bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of the Interobserver Agreement in the Number of Mitotic Figures Breast Carcinoma as Simulation of Quality Monitoring in the Japan National Surgical Adjuvant Study of Breast Cancer (NSAS‐BC) Protocol
Authors: Tsuda Hitoshi , Akiyama Futoshi , Kurosumi Masafumi , Sakamoto Goi , Yamashiro Katsushige , Oyama Tetsunari , Hasebe Takahiro , Kameyama Kaori , Hasegawa Tadashi , Umemura Shinobu , Honma Keiichi , Ozawa Takachika , Sasaki Keiko , Morino Hideo , Ohsumi Shozo
Published: August 2005

In the National Surgical Adjuvant Study for Breast Cancer (NSAS‐BC), node‐negative breast cancers were divided into higher‐ and lower‐risk groups according to the histopathological nuclear grade given at individual collaborating hospitals, and the higher‐risk group was entered into a randomized protocol of adjuvant therapy. Because the nuclear grade was the composite of nuclear atypia and mitotic counts, maintenance of interobserver agreement in mitotic counts was indispensable for the success of the protocol study. Fourteen pathologists participating in the protocol judged whether or not 20 photomicrographs suspected of showing mitotic cancer‐cell figures truly showed mitoses. After standardizing the counting method, these pathologists counted the number of mitotic figures per 10 high‐power fields of hematoxylin‐eosin‐stained main‐tissue sections of 20 tumors. Areas where mitotic counts were considered to be the most frequent by each pathologist were compared for these tumors. For the judgment of whether the photomicrograph indicated mitosis, the level of interobserver agreement was moderate (k=0.569). In the observations of 20 tumors, interobserver agreement level of mitotic counts was moderate (k=0.506), that of nuclear atypia scoring was fair (k=0.265), and that of nuclear grading was substantial (k=0.633). The counted area was almost the same among the observers in 9 tumors, split into two areas in 6, and dispersed in 5. Concordance in judgment was achieved in 7 of the first 9 and in all of the third 5, but only in one of the second 6. The cause of discordance was mostly derived from tumor heterogeneity and the difference in the site where mitoses were counted. Interobserver agreement level was considered to be satisfactory, and it was expected that the case entry would be performed appropriately in the protocol study. The selection of the counting area was confirmed to be important for the acquisition of high‐level agreement level in mitotic counts.
Effects of Three‐month Oral Supplementation of β‐Carotene and Vitamin C on Serum Concentrations of Carotenoids and Vitamins in Middle‐aged Subjects: A Pilot Study for a Randomized Controlled Trial to Prevent Gastric Cancer in High‐risk Japanese Population
Authors: Sasaki Satoshi , Tsubono Yoshitaka , Okubo Shunji , Hayashi Masato , Kakizoe Tadao , Tsugane Shoichiro
Published: August 2005

Prior to a randomized controlled trial to prevent gastric cancer by oral supplementation of β‐carotene and vitamin C in a high‐risk Japanese population, we examined the serum response to threemonth oral supplementation of β‐carotene (0, 3, 30 mg/day) and vitamin C (0, 50, 1000 mg/day) by a three‐by‐three factorial design using 54 subjects (age range=40–69 years). Serum concentrations of carotenoids, α‐tocopherol, and ascorbic acid were examined at baseline, and one, two, and threemonth points. Both serum β‐carotene and ascorbic acid were significantly higher in high‐dose groups than in each placebo group during the supplementation. The serum β‐carotene increased gradually (597–830% increase) during the study, whereas the serum ascorbic acid reached nearly a steady‐state at the one‐month point and remained stable thereafter (88–95% increase). No statistically significant interaction between β‐carotene and vitamin C supplementations was observed either for serum β‐carotene or for serum ascorbic acid. Among carotenoids and α‐tocopherol examined, serum lycopene in the high‐dose β‐carotene group was significantly higher than in the placebo group at all points. No unfavorable change in carotenoids and α‐tocopherol was observed in any group.
Different Mutation Frequencies and Spectra among Organs by N‐Methyl‐N‐nitrosourea in rpsL (strA) Transgenic Mice
Authors: Shioyama Yoshiyuki , Gondo Yoichi , Nakao Kazuki , Katsuki Motoya
Published: August 2005

The frequencies and spectra of N‐methyl‐N‐nitrosourea (MNU)‐induced in vivo somatic mutations were determined in rpsL (strA) transgenic mice. The wild‐type rpsL gene, which exhibits a streptomycin‐sensitive (Sms) phenotype, was used as the rescue marker gene. Studies of mutation spectra among different organs and tissues were simplified using this system because of the short coding sequence (375 bp) of the rpsL gene. MNU administration to transgenic mice significantly elevated the mutation frequencies in various adult organs. Two distinctive patterns of mutation spectrum were observed, depending on the organs tested. Mutations derived from labile organs (spleen and thymus) were predominantly G:C to A:T transitions, as expected for MNU mutagenesis. Stable organs like the liver and brain, however, carried many fewer G:C to A:T transitions but significantly more single base deletions, of which the spectrum was very similar to that of background mutations in the rpsL transgenic mice. This spectrum difference among more and less proliferating organs was confirmed by the predominant occurrence of G:C to A:T transitions in fetal liver cells exposed to transplacental MNU treatment. In addition, most (approximately 90%) of the G:C to A:T transitions induced by MNU were detected in the first nucleotide of some 5′‐G‐(C or G)‐3’ sequences, many of which corresponded to the middle guanine residue of 5′‐purine‐G‐(C or G)‐3’ sequences. It is thus suggested that at particular sites, the neighboring bases in both the 5’ side and 3’ side seem to influence either the susceptibility to DNA damage or the ability to repair MNUinduced lesions.
Changes in Expression of Estrogen Receptors α and β in Relation to Progesterone Receptor and pS2 Status in Normal and Malignant Endometrium
Authors: Saegusa Makoto , Okayasu Isao
Published: August 2005

To clarify changes in estrogen receptor (ER) α and ERβ during endometrial tumorigenesis, 48 endometrial carcinomas (endometrioid type), as well as 40 samples of normal endometrial tissue, were investigated using a combination of reverse‐transcription and polymerase chain reaction with Southern blot hybridization and western blot assays, and the results were compared with findings for progesterone receptor (PR) and pS2 mRNA status. In addition, 166 carcinomas were also examined for immunohistochemistry, along with 171 normal specimens. Relative amounts of ERα at both mRNA and protein levels were significantly greater than those for ERβ in normal and malignant endometrial lesions. ERα mRNA showed a stepwise decrease from normal or grade (G) 1 through to G3 tumor lesions, in line with changes in the protein levels, in contrast to ERβ mRNA or protein expression, which did not alter, suggesting a shift in the ratio of the two ER subtypes during endometrial tumorigenesis. PR mRNA expression was significantly correlated with ERα, but not ERβ mRNA status. Although significantly higher expression of pS2 mRNA or protein was observed in carcinomas than in the normal cases, there was no apparent association with the ER status. The findings suggest that alteration in estrogen signaling pathways may occur during endometrial tumorigenesis, and provide evidence that ERα expression may play an important role in the regulation of PR, but not pS2 expression in normal and malignant endometrium.
BRCA1 Expression Status in Relation to DNA Methylation of the BRCA1 Promoter Region in Sporadic Breast Cancers
Authors: Niwa Youko , Oyama Tetsunari , Nakajima Takashi
Published: August 2005

To understand the biological role of BRCA1 in sporadic breast cancers, the relationship between DNA methylation of the BRCA1 Promoter region and BRCA1 expression was studied using molecular biological and immunohistochemical methods. Furthermore, BRCA1 expression was compared with the expression of various cell cycle regulatory proteins and the morphological nuclear grade of cancer cells. Of 32 sporadic breast cancers investigated in this study, 10 (31%) revealed DNA methylation of the BRCA1 promoter region. The expression of BRCA1 was observed in the nuclei of cancer cells and 18 (56%) of 32 cancers were positive for BRCA1 immunoreactivity. Breast cancers with BRCA1 methylation lacked BRCA1 expression, except for only three cancers, and there was a significant inverse relationship between BRCA1 methylation and its expression in sporadic breast cancers (P=0.043). Compared with the expression of various cell cycle regulatory proteins, breast cancers with BRCA1 methylation showed decreased expression of estrogen receptor (P=0.016) and p27 (P=0.018) and increased expression of p21 (P=0.011). Furthermore, breast cancers without BRCA1 expression or with BRCA1 methylation had a tendency to contain nuclei with higher grade. These findings indicate that BRCA1 methylation might greatly influence its expression and BRCA1 expression might play an important role in cell cycle regulation and influence the grade of malignancy of sporadic breast cancers.
Enhancing Effect of an Inhibitor of Nitric Oxide Synthesis on Bacillus Calmette‐Guérin‐induced Macrophage Cytotoxicity against Murine Bladder Cancer Cell Line MBT‐2 in vitro
Authors: Yamada Hiroshi , Matsumoto Sohkichi , Matsumoto Tetsuro , Yamada Takeshi , Yamashita Uki
Published: August 2005

We studied the effect of an inhibitor of nitric oxide (NO) synthesis, NG‐monomethyl‐L‐arginine (LNMMA), on the Bacillus Calmette‐Guérin (BCG)‐induced antitumor activity of murine peritoneal exudate cells (PEC) against murine bladder cancer cell line MBT‐2 in vitro. L‐NMMA enhanced BCG‐induced cytotoxic activity of PEC, as well as interferon (IFN)‐γ and tumor necrosis factor (TNF)‐α production. The L‐NMMA‐induced enhancement was due to the prolonged survival of BCG in macrophages, because no enhancement of cytotoxicity was observed and neither IFN‐γ nor TNF‐α production was significantly enhanced by killed BCG. Anti‐TNF‐α antibody (Ab) and anti‐IFN‐γAb reduced the L‐NMMA‐induced enhancement of the cytotoxicity. The depletion of T cells from PEC reduced the production of both IFN‐γ and TNF‐α, as well as the enhancement of cytotoxicity induced by viable BCG plus L‐NMMA. These results suggest that L‐NMMA has an enhancing effect on BCG‐induced macrophage cytotoxicity and the enhancement is partially mediated by T cells and their soluble products. Accordingly, NO inhibitor should be a valuable adjunct to BCG immunotherapy for bladder cancer.
Establishment and Characterization of 6‐[[2‐(Dimethylamino)ethyl]amino]‐3‐hydroxy‐7H‐indeno[2,1‐c]quinolin‐7‐one dihydrochloride (TAS‐103)‐resistant Cell Lines
Authors: Aoyagi Yoshimi , Kobunai Takashi , Utsugi Teruhiro , Wierzba Konstanty , Yamada Yuji
Published: August 2005

6‐[[2‐(Dimethylamino)ethyl]amino]‐3‐hydroxy‐7H‐indeno[2,1‐c]quinol in‐7‐one dihydrochloride (TAS‐103) is a novel anticancer agent that was developed to target both topoisomerase (Topo) I and Topo II. To elucidate its mechanism of action, we have established and characterized TAS‐103‐resistant cells, derived from mouse leukemia (P388), human colon cancer (DLD‐1), and human lung adenocarcinoma (A549) cell lines, by exposure to stepwisely increasing concentrations of TAS‐103 in the culture medium. P388/TAS cells showed only cross‐resistance to VP‐16 and adriamycin (ADR). The Topo II activity in these cells was decreased to below one‐fourth of that in the parental cells, while the Topo I activity remained unchanged. DLD/TAS cells appeared to be cross‐resistant to VP‐16, ADR, camptothecin (CPT), SN‐38 and vincristine (VCR). The enzymatic activities of both Topo I and Topo II in these cells were decreased to one‐fourth of that observed in the parental cells. Furthermore, the decreased activities were accompanied by lower expression at the mRNA and protein levels. A549/TAS cells acquired cross‐resistance to VP‐16, ADR and VCR, though the Topo activities were virtually unchanged. In this cell line, the intracellular accumulation of TAS‐103 was significantly decreased and the expression of multidrug resistance associated protein (MRP) was elevated when compared with the parental cells. The results indicate that the affected activities of Topo I and/or Topo II, and in some instances decreased accumulation of TAS‐103, are associated with the development of resistance to TAS‐103, although the main mechanism of resistance to TAS‐103 varied among cell lines.
Identification and Characterization of a Deletion Mutant of DNA Topoisomerase I mRNA in a Camptothecin‐resistant Subline of Human Colon Carcinoma
Authors: Yanase Kae , Sugimoto Yoshikazu , Tsukahara Satomi , Oh‐hara Tomoko , Andoh Toshiwo , Tsuruo Takashi
Published: August 2005

In previous studies, we established two camptothecin (CPT)‐resistant sublines, HT‐29/CPT and St‐4/CPT, from the human colon cancer cell line HT‐29 and the human stomach cancer cell line St‐4, respectively. Cellular contents of DNA topoisomerase I (topo I) in the resistant cells were eight‐fold less than those in the corresponding parental lines. In this study, we have shown expression of two species of the TOP1 mRNA in HT‐29/CPT. The longer mRNA (4.0 kb) is the wild‐type TOP1 mRNA, and the shorter mRNA (3.3 kb) proved to have a deletion of 672 bp (nucleotides 58–729 or 59–730) that caused the in‐frame deletion of amino acids 20–243 of human topo I. The deleted region is identical to exons 3–9 of the TOP1 gene. The expression level of the 3.3‐kb mRNA was similar to that of the wild‐type mRNA in HT‐29/CPT. St‐4/CPT expressed only the wild‐type TOP1 mRNA in lesser amounts than did St‐4. Mouse NIH3T3 cells transfected with the wild‐type TOP1 cDNA showed higher sensitivity to CPT than the parental cells, whereas those transfected with the deleted TOP1 cDNA showed levels similar to those of the parental cells. Expression of the exogenous TOP1 mRNA was confirmed; however, expression of the truncated topo I was not detected in cells transfected with the deleted TOP1 cDNA. These results suggest that the expression of the deleted TOP1 mRNA led to the low expression of CPT‐sensitive topo I in the resistant cells.
Enhanced Efficacy of Radioimmunotherapy Combined with Systemic Chemotherapy and Local Hyperthermia in Xenograft Model
Authors: Kinuya Seigo , Yokoyama Kunihiko , Konishi Shota , Hiramatsu Takashi , Watanabe Naoto , Shuke Noriyuki , Aburano Tamio , Takayama Teruhiko , Michigishi Takatoshi , Tonami Norihisa
Published: August 2005

We previously found that the efficacy of radioimmunotherapy (RIT) with 131I‐A7, an IgG1 against Mr 45000 glycoprotein on colon cancer, was enhanced by local hyperthermia (HT) or chemotherapy with 5‐fluorouracil (5‐FU). In this study, we aimed to further enhance its efficacy by combining these three modalities. Human colon cancer xenografts (146±12 mm3) in Balb/c nu/nu female mice were treated with 9.25 MBq 131I‐A7 i.v. combined with HT (43°C for 1 h) and 5‐FU (30 mg/kg/day i.p. for 5 days). Tumor growth delay, (Tqtreated‐Tqcontrol)/Tqcontrol where Tq is tumor quadrupling time, in mice treated with RIT+HT+5‐FU was improved to 12.7 from 5.90, 7.55 and 10.1 with RIT alone, RIT+5‐FU and RIT+HT, respectively. Complete response was observed in 4 out of 8 tumors with RIT+HT+5‐FU and 3 out of 10 with RIT+HT. No tumor showed complete response with RIT+5‐FU or RIT alone. 5‐FU slightly increased myelotoxicity of RIT, but HT did not affect it. Body weight loss was not enhanced by the combination. These results indicate that the combination of three modalities is a feasible approach to enhance the antitumor efficacy of RIT without serious increase of toxicity.
Avidin Chase Can Reduce Myelotoxicity Associated with Radioimmunotherapy of Experimental Liver Micrometastases in Mice
Authors: Sato Noriko , Saga Tsuneo , Sakahara Harumi , Nakamoto Yuji , Zhao Songji , Kuroki Masahide , Iida Yasuhiko , Endo Keigo , Konishi Junji
Published: August 2005

Myelotoxicity is the main factor which decides the maximum tolerated dose (MTD) in radioimmunotherapy (RIT). Since bone marrow is mostly irradiated from blood radioactivity, enhancing the clearance of unbound circulating radiolabeled antibody is important to reduce myelotoxicity and to increase the MTD. We applied the avidin chase method, which was devised to obtain high tumorto‐background ratios in tumor‐targeting, to RIT of experimental liver micrometastases and evaluated its influence on the side effects and therapeutic outcome. Seven days after intrasplenic injection of human colon cancer LS174T cells, nude mice were intravenously injected with biotinylated 131I‐labeled anti‐CEA monoclonal antibody (MAb) (24–38 μg, 11.1 MBq). Mice of the chase group then received an intravenous injection of avidin twice (24 and 30 h, 72–115 μg each). Biodistribution, side effects (white blood cell counts and body weight change), and short‐ and long‐term therapeutic effects were determined. Avidin chase markedly accelerated the clearance of radiolabeled MAb from the blood (P< 0.0001) and normal tissues, resulting in milder leukocytopenia and body weight loss, both of which recovered earlier than in the non‐chase group (P< 0.01). The tumor uptake of radiolabeled MAb was also decreased by avidin chase, but the metastases‐to‐background ratios were increased. Avidin chase gave the therapeutic gain ratio of 1.89. Treated groups with and without avidin chase showed significant therapeutic effects compared to the non‐treated group. There was no significant difference in the therapeutic effects between the two treated groups. Avidin chase effectively reduced the side effects of RIT and should increase the MTD.
Human T Cell Leukemia Cell Death by Apoptosis‐inducing Nucleosides from CD57+ HLA‐DRbright Natural Suppressor Cell Line
Authors: Mori Tsuneatsu , Li Xiang , Mori Etsuko , Guo Maowu
Published: August 2005

Apoptosis‐inducing nucleosides (AINs) released from CD57+HLA‐DRbright natural suppressor (57.DR‐NS) cell line, derived from human decidual tissue, were isolated from 57.DR‐NS cell culture supernatant by the combination of thin‐layer chromatography (TLC) and high‐performance liquid chromatography (HPLC). Apoptotic cell death was strongly induced in human T cell leukemia Molt4 cells treated with AINs, absolutely depending on DNA strand breaks, with activation of the caspase cascade, especially caspase‐3. The administration of AINs to Molt4 tumor‐bearing severe combined immunodeficiency (SCID) mice resulted in drastic suppression of tumor growth, with a decrease of tumor size and the appearance of apoptotic signals in tumor tissue. Thus, AINs are candidates for development as anticancer agents.
Heterogeneous Nuclear Ribonucleoprotein B1 Expressed in Esophageal Squamous Cell Carcinomas as a New Biomarker for Diagnosis
Authors: Matsuyama Satoru , Goto Yuri , Sueoka Naoko , Ohkura Yasuo , Tanaka Yoichi , Nakachi Kei , Sueoka Eisaburo
Published: August 2005

We recently reported that heterogeneous nuclear ribonucleoprotein (hnRNP) B1 was overexpressed in most human lung cancers, especially squamous cell carcinoma (SCC), as well as human oral SCC. To find the significance of hnRNP B1 in cancer diagnosis, we studied hnRNP B1 expression in 16 paraffinized sections of esophageal SCC, using immunohistochemical staining with antihnRNP B1 polyclonal antibody, raised in a rabbit. We compared the expression of hnRNP B1 in cancerous and noncancerous regions of the same specimen: enhanced expression was observed in 63% of cancerous regions (10/16), whereas none of the noncancerous regions showed enhanced expression. The enhanced expression of hnRNP B1 in cancerous regions was compared with that in noncancerous tissue in relation to histopathological grade: 83% for well differentiated (5/6), 83% for moderately differentiated (5/6) and 0% for poorly differentiated (0/4). Histologically, enhanced expression of hnRNP B1 was observed around cancer pearls, as well as in the cells of nests lacking keratinization in well and moderately differentiated SCC. Western blotting analysis revealed enhanced expression in three frozen specimens of moderately differentiated SCC. Using esophageal cancer cell lines, we further confirmed the decreased expression in poorly differentiated SCC cells, compared with other differentiation types. All our results support the significance of hnRNP B1 expression in esophageal SCC as a unique diagnostic marker with regard to association between expression level and histopathological grading.
Analyses of Oxidative DNA Damage and Its Repair Activity in the Livers of 3′‐Methyl‐4‐dimethylaminoazobenzene‐treated Rodents
Authors: Hirano Takeshi , Higashi Ken , Sakai Akinori , Tsurudome Yosuke , Ootsuyama Yuko , RieKido  , Kasai Hiroshi
Published: August 2005

We measured the levels of 8‐hydroxyguanine (8‐OH‐Gua) and its repair activity in the livers of the Donryu rat, the carcinogen‐resistant DRH rat, and the ddy mouse, which were fed a 0.06% 3′‐ methyl‐4‐dimethylaminoazobenzene (3′‐MeDAB)‐containing diet. In a short‐term rat experiment (maximum 2 months), 3′‐MeDAB did not increase the 8‐OH‐Gua levels in the livers of the two rat strains, although it significantly increased the repair activity in only the Donryu rat liver at 1 and 2 months. After long‐term 3′‐MeDAB administration to the ddy mouse (8 months), the levels of 8‐OH‐Gua and its repair activity were increased in the liver by 3.6‐fold and 1.6‐fold, respectively. These experiments suggest that 3′‐MeDAB increases 8‐OH‐Gua generation in rodent liver DNA and the 8‐OH‐Gua repair assay is a reliable marker of cellular oxidative stress induced by carcinogens.
Preferential Occurrence of Breast Carcinomas with Loss of Chromosome 16q and der(16)t(1;16)/der(1;16) in Middle‐aged Patients with Hyperplasia of Mammary Glands
Authors: Tsuda Hitoshi , Takarabe Teruko , Fukutomi Takashi , Hirohashi Setsuo
Published: August 2005

Structural and numerical alterations of chromosome 16 are considered to be commonly involved in the genesis of breast cancer. To reveal etiological factors that predispose cells to these alterations, we examined the frequencies of chromosome 16 aneusomy, 16q loss and 1;16 fusion indicating der(16)t(1;16)/der(1;16) by multi‐color fluorescence in situ hybridization in 46 tumors resected mostly from young (≤34 years old) or elderly (≥75 years old) women, and compared the results with those in a patient group representing a common age distribution of Japanese patients in whom chromosome 16 status in the tumor had already been studied. The correlation of these chromosome 16 alterations with age, hyperplasia in adjacent mammary glands, cancer history, and obesity indices was investigated in a total of 244 patients. In the present 46 tumors, the frequency of 16q loss and der(16)t(1;16) did not differ between 20 younger patients (30% and 15%) and 23 elderly patients (43% and 13%). However, the incidences of 16q loss and der(16)t(1;16) were low in comparison with the values of 64% and 38% in the 198 Japanese patients representing the common age distribution (P< 0.001). In addition, 16q loss and der(16)t(1;16) were more frequent in tumors arising in hyperplastic mammary glands (68%, 44%) than in those without (52%, 24%) (P< 0.01). Such correlations were not evident for 16cen aneusomy. Other etiological factors examined were not correlated with these chromosome 16 alterations. The 16q loss and der(16)t(1;16) formation were more frequently involved in the development of breast cancer in middle‐aged patients than in young and elderly patients. High‐level estrogens and/or sensitivity of mammary glandular cells to estrogens might induce breast cancers with structural changes of chromosome 16.
Effect of Angiostatin on Liver Metastasis of Pancreatic Cancer in Hamsters
Authors: Yanagi Ken , Onda Masahiko , Uchida Eiji
Published: August 2005

The liver is the most common site of metastasis in pancreatic cancer, and there are no promising strategies to treat it. Angiostatin, a kringle‐containing fragment of plasminogen, is a potent inhibitor of angiogenesis. The effect of angiostatin on liver metastasis in pancreatic cancer was investigated by using our established hamster model of liver metastasis. Pancreatic cancer cells (PGHAM‐1, 1x106) derived from N‐nitrosobis(2‐oxopropyl)amine (BOP)‐induced pancreatic tumor in Syrian golden hamsters were transplanted into the spleen of female hamsters, and the animals were subcutaneously injected with angiostatin and saline. Subsequently, the macroscopic appearance of liver surface metastases was evaluated. In addition, histological sections of the liver metastases were analyzed for neovascularization, proliferation, and apoptosis on the basis of von Willebrand factor, argyrophilic nucleolar organizer region (Ag‐NOR), and TdT‐mediated dUTP‐biotin nick end labeling (TUNEL) staining, respectively. The results showed significant tumor growth retardation and inhibition of angiogenesis in metastatic liver tumors in response to treatment with angiostatin. Moreover, the metastases remained in a nearly dormant state due to a balance between apoptosis and proliferation of the tumor, with no detectable side effects. This is the first experimental trial of angiostatin on pancreatic cancer and liver metastasis. The results suggest that angiostatin therapy could be effective against liver metastases of pancreatic cancer.
Changes in the Sensitivity of Intratumor Cells during Fractionated Tirapazamine Administration
Authors: Masunaga Shin‐ichiro , Ono Koji , Suzuki Minoru , Kinashi Yuko , Takagaki Masao , Kasai Soko , Nagasawa Hideko , Uto Yoshihiro , Hori Hitoshi
Published: August 2005

Mice bearing solid tumors received 10 intraperitoneal administrations of 5‐bromo‐2′‐deoxyuridine (BrdU) to label the proliferating (P) tumor cells. Then, as a priming treatment, tirapazamine (TPZ) was intraperitoneally administered. Further, 0 through 48 h later, the tumor‐bearing mice received TPZ again at various doses. The tumor cells were isolated and incubated with a cytokinesis blocker. The micronucleus (MN) frequencies in cells with and without BrdU labeling, which were regarded as P and quiescent (Q) cells at the priming treatment, respectively, were determined using immunofluorescence staining for BrdU. The MN frequency in the total (P+Q) tumor cells was determined from the tumors that were not pretreated with BrdU. In addition, P cell ratios in the tumors at the second treatment were determined using immunofluorescence staining for P cell nuclear antigen. In each cell fraction, the longer the interval between the two treatments, the higher was the sensitivity to TPZ, except 1 h after the priming treatment. More than 24 h later, total and P cells, especially P cells, showed significantly higher sensitivity to TPZ than in the case of a single TPZ treatment. The longer the period between the two TPZ treatments, the lower was the P cell ratio at the second treatment. These findings were thought to indicate that the use of TPZ in the treatment of solid tumors causes a shift from the P to the Q state in vivo.
Characterization of the Interaction of TZT‐1027, a Potent Antitumor Agent, with Tubulin
Authors: Natsume Tsugitaka , Watanabe Jun‐ichi , Tamaoki Satoru , Fujio Nami , Miyasaka Katsuhiko , Kobayashi Motohiro
Published: August 2005

TZT‐1027, a derivative of dolastatin 10 isolated from the Indian Ocean sea hare Dolabella auricularia in 1987 by Pettit et al., is a potent antimicrotubule agent. We have compared the activity of TZT‐1027 with that of dolastatin 10 as well as the vinca alkaloids vinblastine (VLB), vincristine (VCR) and vindesine (VDS). TZT‐1027 and dolastatin 10 inhibited microtubule polymerization concentration‐dependently at 1–100 μM with IC50 values of 2.2±0.6 and 2.3±0.7 μM, respectively. VLB, VCR and VDS inhibited microtubule polymerization at 1–3 μM with IC50 values of 2.7±0.6, 1.6±0.4 and 1.6±0.2 μM, respectively, but showed a slight decrease in inhibitory effect at concentrations of 10 μM or more. TZT‐1027 also inhibited monosodium glutamate‐induced tubulin polymerization concentration‐dependently at 0.3–10 μM, with an IC50 of 1.2 μM, whereas VLB was only effective at 0.3–3 μM, with an IC50 of 0.6 μM, and caused so‐called “aggregation” of tubulin at 10 μM. Scatchard analysis of the binding data for [3H]VLB suggested one binding site (Kd 0.2±0.04 μM and Bmax 6.0±0.26 nM/mg protein), while that for [3H]TZT‐1027 suggested two binding sites, one of high affinity (Kd 0.2±0.01 μM and Bmax 1.7±0.012 nM/mg protein) and the other of low affinity (Kd 10.3±1.46 μM, and Bmax 11.6±0.83 nM/mg protein). [3H]TZT‐1027 was completely displaced by dolastatin 10 but only incompletely by VLB. [3H]VLB was completely displaced by dolastatin 10 and TZT‐1027. Furthermore, TZT‐1027 prevented [3H]VLB from binding to tubulin in a non‐competitive manner according to Lineweaver‐Burk analysis. TZT‐1027 concentrationdependently inhibited both [3H]guanosine 5′‐triphosphate (GTP) binding to and GTP hydrolysis on tubulin. VLB inhibited the hydrolysis of GTP on tubulin concentration‐dependently to a lesser extent than TZT‐1027, but no inhibitory effect of VLB on [3H]GTP binding to tubulin was evident even at 100 μM. Thus, TZT‐1027 affected the binding of VLB to tubulin, but its binding site was not completely identical to that of VLB. TZT‐1027 had a potent inhibitory effect on tubulin polymerization and differed from vinca alkaloids in its mode of action against tubulin polymerization.
Marked Enhancement by Fish Meal of Helicobacter pylori‐induced Gastritis in Mongolian Gerbils
Authors: Tanigawa Tetsuya , Kawamori Toshihiko , Iimuro Masaki , Ohta Toshihisa , Higuchi Kazuhide , Arakawa Tetsuo , Sugimura Takashi , Wakabayashi Keiji
Published: August 2005

In a search for dietary factors influencing Helicobacter pylori‐induced gastritis, the effects of fish meal in the diet were examined in Mongolian gerbils. When a conventional diet containing 10% fish meal was given to Mongolian gerbils for 4 weeks after inoculation of H. pylori, edematous thickening with severe neutrophil and mononuclear cell infiltration in both the mucosa and submucosa was observed in the glandular stomach of 19 out of the 20 animals, and hemorrhagic spots were evident in 11 cases. These gastric lesions were enhanced by a 20% fish meal supplement, and edema and hemorrhage in the gastric mucosa were observed in 19 and 17 out of 20 animals, respectively. Although almost the same levels of viable bacteria were detected independent of the diet, edema and hemorrhage were seen in only 2 and 1 of 20 gerbils fed a diet containing 10% casein, instead of 10% fish meal, respectively. Neither edema nor hemorrhage was observed in 10% beef diet animals. These results suggest that fish meal contains factors which greatly enhance H. pylori‐induced gastritis in Mongolian gerbils. Since the incidences of gastritis and gastric cancer are very high throughout the world, it is very important to identify these gastritis‐enhancing factors.
Effect of Age on the Relationship between Gastric Cancer and Helicobacter pylori
Authors: Kikuchi Shogo , Nakajima Toshifusa , Kobayashi Osamu , Yamazaki Tatsuo , Kikuichi Masahiro , Mori Kiyoshi , Oura Shinsuke , Watanabe Hideaki , Nagawa Hirokazu , Otani Reiji , Okamoto Naoyuki , Kurosawa Michiko , Anzai Haruyuki , Kubo Takuji , Konishi Toshiro , Futagawa Shunji , Mizobuchi Noboru , Kobori Oichiro , Kaise Ruriko , Sato Tosiya , Inaba Yutaka , Wada Osamu ,   for the Tokyo Research Group of Prevention for Gastric Cancer
Published: August 2005

Helicobacter pylori is thought to be involved in the pathogenesis of gastric cancer, but the time point at which it produces its effects (critical time) is unknown. We measured the serum level of H. pylori antibody in 787 gastric cancer patients and 1007 controls aged 20 to 69. Odds ratios for different gastric cancer types and stages were determined for each 10‐year age class. The overall odds ratio for gastric cancer decreased with age, being 7.0 for those aged 20–29, 14.5 for those aged 30–39, 9.1 for those aged 40–49, 3.5 for those aged 50–59, and 1.5 for those aged 60–69 (trend in odds ratios: P < 0.01). However, there was no such age‐dependent trend for early diffuse‐type cancer; the odds ratios were 12.6, 4.0, 7.2, 6.5, and 18.5 respectively (P=0.29). Early cancer tended to show higher seroprevalence than advanced cancer, especially in older subjects. No significant difference in seroprevalence was observed between diffuse and intestinal cancers within each age‐class. Seroreversion must have occurred in the time interval between the critical time and the diagnosis of the cancer, especially in older patients. The age‐dependent relationship between H. pylori and gastric cancer may be due to seroreversion, which itself may be independent of age. This age‐independence indicates that prolonged exposure to H. pylori does not increase the magnitude of its influence on gastric carcinogenesis. Possible mechanisms through which H. pylori exerts pathogenic effects are continuous inflammation in adulthood and/or irreversible damage to gastric mucosa in childhood or the teenage years.
Bcl‐XL Antisense Sensitizes Human Colon Cancer Cell Line to 5‐Fluorouracil
Authors: Nita Marcelo Eidi , Ono‐Nita Suzane Kioko , Tsuno Nelson , Tominaga Osamu , Takenoue Tomohiro , Sunami Eiji , Kitayama Joji , Nakamura Yusuke , Nagawa Hirokazu
Published: August 2005

Resistance to 5‐fluorouracil (5‐FU) has been frequently found in the treatment of digestive tract cancer patients. Our previous study suggested that high expression of endogenous Bcl‐XL, might be associated with resistance to 5‐FU in colorectal cancer. The aim of this study is to analyze the role of Bcl‐XL in 5‐FU resistance and to explore a new therapeutic strategy using Bcl‐XL antisense. First, western blot analysis shows that Bcl‐XL rather than Bcl‐2 is overexpressed in primary adenocarcinoma of colon. Second, when Colo320 cells, with undetectable endogenous Bcl‐XL expression, were transfected with Bcl‐XL gene, they acquired high resistance to 5‐FU. Finally, antisense oligodeoxynucleotides (ODNs) that targeted the start codon of Bcl‐XL mRNA (ASI) prove to be the most effective in DLD1 cells with high endogenous Bcl‐XL expression. Bcl‐XL protein expression was decreased in a dose‐dependent manner when the cells were treated with AS1 ODNs, while non‐sense and sense controls and 5‐FU had no effect on Bcl‐XL protein. 5‐FU treatment induced a level of apoptosis 10‐fold higher in DLD1 cells than in untreated control cells, while the same dose of 5‐FU induced a 55‐fold higher level of apoptosis in DLD1 cells treated with Bcl‐XL antisense oligodeoxynucleotides (P=0.0003). Moreover, AS1 ODNs coupled with 5‐FU decreased viable colon cancer cells 40% more than did 5‐FU alone (P < 0.05). These results suggest that Bcl‐XL is an important factor for 5‐FU resistance and the suppression of Bcl‐XL expression by the specific antisense ODNs can increase the sensitivity of colon cancer cells to 5‐FU.
A Novel Gene “Niban” Upregulated in Renal Carcinogenesis: Cloning by the cDNA‐amplified Fragment Length Polymorphism Approach
Authors: Majima Shuichi , Kajino Kazunori , Fukuda Tomokazu , Otsuka Fujio , Hino Okio
Published: August 2005

A modified AFLP (amplified fragment length polymorphism) method was employed to isolate genes differentially expressed in renal carcinogenesis of Tsc2 gene mutant (Eker) rats. One gene, selected for further investigation, was named “Niban” (“second” in Japanese), because it is the second new gene to be found after Erc (expressed in renal carcinoma) in our laboratory. Importantly, “Niban” is well expressed even in small primary rat Eker renal tumors, more than in progressed cell lines, and is also expressed in human renal carcinoma cells, but not in normal human or rat kidneys. Chromosome assignment was to RNO 13 in the rat, and HSA 1. This “Niban” gene is a candidate as a marker for renal tumor, especially early‐stage renal carcinogenesis.
Oxidative DNA Damage Induced by an N‐Hydroxy Metabolite of Carcinogenic 4‐Dimethylaminoazobenzene
Authors: Ohnishi Shiho , Murata Mariko , Degawa Masakuni , Kawanishi Shosuke
Published: August 2005

Formation of adducts has been considered to be a major causal factor of DNA damage by carcinogenic aminoazo dyes. We investigated whether a metabolite of hepatocarcinogenic 4‐dimethylaminoazobenzene (DAB) can cause oxidative DNA damage or not, using 32P‐5′‐end‐labeled DNA fragments. The DAB metabolite. N‐hydroxy‐4‐aminoazobenzene (N‐OH‐AAB) was found to cause Cu(II)‐mediated DNA damage, including 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxodG) formation. When an endogenous reductant, β‐nicotinamide adenine dinucleotide (NADH) was added, the DNA damage was greatly enhanced. Very low concentrations of N‐OH‐AAB could induce DNA damage via redox reactions. Catalase and a Cu(I)‐specific chelator inhibited the DNA damage, suggesting the involvement of H2O2 and Cu(I). A typical OH scavenger did not inhibit the DNA damage. The main reactive species are probably DNA‐copper‐hydroperoxo complexes. We conclude that oxidative DNA damage may play an important role in the carcinogenic processes of DAB, in addition to DNA adduct formation.
Differential Effects of Partial Hepatectomy and Carbon Tetrachloride Administration on Induction of Liver Cell Foci in a Model for Detection of Initiation Activity
Authors: Sakai Hiroki , Tsukamoto Tetsuya , Yamamoto Masami , Shirai Norimitsu , lidaka Takeshi , Yanai Tokuma , Masegi Toshiaki , Tatematsu Masae
Published: August 2005

Differential effects of partial hepatectomy (PH) and carbon tetrachloride (CC14) administration on induction of glutathione S‐transferase placental form (GST‐P)‐positive foci were investigated in a model for detection of initiation activity. Firstly, we surveyed cell proliferation kinetics and fluctuation in cytochrome P450 (CYP) mRNA levels by means of relative‐quantitative real‐time reverse transcriptase‐polymerase chain reaction (RT‐PCR) and CYP 2E1 apoprotein amount by immuno‐blotting (experiment I) after PH or CC14 administration. Next, to assess the interrelationships among cell proliferation, fluctuation of CYPs after PH or CC14 administration and induction of liver cell foci, the non‐hepatocarcinogen, 1,2‐dimethylhydrazine (DMH) was administered to 7‐week‐old male F344 rats and initiated populations were selected using the resistant hepatocyte model (experiment II). In experiment I, the values of all CYP isozyme mRNAs after PH or CC14 administration were drastically decreased at the 12‐h tune point. From 72 h, mRNAs for all CYP isozymes began increasing, with complete recovery after 7 days. The CYP 2E1 apoprotein content in the PH group fluctuated weakly, whereas in the CC14 group it had decreased rapidly after 12 h and was still low at the 48 h point. In experiment II, induction of GST‐P‐positive foci was related to cell kinetics in the PH group, with about a 6‐h time lag between tune for carcinogen administration giving greatest induction of GST‐P‐positive foci and peaks in bromodeoxyuridine (BrdU) labeling, presumably due to the necessity for bioactivation of DMH. With CC14 administration, induction of foci appeared dependent on the recovery of CYP 2E1. In conclusion, PH was able to induce cell proliferation with maintenance of CYP 2E1, therefore being advantageous for induction of liver cell foci in models to detect initiation activity.
Sole Expression of Laminin γhain in Invading Tumor Cells and Its Association with Stromal Fibrosis in Lung Adenocarcinomas
Authors: Kagesato Yuumi , Mizushima Hiroto , Koshikawa Naohiko , Kitamura Hitoshi , Hayashi Hiroyuki , Ogawa Nobuo , Tsukuda Mamoru , Miyazaki Kaoru
Published: August 2005

Laminin‐5 (LN‐5), an important basement membrane (BM) protein consisting of laminin α3, β3 and γ2 chains, has been suggested to be involved in tumor cell invasion and tissue repair. In this study, the distribution of the LN‐5 subunits in atypical adenomatous hyperplasia (AAH)and different types of adenocarcinomas of the lung was examined by immunohistochemical analysis. In AAH and non‐sclerosing, well‐differentiated adenocarcinomas, the LN γ2 chain was frequently detected along with the continuous BMs. These BMs were also positive for both LNα3 and β3 chains, suggesting that LN‐5 had been deposited. In contrast, the cytoplasmic staining for the LNγ2 chain was frequently observed in tumor cells of sclerosing, well‐differentiated adenocarcinomas, as well as of moderately and poorly differentiated adenocarcinomas, without any evidence of co‐expression of the LNα3 and β3 chains. This staining pattern of the LNγ2 chain was prominent in carcinoma cells invading into interstitial stroma and was associated with the formation of a central scar in the tumor tissues. These results suggest that the LNγ2 chain monomer could be an important indicator of progression of lung adenocarcinoma.
The Autocrine Loop of Epidermal Growth Factor Receptor‐Epidermal Growth Factor/Transforming Growth Factor‐α in Malignant Rhabdoid Tumor Cell Lines: Heterogeneity of Autocrine Mechanism in TTC549
Authors: Narita Tsutomu , Taga Takashi , Sugita Kanji , Nakazawa Shinpei , Ohta Shigeru
Published: August 2005

To investigate the effects of the autocrine loop of epidermal growth factor receptor (EGFR)‐epidermal growth factor (EGF)/transforming growth factor‐α (TGF‐α) on the proliferation and differentiation of malignant rhabdoid tumor (MRT), we used five MRT cell lines, TM87‐16, STM91‐01, TTC549, TTC642, and YAM‐RTK1. RT‐PCR analyses revealed expression of EGFR mRNA in all MRT cell lines. In contrast, the expression of either EGF or TGF‐α mRNA was detected in all MRT cell lines. Expression of EGF, TGF‐α, and EGFR as determined by immunocytochemical staining and in situ hybridization, correlated with the results of RT‐PCR. Upon differentiation‐induction with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), in TTC549, showing an expression of TGF‐α but not EGF initially, de novo expression of EGF mRNA appeared abruptly on day 2 of TPA treatment. To confirm the EGFR‐EGF/TGF‐α autocrine loop, we used TGF‐α, EGF, and their antibodies in the cultures. Monoclonal antibody (mAb) to EGFR alone significantly inhibited the growth of cell line TTC549. However, mAb to EGF or TGF‐α could inhibit proliferation of this cell line only when administrated together. Our findings would suggest that growth of the TTC549 cell line is constitutionally regulated by TGF‐α/EGFR, but that inhibition of this autocrine mechanism results in transient activation of an autocrine loop involving EGF/EGFR. Our results may indicate the presence of two different autocrine loops of EGFR‐EGF and/or EGFR‐TGF‐α in MRT cell lines. The heterogeneity of autocrine mechanisms found in MRT cell lines would be consistent with the multiphenotypic diversity and aggressive characteristics of this enigmatic tumor.
Cisplatin‐incorporated Polymeric Micelles Eliminate Nephrotoxicity, While Maintaining Antitumor Activity
Authors: Mizumura Yasuo , Matsumura Yasuhiro , Hamaguchi Tetsuya , Nishiyama Nobuhiro , Kataoka Kazunori , Kawaguchi Takanori , Hrushesky William J. M. , Moriyasu Fuminori , Kakizoe Tadao
Published: August 2005

cis‐Diamminedichloroplatinum (II) (cisplatin, CDDP), a potent anticancer agent, was bound to the aspartic acid residues of poly(ethylene glycol)‐poly(aspartic acid) (PEG‐P(ASP)) block copolymer by ligand substitution reaction at the platinum atom of CDDP. The polymeric drug thus obtained was observed to form a micelle structure in aqueous medium, showing excellent water solubility. In the present study, in vitro and in vivo antitumor activity against several human tumor cell lines, toxicity and pharmacokinetic characteristics in rodents of CDDP‐incorporated polymeric micelles (CDDP/m) were evaluated in comparison with those of CDDP. In vitro, CDDP/m exhibited 10‐17% of the cytotoxicity of CDDP against human tumor cell lines. CDDP/m given by intravenous (i.v.) injection yielded higher and more sustained serum levels than CDDP. In vivo CDDP/m treatment resulted in higher and more sustained levels in tumor tissue than CDDP, and showed similar antitumor activity to CDDP against MKN 45 human gastric cancer xenograft. CDDP/m treatment caused much less renal damage than CDDP. These results indicate that CDDP/m treatment can reduce CDDP‐induced nephrotoxicity without compromising the anticancer cytotoxicity of CDDP.
Small GTP‐binding Protein, Rho, Both Increased and Decreased Cellular Motility, Activation of Matrix Metalloproteinase 2 and Invasion of Human Osteosarcoma Cells
Authors: Matsumoto Yoshihiro , Tanaka Kazuhiro , Harimaya Katsumi , Nakatani Fumihiko , Matsuda Shuichi , Iwamoto Yukihide
Published: August 2005

Rho, a member of the small GTP‐binding proteins, and one of its downstream effectors ROCK (Rho‐associated coiled‐coil forming protein kinase) play an important role in the invasion of tumor cells. Lysophosphatidic acid (LPA) activates Rho and ROCK and promotes the organization of stress fibers and focal adhesions. However, the effect of LPA on tumor cell invasion is still controversial. In the present study, human osteosarcoma cells treated with a high concentration of LPA (high LPA) showed considerable formation of stress fibers and focal adhesions compared to the cells treated with a low concentration of LPA (low LPA). C3 (inhibitor of Rho) or Y27632 (an inhibitor of ROCK) inhibited the effects of LPA, indicating that LPA activates the Rho‐ROCK pathway in the cells. In addition, Rho activation assay showed that the activation level of Rho can be altered by changing the concentration of LPA. Low LPA stimulated the motility and invasion of the cells, while high LPA reduced both. The disruption of extracellular matrix (ECM) by matrix metalloproteinase 2 (MMP2) is also critical for tumor cell invasion. MMP2 is activated by membranous type‐1 MMP (MT1‐MMP) and type‐2 tissue inhibitor of MMP (TIMP2). High LPA suppressed the activation of MMP2 through down‐regulation of MT1‐MMP and TIMP2. C3 and Y27632 reversed the suppression of the activation of MMP2 and expression of MT1‐MMP and TIMP2, suggesting the involvement of the Rho‐ROCK pathway in ECM degradation. Tyrosine phosphorylation of focal adhesion kinase (FAK) was also required for the invasion of tumor cells to occur. Low LPA enhanced the tyrosine phosphorylation of FAK whereas high LPA reduced it. In conclusion, we suggest that Rho has a dual effect on the invasion of osteosarcoma cells by modulating the motility, the ability to degrade ECM and tyrosine phosphorylation of FAK.
Mutations in c‐kit Gene Exons 9 and 13 in Gastrointestinal Stromal Tumors among Japanese
Authors: Sakurai Shinji , Oguni Sachiko , Hironaka Mitsugu , Fukayama Masashi , Morinaga Shojiroh , Saito Ken
Published: August 2005

Gain‐of‐function mutation in c‐kit proto‐oncogene exon 11 has been described in about 20‐50% of gastrointestinal stroma tumor (GIST). Recently, additional mutational hot‐spots in exon 9 and exon 13 of the c‐kit gene have been reported in GISTs without mutations of exon 11, but a subsequent report in a Western population indicated that only a small portion of GISTs (eight of 200 GISTs, 4%) showed mutations in these regions. In this study, we evaluated mutations in exon 9 and exon 13 of the c‐kit gene by both polymerase chain reaction‐single strand conformation polymorphism analysis and direct sequencing in 48 GISTs in a Japanese population, for which the clinicopatho‐logical and immunohistochemical features and mutations in exon 11 had previously been reported. C‐kit gene mutation in exon 9, representing insertion of GCC TAT, was identified in only 4 of 48 GISTs (8%), and none of the GISTs had mutations in exon 13. All four GISTs with mutation in exon 9 were high‐risk, and the patients died of multiple tumor metastasis. Mutations in exon 9 and exon 13 of the c‐kit gene were also rare events in Japanese GISTs and were related to a poor prognosis. These results in Japanese are consistent with those in Western populations, although a preferential occurrence of GISTs with exon 9 mutation in the small intestine, which was suggested in a previous report, was not observed.