine oxide, chloroquine, monensin and phenylglyoxal, and by chilling the cells to 4°C. However, ouabain, harmaline, sodium cyanide, probenecid and aspartic acid did not influence the uptake of ATX‐S10(Na), suggesting that cellular uptake of ATX‐S10(Na) was not related to sodium‐potassium pump activity, sodium‐dependent transporter activity, mitochondrial oxidative respiration, organic anion transporter activity or aspartic acid transporter activity. By fluorescence microscopy, lysosomal localization of ATX‐S10(Na) was observed in T.Tn cells. However, electron microscopic observation revealed that many subcellular organelles such as mitochondria, endoplasmic reticulum, ribosomes, Golgi complex and plasma membrane were damaged by PDT using 25 μg/ml ATX‐S10(Na) soon after laser irradiation at 50 J/cm2, and tumor necrosis was rapidly induced. This result indicated that ATX‐S10(Na) was widely distributed within the cell.
Glucose Intolerance, Plasma Insulin Levels, and Colon Adenomas in Japanese Men
Authors: Nishii Takasei , Kono Suminori , Abe Hiroshi , Eguchi Hiroyuki , Shimazaki Kae , Hatano Ben , Hamada Hiroaki
Published: August 2005

Hyperinsulinemia may be related to colon carcinogenesis. Several studies have suggested that diabetes mellitus is related to increased risk of colon cancer. We examined cross‐sectionally the relation of fasting plasma insulin levels and glucose tolerance status to colon adenomas. In a consecutive series of 951 men undergoing total colonoscopy for a health examination at the Japan Self Defense Forces Fukuoka Hospital from April 1998 to August 1999, we identified 233 cases of colon adenomas and 497 controls with normal colonoscopy. Glucose tolerance status was determined by a 75‐g oral glucose tolerance test, and subjects were classified as normal, unpaired glucose tolerance (IGT) or non‐insulin dependent diabetes mellitus (NIDDM). Plasma insulin levels were measured after subjects had fasted overnight. Logistic regression analysis and analysis of covariance was used to control for age and obesity. While plasma insulin levels were unrelated to colon adenomas, NIDDM was associated with a significantly increased risk of colon adenomas. There was no association between IGT and colon adenomas. NIDDM was more strongly associated with proximal colon adenomas. The findings suggest that long‐term hyperinsulinemic status associated with NIDDM may increase the risk of colon adenomas, and subsequently of colon cancer.
Frequent Increase of DNA Copy Number in the 2q24 Chromosomal Region and Its Association with a Poor Clinical Outcome in Hepatoblastoma: Cytogenetic and Comparative Genomic Hybridization Analysis
Authors: Kumon Kazuko , Kobayashi Hirofumi , Namiki Takeshi , Tsunematsu Yukiko , Miyauchi Jun , Kikuta Atsushi , Horikoshi Yasuo , Komada Yoshihiro , Hatae Yoshiro , Eguchi Haruhiko , Kaneko Yasuhiko
Published: August 2005

In a cytogenetic and comparative genomic hybridization (CGH) study of 38 hepatoblastomas, we found gain of Iq in 17 tumors (44.7%), that of 2/2q in 14 (36.8%), that of 20/20q in 9 (23.7%) and that of 8/8q in 8 (21.0%), loss of 4q in 4 (10.5%) and no DNA copy changes with normal karyotype or no mitotic cells in 11 (28.9%). Eleven tumors with 2/2q gain detected by CGH had a total chromosome 2 gain, a partial 2q gain, or a total chromosome 2 gain with an augmented partial 2q region; the common region for DNA copy gain was 2q24. Two‐color fluorescence in situ hybridization (FISH) analyses using probes covering the centromere of chromosome 2 or HOXD13 (2q31) confirmed the CGH findings, and showed that the common region for gain in 2q was centromeric to HOXD13. Event‐free survival (EFS)±standard error (SE) at 5 years was lowest in patients with 2q gain [37±15%], highest in those with no DNA copy changes [82±12%], and intermediate in those with DNA copy changes other than 2q gain [74±13%] (P=0.0549). Multivariate analysis showed that 2q gain was an independent factor predicting a poor outcome. These findings suggest the presence of a growth‐promoting gene or an oncogene in the 2q24 chromosome band, and a tumor suppressor gene in terminal 4q, which have important roles in the development and progression of hepatoblastoma.
Detection and Cloning of a Protein Recognized by Anti‐human Prostate‐specific Antigen (PSA) Antibody in the Rat Ventral Prostate
Authors: Onozawa Mizuki , Fukuda Kazunori , Watanabe Masahiko , Ohtani Mikinobu , Akaza Hideyuki , Sugimura Takashi , Wakabayashi Keiji
Published: August 2005

Prostate‐specific antigen (PSA), a 33 kDa glycoprotein produced in the epithelium of the human prostate, has become established as a useful tumor marker for prostate cancer in man. Since reports of homologous proteins in animals other than primates have been lacking, the present investigation was carried out to identify any PSA‐like protein in rats. Immunoblot analysis using a specific monoclonal anti‐human PSA antibody detected a 32 kDa immunoreactive protein in the ventral lobe of the rat prostate, but not in other lobes or in other tissues. Positive immunostaining was observed only for the luminal surface of the glandular epithelium and the intraductal fluid in the ventral prostate. Sequence analysis of a cDNA for the rat PSA‐like protein, cloned by immunoscreening of an expression cDNA library prepared from the ventral lobe, revealed identity to the rat submaxillary gland S3 kallikrein. Human PSA also belongs to the kallikrein family. Thus, this protein produced in the rat ventral prostate was suggested to be a possible counterpart of human PSA.
SGLT Gene Expression in Primary Lung Cancers and Their Metastatic Lesions
Authors: Ishikawa Nobuhisa , Oguri Tetsuya , Isobe Takeshi , Fujitaka Kazunori , Kohno Nobuoki
Published: August 2005

Cancer cells show increased glucose uptake and utilization in comparison with their normal counterparts. Glucose transporters play an important role in glucose uptake. We previously reported the differential gene expression of the GLUT family in primary and metastatic lesions of lung cancer. To investigate the role of Na+/glucose cotransporter (SGLT) genes in cancers, we examined the levels of expression of SGLT1 and SGLT2 genes in primary lung cancers and their metastatic lesions. Ninety‐six autopsy samples (35 primary lung cancers, 35 corresponding normal lung tissues, 10 metastatic liver lesions, and 16 metastatic lymph nodes) from 35 patients were analyzed for SGLT1 and SGLT2 expression by reverse transcription (RT)‐polymerase chain reaction (PCR). There were no significant differences in the level of expression of either gene between the primary lung cancers and normal lung tissues. The level of SGLT1 expression in the metastatic lesions and primary lung cancers did not differ significantly. The level of SGLT2 expression was, however, significantly higher in the metastatic lesions of both the liver and lymph node than in the primary lung cancers. These results suggest that SGLT2 plays a role in glucose uptake in the metastatic lesions of lung cancer.