bluuebunny/crossref_metadata_2025 huggingface dataset:
Cyclin D1 Overexpression Detected by a Simple Competitive Reverse Transcription‐polymerase Chain Reaction Assay for Lymphoid Malignancies
Authors: Taniguchi Toshiyasu , Fujita Akira , Takahashi Shunji , Uchimaru Kaoru , Yoshikawa Miwa , Asano Shigetaka , Fujita Toshiro , Motokura Toru
Published: August 2005

t(11;14)(q13;q32) observed in B‐cell malignancies is associated with cyclin D1 (bcl‐1, PRAD1, CCND1) overexpression. We devised a simple competitive reverse transcription‐polymerase chain reaction (RT‐PCR) assay for rapid detection of cyclin D1 overexpression. Sharing a single upstream primer derived from a homologous sequence in cyclins D1, D2 and D3, each PCR product serves as a competitor and cyclin D1 overexpression is determined by comparing the intensities of the three amplified products. We analyzed cyclin D1 in clinical specimens from 104 patients with lymphoid malignancies. Cyclin D1 overexpression was evident in 13 of 104 (7/72 non‐Hodgkin's lymphomas, 0/6 adult T‐cell lymphoma/leukemias, 0/4 Hodgkin's diseases, 0/11 acute lymphoblastic leukemias, 3/4 multiple myelomas, 1/2 Waldenströmas, macroglobulinemias, 1/2 prolymphocytic leukemias and 1/3 chronic lymphocytic leukemias). Among 72 patients for whom cytogenetic studies had been done, all 7 patients with t(11;14) were positive. The relative expression levels of D‐type cyclins altered dramatically in the presence of t(11;14). Thus, this RT‐PCR assay can identify tumors with cyclin D1 overexpression. Cyclin D1 overexpression was frequent in extranodal specimens (11 out of 32 vs. 2 of 72 lymph nodes) and was restricted to specific types of lymphoid malignancies, as observed using other methods. This reliable assay should be suitable to provide clinical guidance for the diagnosis and management of lymphoid malignancies, especially in the case of extranodal involvement.
Suppression of Anti‐microtubule Agent‐induced Apoptosis by Nitric Oxide: Possible Mechanism of a New Drug Resistance
Authors: Ogura Tsutomu , DeGeorge George , Tatemichi Masayuki , Esumi Hiroyasu
Published: August 2005

The propensity of a cell to undergo apoptosis has been proposed to be a determinant of sensitivity to anti‐microtubule agents. The anti‐microtubule agents vincristine and paclitaxel induce key features of apoptosis, such as intranucleosomal DNA fragmentation and changes in nuclear morphology in the human neuroblastoma cell line, NB‐39‐nu. Nitric oxide (NO) generated from NO‐releasing drugs prevented anti‐microtubule agent‐induced apoptosis in this cell line. The mechanism of suppression of apoptosis by NO appears to be via the inhibition of an interleukin‐1β converting enzyme‐like protease cascade. This finding reveals a new biological function of NO, as well as a new molecular insight into resistance to chemotherapy with anti‐microtubule agents.
Oral Administration of a Kampo (Japanese Herbal) Medicine Juzen‐taiho‐to Inhibits Liver Metastasis of Colon 26‐L5 Carcinoma Cells
Authors: Ohnishi Yasuharu , Fujii Hideki , Hayakawa Yoshihiro , Sakukawa Rieko , Yamaura Takeshi , Sakamoto Takashi , Tsukada Kazuhiro , Fujimaki Masao , Nunome Shinyu , Komatsu Yasuhiro , Saiki Ikuo
Published: August 2005

We have investigated the inhibitory effect of oral administration of Juzen‐taiho‐to, a Kampo Japanese herbal medicine, on liver metastasis by the inoculation of a liver‐metastatic variant (L5) of murine colon 26 carcinoma cells into the portal vein. Oral administration of Juzen‐taiho‐to for 7 days before tumor inoculation resulted in dose‐dependent inhibition of liver tumor colonies and significant enhancement of survival rate as compared with the untreated control, without side effects. We also found that liver metastasis of L5 cells was enhanced in BALB/c mice pretreated with anti‐asialo GM1 serum or 2‐chloroadenosine, and in BALB/c nu/nu mice, compared to normal mice. This indicates that NK cells, macrophages, and T‐cells play important roles in the prevention of metastasis of tumor cells. Juzen‐taiho‐to significantly inhibited the experimental liver metastasis of colon 26‐L5 cells in mice pretreated with anti‐asialo GM1 serum and untreated normal mice, whereas it did not inhibit metastasis in 2‐chloroadenosine‐pretreated mice or T‐cell‐deficient nude mice. Oral administration of Juzen‐taiho‐to activated peritoneal exudate macrophages (PEM) to become cytostatic against the tumor cells. These results show that oral administration of Juzen‐taiho‐to inhibited liver metastasis of colon 26‐L5 cells, possibly through a mechanism mediated by the activation of macrophages and/or T‐cells in the host immune system. Thus, Juzen‐taiho‐to may be efficacious for the prevention of cancer metastasis.
Role of MDM2 Overexpression in Doxorubicin Resistance of Breast Carcinoma
Authors: Suzuki Akio , Toi Masakazu , Yamamoto Yutaka , Saji Shigehira , Muta Mariko , Tominaga Takeshi
Published: August 2005

Several oncoproteins or tumor suppressor gene products have been indicated to be of value as predictors of the de novo resistance to cytotoxic agents. In this study, we have investigated the role of MDM2 (murine double minutes) overexpression in doxorubicin resistance of breast cancer. Immunocytochemical analysis demonstrated that MDM2‐positive tumors, even with p53‐negative pheno‐type, were significantly more resistant to doxorubicin treatment compared to MDM2‐negative tumors. An in vitro experimental model using stable mdm2‐transfected MCF‐7 cells carrying wild‐type p53 confirmed that the cells become approximately 3‐fold more resistant to doxorubicin as a result of MDM2 overexpression, and the wild‐type p53 function, such as the induction of p21Waf1 following DNA damage, was significantly suppressed. MDM2 overexpression is suggested to be a novel marker for predicting lack of response to doxorubicin treatment in breast cancer patients.
Monitoring of Solar‐UV Exposure among Schoolchildren in Five Japanese Cities Using Spore Dosimeter and UV‐coloring Labels
Authors: Munakata Nobuo , Ono Masashi , Watanabe Shaw
Published: August 2005

To monitor personal exposure to biologically effective solar‐UV radiation, Bacillus subtilis spores on a membrane filter and UV‐coloring labels were incorporated into a monitoring badge. The samples were covered with one of three types of filter sheet, dependent on the season, to reduce the amounts of exposure to measurable levels. Five fifth‐ or sixth‐grade classes of primary schools, each consisting of 30–40 children, were chosen in northern (Sapporo), central (Tsukuba and Tokyo), and southern (Miyazaki and Naha) cities in Japan. In all four seasons, each child wore a badge on an upper arm for the entire waking hours, changing it daily, for a week. Upon collection of the badges, the survival of spores and the extent of coloration of the label were determined. The results were used to estimate the amount of daily exposure to biologically effective UV radiation, expressed as the value of spore inactivation dose. Unexpectedly, the average amounts of exposure were not directly correlated with the outdoor UV irradiance: in the two southern cities, despite high outdoor irradiance from spring to autumn, the average amounts of exposure were less than 3.1% of the average irradiance. Highly concentrated exposures occurred in two central cities on three days when extensive outdoor exercise took place. These results contradict the simple notion that childrens' exposure is in proportion to the outdoor UV irradiance, and support the view that the extent of solar‐UV exposure is primarily determined by life‐style rather than living location.
Influence of Drinking Green Tea on Breast Cancer Malignancy among Japanese Patients
Authors: Nakachi Kei , Suemasu Kimito , Suga Kenji , Takeo Takeshi , Imai Kazue , Higashi Yasuhiro
Published: August 2005

Inhibitory effects of green tea on carcinogenesis have been investigated in numerous laboratory studies using (–)‐epigallocatechin gallate (EGCG) or crude green tea extract, and there is also some epidemiologic evidence. Further, EGCG has been reported to inhibit the growth of cancer cells, lung metastasis in an animal model, and urokinase activity. In this study, we first examined the association between consumption of green tea prior to clinical cancer onset and various clinical parameters assessed at surgery among 472 patients with stage I, II, and III breast cancer. We found that increased consumption of green tea was closely associated with decreased numbers of axillary lymph node metastases among premenopausal patients with stage I and II breast cancer and with increased expression of progesterone receptor (PgR) and estrogen receptor (ER) among postmenopausal ones. Since these are potential prognostic factors, we then investigated the prognosis of breast cancer with special reference to consumption of green tea, in a follow‐up study of these patients. We found that increased consumption of green tea was correlated with decreased recurrence of stage I and II breast cancer (P<0.05 for crude disease‐free survival); the recurrence rate was 16.7 or 24.3% among those consuming ≥5 cups or ≥4 cups per day, respectively, in a seven‐year follow‐up of stage I and II breast cancer, and the relative risk of recurrence was 0.564 (95% confidence interval, 0.350–0.911) after adjustment for other lifestyle factors. However, no improvement in prognosis was observed in stage III breast cancer. Our results indicate that increased consumption of green tea prior to clinical cancer onset is significantly associated with improved prognosis of stage I and II breast cancer, and this association may be related to a modifying effect of green tea on the clinical characteristics of the cancer.
Early Phase Tumor Accumulation of Macromolecules: A Great Difference in Clearance Rate between Tumor and Normal Tissues
Authors: Noguchi Youichiro , Wu Jun , Duncan Ruth , Strohalm Jiří , Ulbrich Karel , Akaike Takaaki , Maeda Hiroshi
Published: August 2005

The objective of this study was to investigate the molecular weight (MW) and time‐dependence of the phenomenon termed “the enhanced permeability and retention”(EPR) effect in solid tumor, in particular to determine and define the early phase accumulation of macromolecules in tumor and normal tissues and the relationship between blood concentration and tissue clearance. As a model, radioiodinated N‐(2‐hydroxypropyl)methacrylamide (HPMA) copolymers of MW ranging from 4.5 K to 800 K were administered i.v. to mice bearing sarcoma 180 tumor. Within 10 min all HPMA copolymers accumulated effectively in the tumor regardless of MW (1.0–1.5% of injected dose per g of tumor). However, higher MW copolymers (>50 K) showed significantly increased tumor accumulation after 6 h, while the lower MW copolymers (<40 K) were cleared rapidly from tumor tissue due to rapid diffusion back into the bloodstream. Blood clearance was also MW‐dependent; the lower MW copolymers displayed rapid clearance, with kidney radioactivity of the copolymers of MW <20 K representing 24% of injected dose per g kidney at 1 min after i.v. administration. Within 10 min these copolymers passed through the kidney and were excreted in the urine. Higher MW copolymers consistently showed kidney levels of 3–5% dose per g kidney in the early phase with no time‐dependent accumulation in kidney. There was also no progressive accumulation in muscle or liver, regardless of polymer MW. These results suggest the “EPR effect” in solid tumor primarily arises from in the difference in clearance rate between the solid tumor and the normal tissues after initial penetration of the polymers into these tissues.
Intrinsic Sensitivity of Tumor Cells to Bleomycin as an Indicator of Tumor Response to Electrochemotherapy
Authors: Čemažar Maja , Miklavčič Damijan , Serša Gregor
Published: August 2005

Electrochemotherapy (ECT) involves the use of locally applied electric pulses to increase delivery of chemotherapeutic drugs into cells in tissues. ECT with bleomycin (BLM) is a very effective local treatment, but different tumors have different response rates to ECT. The aim of our study was to compare the responsiveness of SA‐1 and EAT tumors to BLM and ECT in vitro and in vivo, in order to find possible reasons for the observed difference in response rate. The difference in sensitivity to ECT in vitro between the SA‐1 and EAT cells was 10‐fold and was the same as the difference in sensitivity to chronic BLM exposure, as measured by tetrazolium‐based colorimetric (MTT) assay. This difference in sensitivity between SA‐1 and EAT to ECT was also reflected in tumor cure rate. A six‐times lower dose of BLM was needed to obtain local tumor control in SA‐1 than in EAT tumors. Therefore, we suggest that the difference in sensitivity to BLM and ECT predominantly reflects the difference in intrinsic sensitivity of the cells to BLM.
Effects of Boron Neutron Capture Therapy Using Borocaptate Sodium in Combination with a Tumor‐selective Vasoactive Agent in Mice
Authors: Ono Koji , Masunaga Shin‐ichiro , Kinashi Yuko , Takagaki Masao , Akaboshi Mitsuhiko , Suzuki Minoru , Baba Hideo
Published: August 2005

Boron neutron capture therapy (BNCT) destroys tumor cells by means of α particles and recoil protons emitted by 10B(n,α)7Li reaction. For BNCT to be effective, the tumor/normal tissue concentration ratio of 10B must be larger than 1.0, because neutron distribution is not selective. We examined the combination of 10B‐enriched borocaptate sodium (BSH) with flavone acetic acid (FAA) as a model compound which causes vascular collapse in squamous cell carcinoma in mice (SCCVII tumors) and would increase the tumor/normal tissue concentration ratio of 10B. FAA (200 mg/kg, i.p.) was injected, and 5 min later BSH (75 mg/kg, i.v.) was administered, followed 15 to 180 min later by irradiation with thermal neutrons. The 10B concentrations were measured by prompt gamma ray spectrometry. Without FAA, tumor 10B concentrations were less than or equal to normal tissue concentrations at all time intervals, except that the concentrations were 1.7‐ to 2.7‐fold greater in tumor than muscle at 15 and 180 min after injection of BSH. With FAA, 10B concentrations 2.1‐ to 6.9‐fold greater in tumor than in muscle were achieved at all intervals tested. For blood and skin, significant differential accumulations were found in tumors at 120 and 180 min. Tumor/liver ratios were less than 1 at all times. Cell survival was determined by in vivo/in vitro colony assay, and increasing radiosensitization correlated with increasing tumor 10B concentrations, whether or not they were achieved with FAA. Tumor control rates, determined at 180 days after BNCT, similarly appeared to depend only on 10B levels at the time of irradiation. Because 10B levels correlate with the radiation response of tissues, a therapeutic gain would be expected whenever the tumor levels exceed normal tissue levels, such as in tumors located in muscle irradiated at 15–180 min after FAA+BSH, or in those in skin irradiated at 120 and 180 min.
Patients' Views on Residual Blood Use for Research Purposes
Authors: Hamajima Nobuyuki , Tajima Kazuo , Oya Haruji , Kato Taketoshi , Okuma Katashi , Kaneda Hisae , Moritaka Shigeko , Kato Tomoyuki
Published: August 2005

In order to document patients' views on residual blood use for research purposes, a questionnaire survey was conducted at Aichi Cancer Center Hospital in October 1997. Subjects were patients who had undergone blood tests at a central blood sampling room in the morning during the week of the study enrollment. The questionnaire was handed out and collected at a waiting area in front of the blood sampling room. Of the 583 patients to whom we tried to hand out the questionnaire, 558 participated (258 males, 294 females, and 6 of sex unknown) and 25 refused. Those who regarded research to improve health care as important were 76.7% of those sampled. Only 28 patients (5.0%) answered that they would not permit the use of their residual blood for research purposes. Although logistic analysis did not detect significant factors influencing the giving of permission, the percentage who would not permit the use of their residual blood for research purposes was significantly higher in cancer outpatients (6.7%) than in inpatients (1.0%). It seems desirable for hospitals to establish an open policy concerning residual blood use for research purposes.
Suppressive Effect of Irsogladine Maleate on Diethylnitrosamine‐initiated and Phenobarbital‐promoted Hepatocarcinogenesis in Male F344 Rats
Authors: Sugie Shigeyuki , Okamoto Kiyohisa , Ueda Fusao , Watanabe Tomoyuki , Tanaka Takuji , Mori Hideki
Published: August 2005

Modifying effects of irsogladine maleate (IRG) on diethylnitrosamine (DEN)‐induced hepatocarcinogenesis were examined in male F344 rats. Six‐week‐old rats were divided into 8 groups. Groups 1 through 4 were given a single i.p. injection of DEN (200 mg/kg body weight) at the start of the experiment, whereas groups 5 through 8 received a single i.p. injection of saline as the vehicle treatment. Groups 1 and 8 were kept on the basal diet and distilled water throughout the experiment (36 weeks). Groups 2 and 7 were exposed to 500 ppm phenobarbital (PB) in the drinking water, starting one week after the carcinogen or vehicle treatment. Groups 3 and 5 were fed the diet mixed with 125 ppm IRG from one week after DEN or vehicle treatment. Groups 4 and 6 were given 125 ppm IRG‐containing diet and drinking water with 500 ppm PB after the carcinogen or vehicle treatment. Liver neoplasms developed in groups 1 (1/15 rats, 7%) and 2 (14/14 rats, 100%). However, no liver tumors were found in rats of groups 3 through 8. Incidence and average number of liver neoplasms in group 4 (0/14 rats, 0%) were less than those in group 2 (P<0.0001). The number of glutathione S‐transferase placental form (GST‐P)‐positive liver cell foci in group 3 or 4 was significantly smaller than that in the appropriate control (P<0.01, P<0.001, respectively). The average and unit areas of these foci in group 4 were also significantly smaller than those in group 2 (P<0.001, P<0.05, respectively). These results suggest that IRG could be a chemopreventive agent for rat liver carcinogenesis.
Reduced Invasive and Metastatic Potentials of KAI1‐transfected Melanoma Cells
Authors: Takaoka Akinori , Hinoda Yuji , Sato Shuji , Itoh Fumio , Adachi Masaaki , Hareyama Masato , Imai Kohzoh
Published: August 2005

KAI1 is a metastasis suppressor gene for human prostate cancer. To reveal the effect of KAI1 on the in vivo metastasis of tumors other than prostatic cancer, we transfected a human KAI1 cDNA into highly metastatic B16‐BL6 murine melanoma cells and established stable transfectant clones with different expression levels of KAI1 message. The following results were obtained with the use of those transfectants. (1) Cell aggregation assay revealed a significantly enhanced Ca2+‐independent aggregation of B16‐BL6 cells by KAI1 cDNA transfection compared with mock transfectants (P<0.01). (2) The in vivo phagokinetic activity and invasive ability of KAI1 transfectants were clearly decreased as compared with those of mock transfectants (P<0.01). There was no significant effect of KAI1 expression on the in vitro or in vivo proliferation of B16‐BL6 cells. (3) Lung colony formation of intravenously injected KAI1 transfectants in nude mice was significantly reduced as compared with mock transfectants or parental B16‐BL6 cells (P<0.01). These data suggest that KAI1 expression gives rise to the suppression of invasive and metastatic potentials of B16‐BL6 cells.
In vivo Adenovirus‐mediated Prodrug Gene Therapy for Carcinoembryonic Antigen‐producing Pancreatic Cancer
Authors: Ohashi Makoto , Kanai Fumihiko , Tanaka Torao , Lan Keng‐Hsin , Shiratori Yasushi , Komatsu Yutaka , Kawabe Takao , Yoshida Haruhiko , Hamada Hirofumi , Omata Masao
Published: August 2005

In gene therapy for malignancy, the herpes simplex virus thymidine kinase (HSVtk)‐ganciclovir (GCV) system has been widely used. For pancreatic cancer targeting, we estimated the therapeutic efficacy of gene transduction by an adenovirus‐carrying HSVtk gene under the control of a carci‐noembryonic antigen (CEA) promoter (AdCEAtk) followed by systemic administration of GCV. Four cell lines, CEA‐producing Su.86.86, BxPC‐3 (pancreatic cancer cells), MKN45 (gastric cancer cells) and CEA‐nonproducing HeLa, were used for analysis of GCV sensitivity induced by adeno‐viral gene transduction. To evaluate the therapeutic efficacy of AdCEAtk and GCV administration in human CEA‐positive pancreatic cancer in vivo, a subcutaneously implanted tumor‐bearing nude mouse model was used. When the HSVtk gene was transduced with a ubiquitous promoter into these cells, increase of the GCV sensitivity was independent of CEA‐production. In contrast, when the cells were transduced with a CEA promoter, the cell‐killing effect of GCV was increased in only CEA‐producing cells. For in vivo analysis, AdCEAtk was delivered into subcutaneously established tumors of Su.86.86 cells. Immunohistochemical staining of the tumor showed that HSVtk protein was expressed only in tumor cells, and tumor growth was markedly suppressed by administration of GCV. These results suggest that the adenovirus‐mediated transfer of HSVtk gene with CEA promoter specifically increases the GCV sensitivity of CEA‐producing pancreatic cancer cells in vitro and in vivo. This strategy may provide a useful tool for treating pancreatic cancer, especially CEA‐producing tumor cells.
Development of High‐grade Renal Cell Carcinomas in Rats Independently of Somatic Mutations in the Tsc2 and VHL Tumor Suppressor Genes
Authors: Toyokuni Shinya , Okada Kunihiko , Kondo Shohei , Nishioka Hiroaki , Tanaka Tomoyuki , Nishiyama Yasuyuki , Hino Okio , Hiai Hiroshi
Published: August 2005

Ferric nitrilotriacetate (Fe‐NTA) induces renal proximal tubular damage that ultimately leads to a high incidence of renal cell carcinoma (RCC) in rats. The RCCs are characterized by 1) high incidence of pulmonary metastasis and peritoneal invasion, 2) high incidence of tumor‐associated mortality and 3) possible involvement of reactive oxygen species in carcinogenesis. The present study investigated the possible role of Tsc2 and VHL tumor suppressor genes in this model. Thirty‐four Fe‐NTA‐induced primary RCCs and 20 other primary or metastatic tumors of rats were searched for genetic alteration in all the coding exons of both genes by polymerase chain reaction‐single‐strand‐conformation polymorphism analysis and sequencing in conjunction with morphological evaluation. In the Fe‐NTA‐induced RCCs, frequency of metastasis or invasion was proportionally associated with the nuclear grade of the tumor (grades 1–3). Only one Fe‐NTA‐induced RCC of grade 1 revealed missense mutations with loss of heterozygosity in exon 10 of the Tsc2 gene (codons 334, GTG (Val) to GCG (Ala), and 336, TAT (Tyr) to CAT (His)). No mutation was found in the VHL gene. The results suggest that 1) high‐grade RCCs can develop in the absence of mutations in the Tsc2 and VHL genes in rats, and that 2) Tsc2 gene somatic mutation can nonetheless be one of the causes of non‐Eker rat RCCs.
Mutation Analysis of the WT1 Gene in Myelodysplastic Syndromes
Authors: Hosoya Noriko , Miyagawa Kiyoshi , Mitani Kinuko , Yazaki Yoshio , Hirai Hisamaru
Published: August 2005

The WT1 tumor suppressor gene was examined for mutations in a panel of 44 patients with myelo‐dysplastic syndromes (MDS) including acute myelogenous leukemias (AML) secondary to MDS, using polymerase chain reaction single‐strand conformation polymorphism (PCR‐SSCP) analysis and sequencing analysis. A WT1 mutation was detected in one out of 17 cases of AML secondary to MDS. This mutation exists upstream of the zinc finger region and is predicted to produce a truncated WT1 protein lacking the zinc finger region. No mutations were detected in 27 MDS patients who had not progressed to AML. This is the first report of analysis for WT1 mutations in a large number of MDS patients, suggesting that WT1 mutations are uncommon in MDS. Abnormalities in this gene may, however, contribute to a small proportion of cases showing progression from MDS into AML.
Infrequent Mutations of the hOGG1 Gene, That Is Involved in the Excision of 8‐Hydroxyguanine in Damaged DNA, in Human Gastric Cancer
Authors: Shinmura Kazuya , Kohno Takashi , Kasai Hiroshi , Koda Kenji , Sugimura Haruhiko , Yokota Jun
Published: August 2005

DNA glycosylase, encoded by the hOGG1 gene, repairs 8‐hydroxyguanine (oh8Gua), which is an oxidatively damaged mutagenic base. To clarify whether the DNA repair activity of hOGG1 protein is involved in gastric carcinogenesis, we examined 9 gastric cancer cell lines and 35 primary gastric cancers for mutations and genetic polymorphisms of the hOGG1 gene by polymerase chain reaction‐single strand conformation polymorphism analysis. A G‐to‐A transition was detected in a gastric cancer cell line, MKN1. This nucleotide change caused the conversion of the amino acid from Arg to His at codon 154, which is located in a domain highly conserved among human, mouse, and yeast OGG1 proteins. No mutation was detected in primary gastric cancers. We compared the distribution of the polymorphic alleles associated with enzymatic activity (hOGG1‐Ser326 vs. hOGG1‐Cys326) between 35 gastric cancer patients and 42 healthy individuals. Although the frequency of the Cys326 allele, associated with low enzymatic activity, in gastric cancer patients was a little higher than that in healthy individuals, the difference did not reach statistical significance. These results suggest that low hOGG1 activity due to mutations and genetic polymorphisms is involved in the development of only a small subset of gastric cancers.
Differential Expression of DNA Topoisomerase IIα and IIβ Genes between Small Cell and Non‐small Cell Lung Cancer
Authors: Syahruddin Elisna , Oguri Tetsuya , Takahashi Toshiaki , Isobe Takeshi , Fujiwara Yasuhiro , Yamakido Michio
Published: August 2005

DNA topoisomerase II (Topo II) inhibitors are widely used in lung cancer chemotherapy, but small cell lung cancer (SCLC) and non‐small cell lung cancer (NSCLC) show different sensitivity to them. In this study, we examined the gene expression levels of both isoforms of Topo II (IIα and IIβ) in lung cancer specimens to investigate the differential expression between SCLC and NSCLC. The expression levels of the Topo IIα and Topo IIβ genes were assessed in 80 autopsy samples (40 primary tumors and 40 corresponding normal lung tissues) by using the reverse transcription polymerase chain reaction. We found that the expression levels of the Topo IIα gene in tumors were significantly higher than those in normal lung tissues, and that those in SCLC were significantly higher than those in NSCLC. There were no significant differences in Topo IIβ gene expression between tumors and normal lung tissues and between SCLC and NSCLC. Furthermore, correlation analysis revealed that Topo IIα expression was correlated with Topo IIβ expression in both tumor and normal lung tissues. These results indicate that a difference exists in the regulation of the Topo II gene between lung tumors and normal lung tissues. Our finding of differential expression of Topo IIα between SCLC and NSCLC also suggests that the Topo IIα expression level is associated with sensitivity to Topo II inhibitors.
Allelic Loss on Chromosome 9q Is Associated with Lymph Node Metastasis of Primary Breast Cancer
Authors: Minobe Kaori , Onda Masahiko , Iida Aritoshi , Kasumi Fujio , Sakamoto Goi , Nakamura Yusuke , Emi Mitsuru
Published: August 2005

Frequent allelic losses on chromosome 9 are seen in a wide variety of human tumors; moreover, two genes (P16 and PTC) whose mutant alleles confer predispositions to some inherited cancer syndromes have been identified on this chromosome. Using 15 highly polymorphic microsatellite markers distributed on both arms of chromosome 9, we tested 96 primary breast carcinomas for allelic loss in order to define the locations of genes that might be involved in this type of tumor. Allelic loss was observed in 37 of the tumors (39%) and detailed deletion mapping identified target regions at 9p21, 9q22.3 and 9q33. Losses at 9q22.3 and 9q33 were correlated with the presence of lymph node metastasis, and allelic loss at 9q22.3 was observed more frequently in scirrhous tumors than in less aggressive histologic types. Therefore, inactivation of tumor suppressor genes in 9q22.3 and 9q33 regions might play a role in progression of breast cancers, especially in metastasis to lymph nodes and in development of scirrhous tumors.
Selection of Human Ovarian Carcinoma Cells with High Dissemination Potential by Repeated Passage of the Cells in vivo into Nude Mice, and Involvement of Lex‐determinant in the Dissemination Potential
Authors: Kiguchi Kazushige , Iwamori Masao , Mochizuki Yukari , Kishikawa Takeshi , Tsukazaki Katsumi , Saga Masahiko , Amemiya Akira , Nozawa Shiro
Published: August 2005

Cells of the human tumor cell line RMG‐1, derived from a clear‐cell adenocarcinoma of the ovary, were injected intraperitoneally into nude mice, and the cells obtained from the tumor nodules in the mesenterium were found to form a larger number of, and larger‐sized, tumor nodules than the original RMG‐1 cells. The RMG‐1‐h cells, transferred into culture from the tumor nodules after a 4th in vivo passage, showed a dissemination potential as high as that of cells disseminating directly from the tissues, and exceedingly higher than that of RMG‐1 cells. To assess the molecular bases of the different biological properties of RMG‐1 and RMG‐1‐h cells, we compared the content and expression of various carbohydrate antigens in both cells. The chromosomal profile of RMG‐1‐h cells revealed their human origin and was identical to that of the original RMG‐1 cells. In contrast to the broad histogram for the Lex‐bearing cells among RMG‐1 cells in flow cytometry, the weakly and moderately positive cells toward anti‐Lex antibody were found to be eliminated from the histogram for the RMG‐1‐h cells, resulting in the enrichment of cells strongly expressing Lex, which may account for the high dissemination potential. In addition, the adhesion of RMG‐1 cells to mesothelial cells was found to be significantly inhibited by pretreatment of the cells with anti‐Lex antibody, indicating Lex‐mediated cell‐to‐cell interaction between ovarian cancer cells and mesothelial cells. By TLC‐immunostaining, two Lex‐glycolipids, III3Fucα‐nLc4Cer and V3Fucα‐nLc6Cer were detected in both RMG‐1 and RMG‐1‐h cells, and their total concentrations were not significantly different from each other. However, the hydrophobic moieties of Lex‐glycolipids in RMG‐1‐h cells were different from those in RMG‐1 cells, suggesting that a difference in the structure of the hydrophobic moieties of Lex is partly involved in the enhanced reactivity of RMG‐1‐h cells toward anti‐Lex antibody. Thus, the high dissemination potential of ovarian cancer cells was shown to be mediated by the Lex‐determinant and the Lex‐bearing cells are enriched by repeated in vivo passage of the cells into nude mice.
Antitumor Effect of a Neutralizing Antibody to Vascular Endothelial Growth Factor on Liver Metastasis of Endocrine Neoplasm
Authors: Konno Hiroyuki , Arai Tomio , Tanaka Tatsuo , Baba Megumi , Matsumoto Keigo , Kanai Toshikazu , Nakamura Satoshi , Baba Shozo , Naito Yasuhisa , Sugimura Haruhiko , Yukita Ayako , Asano Makoto , Suzuki Hideo
Published: August 2005

Distant metastasis of gastrointestinal endocrine neoplasm is resistant to currently available treatments. Because hematogenic metastasis is dominant, anti‐angiogenic drugs are expected to be a novel therapy for this neoplasm. In the present study, the therapeutic effect of vascular endothelial growth factor neutralizing antibody (VEGFAb) on liver metastasis of an endocrine neoplasm was investigated experimentally. Cecal transplantation into nude mice of small pieces of EN‐1, a xenotransplanted human intestinal endocrine neoplasm, resulted in liver metastasis. A treated group (n=19) received 100 μg/mouse of VEGFAb intraperitoneally on alternate days from day 10 after tumor transplantation, and the control group (n=19) received saline. Five of the 19 control mice died of tumor progression, of which 2 could not be evaluated. The cecal tumor weighed 6316±2333 mg (n=17) in the control group and 1209±837 mg (n=19) in the treated group (P<0.01) 6 weeks after transplantation. Liver metastasis developed in 16 of 17 control mice and in 2 of 19 treated mice (P<0.01). The VEGF level of the whole cecal tumor in the control group was significantly higher than that in the treated group (305.1±174.1 vs. 54.7±41.2 mg; P<0.001). VEGFAb did not cause any body weight loss (28.52±1.63 in the control vs. 28.44±1.71 g in the treated group). These results indicate that VEGFAb may be a novel therapeutic agent for endocrine neoplasm with distant metastasis.
Caspase‐3 Activation Is Not Responsible for Vinblastine‐induced Bcl‐2 Phosphorylation and G2/M Arrest in Human Small Cell Lung Carcinoma Ms‐1 Cells
Authors: Tashiro Etsu , Simizu Siro , Takada Minoru , Umezawa Kazuo , Imoto Masaya
Published: August 2005

Vinblastine arrests cells in the G2/M phase of the cell cycle and subsequently induces cell death by apoptosis. We found that treatment of cells with vinblastine induced phosphorylation of Bcl‐2, resulting in the dissociation of Bcl‐2 and Bax. Moreover, vinblastine‐induced apoptosis was suppressed by an inhibitor of caspase‐3, Ac‐DEVD‐CHO; and a 17‐kDa active fragment of caspase‐3 was detected following vinblastine treatment, suggesting that caspase‐3 is involved in vinblastine‐induced apoptosis. However, Ac‐DEVD‐CHO affected neither vinblastine‐induced Bcl‐2 phosphorylation nor vinblastine‐induced G2/M arrest. Vinblastine caused G2/M arrest prior to apoptosis, whereas vinblastine‐induced apoptosis was not dependent on the duration of the G2/M phase. Thus, vinblastine‐induced apoptosis might be mediated by the phosphorylation of Bcl‐2, resulting in Bcl‐2 inactivation, and by subsequent activation of caspase‐3.
Inhibition of Liver Metastasis of Human Pancreatic Carcinoma by Angiogenesis Inhibitor TNP‐470 in Combination with Cisplatin
Authors: Shishido Takayuki , Yasoshima Takahiro , Denno Ryuichi , Mukaiya Mitsuhiro , Sato Noriyuki , Hirata Koichi
Published: August 2005

The anti‐tumor and anti‐metastatic effects of O‐(chloroacetyl‐carbamoyl) fumagillol (TNP‐470), an angiogenesis inhibitor, and cisplatin (CDDP), an anti‐neoplastic agent, were investigated using our established liver‐metastasizing pancreatic carcinoma line, HPC‐3H4. HPC‐3H4 was injected into the spleens of nude mice. Mice were randomly divided into 5 groups; a control group given saline solution, a group receiving 45 mg/kg TNP‐470, a group receiving 90 mg/kg TNP‐470, a group receiving 90 mg/kg TNP‐470 in combination with 0.25 mg/kg CDDP, and a group receiving 0.25 mg/kg CDDP. In the control group, liver metastasis developed in 14 of 15 mice (93.3%). Liver metastasis developed in 9 of 11 mice (81.8%) receiving 0.25 mg/kg CDDP. It developed in 11 of 15 mice (73.3%) receiving 45 mg/kg TNP‐470, in 17 of 18 mice (94.4%) receiving 90 mg/kg TNP‐470, and in 4 of 10 mice (40%) receiving 90 mg/kg TNP‐470 in combination with 0.25 mg/kg CDDP. TNP‐470 in combination with CDDP displayed a significant inhibitory effect on liver metastasis compared to the control. Although TNP‐470 alone and CDDP alone had no effect on the tumor growth in vivo, 90 mg/kg TNP‐470 in combination with 0.25 mg/kg CDDP had a significant effect. In vitro examinations demonstrated that the growth of HPC‐3H4 cells was only mildly inhibited by TNP‐470, but the production of vascular endothelial growth factor (VEGF) by HPC‐3H4 was clearly inhibited by TNP‐470. The inhibitory effect on the production of VEGF was not strong with CDDP treatment. These results indicate that the angiogenesis inhibitor TNP‐470 in combination with low‐dose CDDP has inhibitory activity against liver metastasis of human pancreatic carcinoma.
Potentiation of Paclitaxel Cytotoxicity by Inostamycin in Human Small Cell Lung Carcinoma, Ms‐1 Cells
Authors: Simizu Siro , Tanabe Keiko , Tashiro Etsu , Takada Minoru , Umezawa Kazuo , Imoto Masaya
Published: August 2005

In the present study, we found that inostamycin increased the ability of paclitaxel to induce apoptosis in Ms‐1 cells. A considerably higher concentration of paclitaxel was required for the induction of apoptosis in Ms‐1 cells than in other cell lines tested. Treatment of Ms‐1 cells with inostamycin, an inhibitor of phosphatidylinositol (PI) synthesis, reduced the dosage of paclitaxel required to induce cell death by apoptosis. This effect of inostamycin is specific to Ms‐1 cells, and inostamycin did not increase the cytotoxicity of other antitumor drugs such as adriamycin, vinblastine, methotrexate, cisplatin, etoposide, or camptothecin in Ms‐1 cells. Addition of inostamycin to paclitaxel‐treated cells caused a significant increase in the sub G1 peak, representing apoptosis, which was accompanied by a decrease in the G2/M peak seen in paclitaxel‐treated Ms‐1 cells, without affecting paclitaxel‐inhibited tubulin depolymerization. Moreover, paclitaxel did not enhance inostamycin‐inhibited PI synthesis. The expression levels of Bcl‐2, Bax, and Bcl‐XL were not changed following the co‐treatment with inostamycin plus paclitaxel, whereas the activated form of caspase‐3 was markedly increased. Thus, inostamycin is a chemosensitizer of paclitaxel in small cell lung carcinoma Ms‐1 cells.
Effects of a Gonadotropin‐releasing Hormone Agonist on Rat Ovarian Adenocarcinoma Cell Lines in vitro and in vivo
Authors: Maruuchi Tatsuhiro , Sugiyama Toru , Kataoka Akio , Nishida Takashi , Yakushiji Michiaki
Published: August 2005

To evaluate the biologic effects of the gonadotropin‐releasing hormone (GnRH) agonist buserelin on rat ovarian adenocarcinoma cells in vivo and in vitro, female Wistar rats with primary ovarian adenocarcinoma induced by 7, 12‐dimethylbenz(a)anthracene (DMBA) and the DMBA‐OC‐1 cell line established from a DMBA‐induced rat tumor were used in this study. In vivo, daily administration of buserelin significantly suppressed the release of follicle‐stimulating hormone (FSH), luteinizing hormone (LH), and progesterone as compared with controls. Buserelin did not inhibit the growth of DMBA‐induced tumors. However, histopathologically, there was increased central necrosis and a decrease in the number of neoplastic cells, with proliferation of connective tissue, in the group treated with buserelin. In vitro, FSH‐induced proliferation of DMBA‐OC‐1 cells was suppressed by buserelin. Thus, this basic experimental study supports the potential use of a GnRH agonist to suppress the growth of ovarian cancer.
Neoplastic Transformation of Rat Colon Epithelial Cells by Expression of Activated Human K‐ras
Authors: Sugiyama Kenji , Otori Kazuhiko , Esumi Hiroyasu
Published: August 2005

Somatic mutations of the K‐ras oncogene play an important role in colorectal carcinogenesis. We determined whether rat colon epithelial cells could be transformed by introducing retroviruses carrying the activated human K‐ras oncogene alone. Primary epithelial cells from the rat distal colon were infected with retroviruses carrying wild‐type and two types of activated K‐ras (asp and val at codon 12) cDNAs. Cells infected with the wild‐type K‐ras virus showed no change in morphology and died within 3 weeks, whereas the activated K‐ras virus‐infected cells underwent morphological changes within 3 days and continued to proliferate. From these cells, several cell lines were subsequently established. Epithelial cells transformed by activated K‐ras formed colonies in soft agar culture and tumors in athymic nude mice. Multiple copies of human K‐ras genes and large amounts of K‐ras mRNAs and proteins were found in the transformed cells. These data suggest that overexpression of activated K‐ras transforms rat colon epithelial cells.
Increased Hepatic Nicotinamide N‐Methyltransferase Activity as a Marker of Cancer Cachexia in Mice Bearing Colon 26 Adenocarcinoma
Authors: Okamura Atsushi , Ohmura Yoshihisa , Islam Md. Muzharul , Tagawa Masatoshi , Horitsu Keisuke , Moriyama Yoichi , Fujimura Shinji
Published: August 2005

When a cachexigenic subclone (clone 20) of murine colon 26 adenocarcinoma was transplanted into female BALB/c mice, hepatic NNMT activity continued to increase until death in proportion to progressive carcass weight loss, a marker of cancer cachexia. On the other hand, noncachexigenic subclone (clone 5)‐transplanted mice showed neither increase of NNMT activity nor carcass weight loss. Among cytostatic fluorinated pyrimidines, 5′‐dFUrd could inhibit the increase of NNMT activity and prevent weight loss in mice bearing clone 20. On the other hand, 2′‐dFUrd did not show these effects. 5‐FUra and Tegafur inhibited the increase of NNMT activity at higher concentrations. These findings suggest that the levels of hepatic NNMT activity are closely associated with the degree of weight loss, and they appear to be a useful marker of cancer cachexia.
Possibility of Contribution of Nucleoside Transport Systems to Pirarubicin Uptake by HL60 Cells but Not Mononuclear Cells
Authors: Nagasawa Kazuki , Ohnishi Noriaki , Yokoyama Teruyoshi
Published: August 2005

Previously, we reported that pirarubicin (THP), an anthracycline, was taken up, at least in part, by both human leukemic HL60 cells and mononuclear cells (MNCs) via a carrier‐mediated system. In this study, the possibility of a contribution of nucleoside transport systems to the uptake of THP by HL60 cells and MNCs was investigated. The experiments were performed after both types of cells had been pretreated with a metabolic inhibitor, 2, 4‐dinitrophenol, to deplete cellular ATP. In HL60 cells, THP uptake was increased and decreased significantly by treatment with equilibrative nucleoside transport inhibitors, nitrobenzylthioinosine (NBMPR), nitrobenzylthioguanosine and dilazep, in the presence and absence, respectively, of an inwardly directed Na+‐gradient. THP uptake by HL60 cells showed an overshoot in the presence of the gradient, and was decreased by treatment of the cells with monensin, indicating that the uptake partially depended on the Na+‐gradient. In HL60 cells in which equilibrative nucleoside transport was inhibited by NBMPR, THP uptake in the presence of the gradient was inhibited by Na+‐dependent concentrative nucleoside transport inhibitors, but no inhibition was observed in the absence of the gradient. In MNCs, conversely, there was no effect of any equilibrative nucleoside transport inhibitor or the Na+‐gradient on THP uptake. These results suggested that THP was taken up, at least in part, via both equilibrative and concentrative nucleoside transport systems in HL60 cells, but not in MNCs.
Molecular Analysis of Ewing's Sarcoma: Another Fusion Gene, EWS‐E1AF, Available for Diagnosis
Authors: Urano Fumihiko , Umezawa Akihiro , Yabe Hiroki , Hong Wei , Yoshida Koichi , Fujinaga Kei , Hata Jun‐ichi
Published: August 2005

Ewing's sarcoma, one of the most malignant tumors of children and young adults, expresses specific chimeric genes, e.g. EWS‐FLI‐1, EWS‐ERG, EWS‐ETV1 and EWS‐FEV. In this paper, we extensively characterized a new fusion gene, EWS‐E1AF by means of whole cDNA sequencing, RNA blot analysis, DNA blot analysis and chromosomal analysis, and showed it to be available for the diagnosis of Ewing's sarcoma and to participate in the oncogenesis of Ewing's sarcoma. Furthermore, we conducted a genetic analysis of Ewing family tumors in conjuction with immunohistochemical analysis and ultrastructural analysis. Our results demonstrate some limitations of both genetic analysis and histopathological analysis, and establish the relationship between neurogenic phenotypes and chimera genes.
Germline Mutations of the PTEN/MMAC1 Gene in Japanese Patients with Cowden Disease
Authors: Kohno Takashi , Takahashi Mina , Fukutomi Takashi , Ushio Kyosuke , Yokota Jun
Published: August 2005

Cowden disease (CD) is an autosomal dominant disorder which confers a high susceptibility to diverse benign and malignant tumors. The PTEN/MMAC1 gene was identified as being responsible for CD, since its germline mutations have been identified in affected individuals in the United States and Europe. We identified three novel germline PTEN mutations, a 2‐bp deletion, a 1‐bp insertion and a missense mutation, in three of five Japanese patients with CD. The missense mutation resided outside of the region encoding a putative phosphatase domain of the predicted PTEN protein, where previously reported missense mutations in CD patients have been clustered. The present result suggests that a wide range of germline PTEN mutations may play a role in the pathogenesis of CD.
Chemoprevention of N‐Nitroso‐N‐methylurea‐induced Rat Mammary Cancer by Miso and Tamoxifen, Alone and in Combination
Authors: Gotoh Takahiko , Yamada Kazumasa , Ito Akihiro , Yin Hong , Kataoka Tsuyoshi , Dohi Kiyohiko
Published: August 2005

We examined the effects of a Japanese fermented soybean product, miso, and tamoxifen (TAM), alone and in combination, on N‐nitroso‐N‐methylurea (MNU)‐induced rat mammary cancer. Seven‐week‐old female CD/Crj rats received a single i.v. dose (50 mg/kg body weight) of MNU. After administration of MNU, the rats were divided into 4 groups: regular diet (control), 10% miso diet, regular diet+TAM, and 10% miso diet+TAM. TAM was implanted s.c. in the form of pellets containing 2.5 mg at the same time as MNU was administered. All rats were observed for 18 weeks after MNU administration. Incidence (percentage of rats with tumors) and multiplicity (mean tumors/rat) of mammary tumors were 91% and 4.5 in the control, 77% and 2.4 (P<0.05) in the 10% miso group, 68% and 1.4 (P<0.01) in the TAM group, and 10% (P<0.0001 or less) and 0.2 (P<0.0001) in the 10% miso+TAM group. In the second experiment, the effect of the combination of miso and TAM on established rat mammary tumors was investigated. When the mammary tumors induced by MNU reached 10 to 25 mm, the rats were divided into 3 treatment groups: regular diet, regular diet+TAM, and 10% miso diet+TAM. At 6 weeks after the start of treatment, the mean tumor size in the control and TAM groups was 160% and 141% of the pretreatment value, but a decrease to 85% of the pretreatment value was produced by the combination of miso and TAM, and this was significantly different from both the control and TAM groups (P<0.01 and P<0.05, respectively). These results indicate that miso is useful in protecting against mammary cancer and it can be expected to have a potent antitumor effect, especially when used in combination with TAM.
Establishment and Characterization of Two Cell Lines from N‐Methyl‐N‐nitrosourea‐induced Mouse Glandular Stomach Carcinomas
Authors: Ichinose Masutaro , Nakanishi Hayao , Fujino Shozo , Tatematsu Masae
Published: August 2005

We previously reported the induction with N‐methyl‐N‐nitrosourea (MNU) of mouse glandular stomach carcinomas showing a gastric phenotype but variation in histologic appearance, as with human gastric carcinomas. In the present study, we established two cell lines, designated MGT‐40 and MGT‐93, from MNU‐induced mouse glandular stomach carcinomas. These cell lines are keratin‐positive and grow as epithelial monolayers in culture, requiring transforming growth factor α, epidermal growth factor or insulin/transferrin for optimal growth in addition to 10% fetal bovine serum. Retention of the differentiated phenotype for gastric surface mucous cells has been confirmed by cathepsin E immunohistochemistry and reverse transcriptase‐polymerase chain reaction for mouse spasmolytic polypeptide. Neither transplantability in nude mice nor colony formation on soft agar was observed, except in one subline. Chromosome analysis revealed aneuploidy with modal chromosome numbers ranging from 58 to 78 and no specific structural abnormalities. This is the first report of cell lines derived from mouse glandular stomach carcinomas. They should prove useful for studies of the mechanisms of regulation of growth and differentiation.