bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunohistochemical Analysis of nm23/NDP Kinase Expression in Human Lung Adenocarcinoma: Association with Tumor Progression in Clara Cell Type
Authors: Ozeki Yuichi , Takishima Kunio , Mamiya Gunji
Published: August 2005

Levels of nm23‐H1/nucleoside diphosphate kinase (NDP kinase) expression have been reported to correlate inversely with mctastatic potential in some tumors but not in others. Whether or not nm23 gene product is associated with metastatic potential in lung cancer is not clear as yet. We therefore immunohistochemically examined the expression of nm23 gene products in primary lung adenocarci‐no mas according to cytologic subtypes in order to clarify the association of its expression with the clinical features of the disease. Seventy‐two (64.9%) of the 111 lung adenocarcinomas were positive for nm23 protein. In lung adenocarcinoma of Clara cell type, high levels of nm23 expression were associated with advanced pathologic stage, positive lymph node status, and poorer prognosis (P<0.05). However, no correlation with clinical outcome was observed in other cell types. Our data suggest that higher levels of nm23 expression are associated with tumor progression in lung adenocarcinoma of Clara cell type.
Expression of Aminopeptidase N on Human Choriocarcinoma Cells and Cell Growth Suppression by the Inhibition of Aminopeptidase N Activity
Authors: Ino Kazuhiko , Goto Setsuko , Okamoto Tomomitsu , Nomura Seiji , Nawa Akihiro , Isobe Ken‐ichi , Mizutani Shigehiko , Tomoda Yutaka
Published: August 2005

We previously found that an aminopeptidase inhibitor, ubenimex (bestatin), had a growth‐suppressive effect on Choriocarcinoma cell lines in vitro. To clarify the mechanism of this action, we investigated the expression of aminopeptidase N (AP‐N/CD13) on Choriocarcinoma cells and other human tumor cells. Two Choriocarcinoma cell lines, NaUCC‐4 and Be Wo, had higher AP‐N activity than other cell lines (358.8 and 340.2 nmol/h/106 cells, respectively), as did the human myeloid leukemia cell line, HL‐60 (373.8 nmol/h/106 cells). These Choriocarcinoma and leukemia cell lines with abundant AP‐N activity showed much higher sensitivity to bestatin (IC50 = 0.5, 2.1 and l.0μg/ml, respectively) than the other cell lines. By imniuiioblotting and immunocytochemical staining, AP‐N was detected as an approximately 165‐kDa protein and localized on the cell membrane in Choriocarcinoma cells. We also examined the effects of two other aminopeptidase inhibitors and three anti‐CD13 monoclonal antibodies (MAbs) (WM15, MCS2 and MY7) on the growth of NaUCC‐4 cells. Cell growth was markedly suppressed by the AP‐N inhibitor actinonin as well as bestatin, but not by the AP‐B inhibitor arphamenine. Of the three MAbs, only WM15, which is able to inhibit AP‐N activity, suppressed cell growth in a dose‐dependent manner. These results indicate that AP‐N inhibitors show a growth‐suppressive effect, presumably through inhibition of the enzymatic activity of AP‐N on tumor cells, and suggest that AP‐N may play important roles in the growth of certain tumors, such as Choriocarcinoma and leukemia.
Hepatocyte Growth Factor Enhancement of Preneoplastic Hepatic Foci Development in Rats Treated with Diethylnitrosamine and N‐Ethyl‐N‐hydroxyethylnitrosamine
Authors: Yaono Makoto , Hasegawa Ryohei , Mizoguchi Yasumoto , Futakuchi Mitsuru , Nakamura Toshikazu , Ito Nobuyuki , Shirai Tomoyuki
Published: August 2005

Effects of hepatocyte growth factor were investigated in a two‐stage rat liver carcinogenesis protocol. Male F344 rats were first treated with diethylnitrosamine (200 mg/kg, i.p.) and then, starting two weeks later, with N‐ethyl‐N‐hydroxyethylnitrosamine (EHEN) for 6 weeks at a dose of 0.01% in drinking water. Hepatocyte growth factor, which was injected i.v. at a dose of 200 μg/kg body weight one (at week 3) or two times (at weeks 3 and 4) during EHEN administration, significantly increased the development of preneoplastic glutathione S‐transferase placental form‐positive foci. Although the observed effects of hepatocyte growth factor were weaker than that of the two‐thirds partial hepatectomy (PH) performed at week 3, the present results suggest that the enhancing effects of PH performed during the promotion stage may be largely mediated through induction of hepatocyte growth factor.
Increased Prevalence of HTLV‐I Infection in Patients with Hepatocellular Carcinoma Associated with Hepatitis C Virus
Authors: Okayama Akihiko , Maruyama Toshihiro , Tachibana Nobuyoshi , Hayashi Katsuhiro , Kitamura Tohru , Mueller Nancy , Tsubouchi Hirohito
Published: August 2005

The progression from chronic hepatitis C virus (HCV) infection to hepatocellular carcinoma (HCC) has been reported. We evaluated whether co‐infection with the human T‐lymphotropic virus type I (HTLV‐I) might be associated with this transition in a cross‐sectional analysis of 127 patients with HCV‐chronic hepatitis (mean age=51.7) and 43 patients with HCV‐associated HCC (mean age=62.4); the seroprevalence of anti‐HTLV‐I was 9.5% and 30.2%, respectively. For subjects 50 years or older, the seroprevalence of anti‐HTLV‐I in HCC patients was 13/41 (31.7%) which was significantly higher than that in chronic hepatitis patients (6/82, 7.3%) (P=0.001). The relative risk (RR) of association was 12.8 (P=0.0004) among the males, however, no association was evident among the females, RR=1.3 (P=0.80). The increased prevalence of HTLV‐I positivity among the HCC cases could not be attributed to a higher rate of prior transfusion. These data suggest that co‐infection with HTLV‐I may contribute to the development of HCC among patients with HCV‐induced chronic liver diseases in a highly HTLV‐I‐endemic area.
Comparison of Calbindin D‐28k and S‐100 Protein B in Neuroblastoma as Determined by Enzyme Immunoassay
Authors: Ishiguro Yukio , Kato Kanefusa , Akatsuka Hiroshi , Iwata Hiroyuki , Ito Fujio , Watanabe Yoshio , Nagaya Masahiro
Published: August 2005

Levels of two calcium‐binding proteins, calbindin D‐28k (calbindin‐D) and S‐100 protein B (S‐100b), were measured by immunoassay in solid tumors obtained surgically from pediatric patients. Mean concentrations of calbindin‐D and S‐100b in 73 neuroblastomas (23 ganglioneuroblastomas and 50 neuroblastomas) were 10‐ or 25‐fold higher, respectively, than those in other types of solid tumors in pediatric patients (n=15). The mean tumor concentration of calbindin‐D in patients with neuroblastoma (n=73) was 25.1 ng/mg (range 0.20 to 317.0 ng/mg soluble protein, SE=6.26); that of S‐100b was 278.3 ng/mg (range 0.93 to 2521 ng/mg soluble protein, SE=71.7). The mean concentration of calbindin‐D (4.4 ng/mg soluble protein) was significantly (P < 0.05) lower in stage IV, the most advanced stage. The mean concentration of S‐100b (74.0 ng/mg soluble protein) was lower in patients with undifferentiated neuroblastomas (P < 0.01). Tumor levels of the two calcium‐binding proteins were not correlated in patients with neuroblastoma, but each was strongly correlated with outcome in patients with neuroblastoma. The evidence suggests that measurements of the calcium‐binding proteins calbindin‐D and S‐100b would be useful for evaluating the prognosis of patients with neuroblastoma.
Second Primary Neoplasms in Thyroid Cancer Patients
Authors: Ishikawa Kazuhiro , Noguchi Shiro , Tanaka Keitaro , Fukuda Atsushi , Hirohata Tomio
Published: August 2005

To determine risk patterns for second primary neoplasms after the occurrence of thyroid cancer, we conducted a retrospective cohort study of 3321 thyroid cancer patients who were operated and histologically confirmed at the Noguchi Thyroid Clinic and Hospital Foundation between 1946 and 1985. They were followed from the date of operation through the end of 1990 with an observation period from 45 to 5 years. The average observation period of the patients was 13.4 years and the follow‐up rate reached 98%. The standardized mortality ratio (SMR) was computed to assess possible risk increase by cancer site. In this computation, the time period less than 5 years after operation was omitted to reduce the influence of deaths related to the original thyroid cancer. A total of 103 deaths from malignant neoplasms other than thyroid cancer were observed during this time period (SMR=1.6, 95% confidence interval [CI]=1.3–2.0). Analyses of site‐specific cancer mortality revealed significantly elevated risks for the central nervous system (SMR=16.1, CI=5,2–37.6) and respiratory organs (SMR=2.6, CI=1.5–4.1). Based on a review of available medical records with histological findings, we concluded that the risk increases for these sites were most likely to be attributable to second primary neoplasms. Whether or not the patients had received radiotherapy was not significantly associated with elevated risk. Further investigations are needed to clarify the risk factors responsible for the above findings.
Characterization of Transendothelial Migratory Lymphokine‐activated Killer Cells
Authors: Nakano Koji , Eura Masao , Chikamatsu Kazuaki , Masuyama Keisuke , Ishikawa Takeru
Published: August 2005

We examined the killing activity of transmigrated lymphokine‐activated killer (LAK) cells and their surface molecules associated with both transendothelial migration and cytotoxicity, using human umbilical vein‐derived endothelial cell (HUVEC) monolayers on fibronectin with gelatin separating the upper chamber from the lower chamber. Migratory LAK cells were significantly more cytotoxic to Daudi target cells, expressed more LFA‐1, and were more likely to be positive for CD2, compared to those LAK cells not adherent to the HUVEC monolayer. In contrast, in the absence of the HUVEC monolayer, there was no difference in LAK activity between migratory and non‐adherent LAK cells. These results indicate that the interaction between LAK cells and the HUVEC monolayer allows selective migration of LAK cells with cytotoxic activity that is enhanced with respect to some surface molecules.
Molecular Genetic Diagnosis of von Hippel‐Lindau Disease: Analysis of Five Japanese Families
Authors: Kanno Hiroshi , Shuin Taro , Kondo Keiichi , Ito Susumu , Hosaka Masahiko , Torigoe Soichiro , Fujii Satoshi , Tanaka Yoshihide , Yamamoto Isao , Kim Ilu , Yao Masahiro
Published: August 2005

We analyzed deoxyribonucleic acids from blood samples of five Japanese von Hippel‐Lindau (VHL) disease families (three familial cases, two new mutations) for the presence of VHL gene mutations by single‐strand conformational polymorphism analysis and direct sequencing. Four of the five families showed germ line mutations in VHL gene, comprising 2 missense mutations, 1 deletion, and 1 splice‐site mutation. Two families had VHL gene mutations at exon 1; 1 family at exon 3; and 1 family at the splice‐site adjacent to exon 3. Presymptomatic patients were accurately diagnosed by these methods. However, one family did not show a VHL gene mutation in the germ line but showed a somatic mutation at exon 2 in the hemangioblastoma tissue. The consequence of the somatic mutation was a microdeletion leading to a frameshift mutation. Our study is the first report of VHL gene analyses of Japanese VHL disease families, and suggests that not only germ line mutation, but also somatic mutation can lead to development of a tumor associated with the VHL disease.
Establishment of a Clonal Cell Line Producing Granulocyte Colony‐Stimulating Factor and Parathyroid Hormone‐Related Protein from a Lung Cancer Patient with Leukocytosis and Hypercalcemia
Authors: Asahi Yasutomo , Kubonishi Ichiro , Imamura Jun , Kamioka Mikio , Matsushita Hiroshi , Furihata Mutsuo , Ohtsuki Yuji , Miyoshi Isao
Published: August 2005

Squamous cell lung carcinoma cells obtained from a patient who presented with leukocytosis and hypercalcemia were transplanted into nude mice anda serially transplantable cell line, OKa‐N‐1, was established. The nude mice transplanted with OKa‐N‐1 cells displayed leukocytosis and hypercalcemia. Serum levels of granulocyte colony‐stimulating factor (G‐CSF) and parathyroid hormone‐related protein (PTHrP) were both elevated in these mice. In vitro cultivation of this tumor cell line gave rise to a clonal cell line, OKa‐C‐1. Nude mice transplanted with the OKa‐C‐1 cell line also showed leukocytosis and hypercalcemia with high serum G‐CSF and PTHrP levels. The culture supernatant of OKa‐C‐1 contained high levels of G‐CSF and PTHrP. Immunohistochemical studies showed the expression of PTHrP in OKa‐C‐1 cells. Reverse transcription polymerase chain reaction revealed the presence of G‐CSF and PTHrP mRNA in this cell line. Dexamethasone treatment inhibited the transcription of G‐CSF and PTHrP genes. This new human squamous carcinoma cell line, OKa‐C‐1, would be useful for studying the mechanism of simultaneous production of G‐CSF and PTHrP and their control in cancer patients with leukocytosis and hypercalcemia.
Specific Growth Inhibition of Small‐cell Lung Cancer Cells by Adenovirus Vector Expressing Antisense c‐kit Transcripts
Authors: Yamanishi Yuji , Maeda Hiroyuki , Hiyama Keiko , Ishioka Shinichi , Yamakido Michio
Published: August 2005

Antisense methods to control aberrant gene expression have been investigated as therapeutic strategies. A proto‐oncogene c‐kit, which encodes a transmembrane tyrosine kinase, is overexpressed in some malignancies, including small‐cell lung cancer (SCLC), and is thought to be involved in their pathogenesis. To test the feasibility of using adenovirus vectors for antisense strategies and to target c‐kit in SCLC therapy, we constructed replication‐deficient recombinant adenovirus vectors which express fragments of c‐kit transcripts in antisense (Ad.kitAS) or sense orientation (Ad.kitS: control). In vitro infection of SBC‐1 cells, which are c‐Kit protein‐producing SCLC cells, by these vectors resulted in the expression of artificial c‐kit transcripts. The Ad.kitAS‐infected SBC‐1 cells showed reductions in the amount of c‐Kit protein. As expected, at 10 days after infection (1 multiplicity of infection), Ad.kitAS‐infected SBC‐1 cells showed approximately 40% growth inhibition compared to uninfected or Ad.kitS‐infected cells in vitro. Such a significant growth inhibition by Ad.kitAS was not induced in SBC‐5 cells, which are SCLC cells producing no c‐Kit protein. These results demonstrate the usefulness of adenovirus vectors in antisense strategies, and the feasibility of targeting c‐kit in the therapy of c‐Kit‐producing SCLC.
Genetic Status and Expression of the Cyclin‐dependent Kinase Inhibitors in Human Gastric Carcinoma Cell Lines
Authors: Akama Yoshihiko , Yasui Wataru , Kuniyasu Hiroki , Yokozaki Hiroshi , Akagi Morihisa , Tahara Hidetoshi , Ishikawa Takenori , Tahara Eiichi
Published: August 2005

Deregulation of cyclin, cyclin‐dependent kinases (CDKs) and their inhibitors could have a pivotal role in the development of diverse human cancers. We examined the genetic status and the expression of CDK inhibitors (p21, p27, pl6 and p15), CDK2 and cyclins (A, D1 and E) in eight gastric carcinoma cell lines, in comparison with the status of p53 gene alterations. All the cell lines (except MKN‐28) that contained a p53 gene abnormality expressed very low or undetectable levels of p21 mRNA, while the cell lines (MKN‐45 and ‐74) with wild‐type p53 gene expressed high levels of p21 mRNA. An inverse correlation was found between the level of p21 mRNA and the expression of mRNAs for CDK2 and G1 cyclins. MKN‐28 was an exception; it contained mutated p53, and expressed mRNAs for p21, CDK2 and G1 cyclins at high levels. Only MKN‐45 and ‐74, with wild‐type p53, expressed considerable levels of p21 protein. Homozygous deletion of the p16 and p15 genes was detected in two (MKN‐45 and HSC‐39) of the eight gastric carcinoma cell lines. p16 protein was not expressed in three cell lines (MKN‐28, MKN‐74 and KATO‐III), as well as MKN‐45 and HSC‐39. Rearrangement of the p15 gene was found in TMK‐1. Rearrangement of the p27 gene was detected in MKN‐45, although the expression of p27 protein was well preserved in all the gastric carcinoma cell lines. The expression of pRb was also preserved in all the cell lines except KATO‐III. No obvious correlation was observed between the p53 gene status and the expression of p27 and p16. These findings suggest that abnormal regulation of CDK2/cyclins and CDK inhibitors might be involved in deregulated growth of gastric carcinomas.
Inhibition of Nucleolar Function and Morphological Change by Adriamycin Associated with Heat Shock Protein 70 Accumulation
Authors: Abe Tetsuya , Fukamachi Yukiyo , Kanazawa Yohsuke , Furukawa Hiroshi , Shimizu Kenji , Hirano Takeshi , Kasai Hiroshi , Kashimura Masamichi , Higashi Ken
Published: August 2005

Adriamycin (ADR) has been considered to target mainly DNA metabolism in the nucleus. Recently, we observed the nuclear translocation of heat shock protein 70 (HSP70) after ADR treatment. We examined which intranuclear changes might be related to this alteration of HSP70 localization. We found considerable alternations in the nucleolar morphology and function in ADR‐treated tumor cells, i.e., a ring‐shaped segregation of granular components of almost all nucleoli and a dramatic reduction of nucleolar 45S ribosomal precursor RNA biosynthesis in HeLa cells exposed to 100 μM ADR for 2 h. Concomitantly with these changes, HSP70 was concentrated into the nucleoli, as in the case of heat shock treatment. These results indicate a novel anticancer effect of ADR via the suppression of cellular protein biosynthesis, in addition to its effect on DNA.
A Topoisomerase II Inhibitor, NK109, Induces DNA Single‐ and Double‐strand Breaks and Apoptosis
Authors: Fukuda Minoru , Inomata Motoko , Nishio Kazuto , Fukuoka Kazuya , Kanzawa Fumihiko , Arioka Hitoshi , Ishida Tomoyuki , Fukumoto Hisao , Kurokawa Hirokazu , Oka Mikio , Saijo Nagahiro
Published: August 2005

2,3‐(Methylenedioxy)‐5‐methyl‐7‐hydroxy‐8‐methoxybenzo[c]phenanthridinium hydrogensulfate di‐hydrate, called NK109, is a benzo[c]phenanthridine derivative, which inhibits DNA topoisomerase II activity by stabilizing the DNA‐enzyme‐drug complex, and shows strong growth‐inhibitory effects on several human cancer cells. In the present study, NK109 treatment induced DNA fragmentation and a rise in the level of cytoplasmic nucleosomes, which are markers of apoptosis, in human small‐cell lung carcinoma SBC‐3 cells. These effects were inhibited by zinc ions and enhanced by cycloheximide or actinomycin D. Dose‐dependent single‐ and double‐strand DNA breaks were observed, using alkaline and neutral elution assays, in SBC‐3 cells treated with more than 0.2 μM NK109 for 4 h. Treatment with NK109 caused more DNA single‐ and double‐strand breaks than treatment with an equimolar amount of VP‐16. These results suggest that NK109 induces DNA strand breaks and apoptosis. In addition, it appears that this process does not require protein or RNA synthesis, but involves a specific endonuclease which is inhibited by zinc ions.
Primary Human Fibroblasts Induce Diverse Tumor Invasiveness: Involvement of HGF as an Important Paracrine Factor
Authors: Iwazawa Takashi , Shiozaki Hitoshi , Doki Yuichiro , Inoue Masatoshi , Tamura Shigeyuki , Matsui Shigeo , Monden Takushi , Matsumoto Kunio , Nakamura Toshikazu , Monden Morito
Published: August 2005

Fibroblasts have been considered to play an important role in tumor progression. In order to evaluate the contribution of fibroblasts to tumor invasion, TE2‐NS, an esophageal cancer cell line, was cultured on collagen gel containing primary fibroblasts derived from normal esophageal submucosa or cancerous tissues of seven esophageal cancer patients. TE2‐NS showed diverse invasiveness into the underlying gel containing fibroblasts, but did not invade the gel not containing fibroblasts. The invasiveness of TE2‐NS, which expressed hepatocyte growth factor (HGF) receptor, was well‐correlated with the concentration of HGF in conditioned medium. Administration of neutralizing antibody against HGF effectively suppressed the invasion, but application of recombinant HGF without fibroblasts induced little invasion into the gel. Fibroblasts from non‐cancerous tissue generally secreted a larger amount of HGF and induced tumor invasion to a greater extent than those from cancer tissue, with large diversity. Interestingly, HGF secretion of fibroblasts from non‐cancerous tissue was stimulated by co‐culture with TE2‐NS in two lines, but not in the other four. These results indicate that HGF is an important paracrine factor which induces tumor cell invasion, and the diversity of HGF production by fibroblasts might suggest different potentiality to induce tumor invasion among patients.
Aberrant DNA Methylation on Chromosome 16 Is an Early Event in Hepatocarcinogenesis
Authors: Kanai Yae , Ushijima Saori , Tsuda Hitoshi , Sakamoto Michiie , Sugimura Takashi , Hirohashi Setsuo
Published: August 2005

In order to clarify the significance of DNA methylation in both earlier and later stages of hepatocarcinogenesis, the DNA methylation state on chromosome 16, on which loss of heterozygosity (LOH) has frequently been detected in human hepatocellular carcinomas (HCCs), was examined. DNA from primary HCCs and tissues showing chronic hepatitis and liver cirrhosis, which are considered to be precancerous conditions, was analyzed by digestion with methylation‐sensitive and non‐sensitive restriction enzymes. DNA hypermethylation at the D16S32, tyrosine aminotransferase (TAT) and D16S7 loci and hypomethylation at the D16S4 locus were detected in 18%, 58%, 20% and 48% of examined HCCs, respectively. Aberrant DNA methylation occurred more frequently in advanced HCCs than in early HCCs. Moreover, DNA hypermethylation at the D16S32, TAT and D16S7 loci was frequently observed in chronic hepatitis and liver cirrhosis. The incidence of DNA hypermethylation was higher than that of LOH (42% at the TAT locus). These data suggest that DNA hypermethylation might predispose the locus to allelic loss. Aberrant DNA methylation is a significant change which may participate in the early developmental stages of HCCs.
Presence in Pieris rapae of Cytotoxic Activity against Human Carcinoma Cells
Authors: Koyama Kotaro , Wakabayashi Keiji , Masutani Mitsuko , Koiwai Kazunori , Watanabe Masahiko , Yamazaki Shigeru , Kono Takuo , Miki Keizaburo , Sugimura Takashi
Published: August 2005

Cytotoxic activity in extracts of pupae and adults of various kinds of butterflies and moths was tested in vitro against the human gastric carcinoma cell line, TMK‐1, which was chosen as an example of human carcinoma cells. Among the species examined, cytotoxicity was limited to Pieris rapae, Pieris napi and Pieris brassicae. Activity was found down to a dilution of 1/104, while with the other butterflies and moths no activity was observed, even at 1/102. When the cytotoxicity of the three developmental stages, larvae, pupae and adults, of Pieris rapae was compared, the pupae showed the strongest activity, the IC50 against TMK‐1 cells being at the 1/106 dilution. For larvae and adults, the respective IC50 values were at the 1/105 and 5/105 dilutions. The active principle in the pupae of Pieris rapae was found to be heat‐labile and not extractable with organic solvents, but precipitated with ammonium sulfate and digested by proteases, suggesting that it is a protein. This cytotoxic factor was named pierisin.
p16/CDKN2 Gene and p53 Gene Alterations in Japanese Non‐smoking Female Lung Adenocarcinoma
Authors: Takeshima Yukio , Nishisaka Takashi , Kawano Ryoji , Kishizuchi Kentaro , Fujii Satoshi , Kitaguchi Souichi , Inai Kouki
Published: August 2005

Primary lung adenocarcinomas in non‐smoking females are increasing in the USA and Japan. Environmental factors such as passive smoking, asbestos, domestic radon, and hormonal effects have been implicated, but the etiology is still uncertain. We therefore analyzed point mutations of p16 gene, a newly characterized tumor suppressor gene, and compared the results with alterations of p53 gene in 28 primary lung adenocarcinomas in non‐smoking Japanese females. There were no cases with somatic point mutation of p16 gene, except for one case with two germline mutations (silent mutations). In contrast, six out of 16 informative cases showed loss of heterozygosity of p53 gene using a TP53 microsatellite marker and 19 out of 28 cases showed expression of oncoprotein using DO‐7 immunohistochemistry. These findings suggest that p16 gene alteration is a rare event in primary lung adenocarcinomas in Japanese non‐smoking females, compared with alterations of the p53 gene.
Mutational Analysis of Mismatch Repair Genes, hMLH1 and hMSH2, in Sporadic Endometrial Carcinomas with Microsatellite Instability
Authors: Kobayashi Kanji , Matsushima Mieko , Koi Sumiko , Saito Hiroko , Sagae Satoru , Kudo Ryuichi , Nakamura Yusuke
Published: August 2005

Microsatellite instability, monitored by replication error (RER), bas been observed in both sporadic and hereditary types of endometrial carcinoma. In the hereditary tumors, this instability is considered to be caused by a germline defect in the DNA mismatch‐repair system. We previously reported that nearly one‐quarter of sporadic endometrial carcinomas examined revealed an RER‐positive phenotype at multiple microsatellite loci. To investigate the role of genetic alterations of DNA mismatch‐repair genes in sporadic endometrial carcinomas, we screened 18 RER(+) endometrial carcinomas for mutations of hMLH1 and hMSH2. Although we found no germline mutations, we detected two somatic mutations of hMLH1 in a single endometrial cancer; these two mutations had occurred on different alleles, suggesting that two separate mutational events had affected both copies of hMLH1 in this particular tumor. These data implied that mutations of hMLH1 or hMSH2 play limited roles in the development of sporadic endometrial carcinomas, and that the tumors with genetic instability might have alterations of other mismatch‐repair genes, such as hPMS1 and hPMS2, or of unknown genes related to the mismatch‐repair system.
Enhanced Anticancer Effect of Vincristine with Methionine Infusion after Methionine‐depleting Total Parenteral Nutrition in Tumor‐bearing Rats
Authors: Goseki Narihide , Nagahama Takeshi , Maruyama Michio , Endo Mitsuo
Published: August 2005

Methionine‐depleting total parenteral nutrition (Met(−) TPN), in which an amino acid solution devoid of L‐methionine and L‐cysteine is infused, is thought to reduce tumor cell growth through acting as a partial late S‐G2 (i.e., late‐S and G2 phases) blocker. The antitumor effect of vincristine (VCR), which acts on mitotic phase cells, was examined with methionine infusion immediately after Met(−) TPN in Yoshida sarcoma (YS)‐bearing rats. Rats were given Met(−) TPN for 8 days immediately after inoculation with YS cells (days 0 to 8), which was followed by methionine‐containing (Met(+)) regular TPN for 3 days (days 9 to 11) along with intraperitoneal administration of 0.05 mg/kg/day VCR. All rats were then fed solid food and water ad libitum until they died, with 0.1 mg/kg VCR administration on days 12 and 13. As controls, a Met(−) TPN only group, Met(+) TPN groups with and without VCR, and freely fed groups with and without VCR were studied. The progression of YS was markedly suppressed by Met(−) TPN with VCR. The median survival time in days was 25 days, significantly longer (P<0.001) (generalized Wilcoxon's tests) by 11 to 14 days than that of any of the other groups. In conclusion, VCR appears to have greater efficacy as an anticancer agent when administered together with methionine after Met(−) TPN.
Triazine Derivatives Inhibit Rat Hepatocarcinogenesis but Do Not Enhance Gap Junctional Intercellular Communication
Authors: Hori Takaaki , Asamoto Makoto , Krutovskikh Vladimir , Iwahori Yoshio , Maeda Mitsuaki , Toriyama‐Baba Hiroyasu , Takasuka Nobuo , Tsuda Hiroyuki
Published: August 2005

We report here novel candidate chemopreventive agents active against experimental hepatocarcino‐genesis. The triazine derivatives 6‐(2‐chlorophenyl)‐2,4‐diamino‐l,3,5‐triazine (2CPDAT), 6‐(3‐chlorophenyl)‐2,4‐diamino‐l,3,5‐triazine (3CPDAT), 6‐(4‐chlorophenyl)‐2,4‐diamino‐l,3,5‐triazine (4CPDAT), 6‐(4‐pyridyl)‐2,4‐diamino‐l,3,5‐triazine (PyDAT), and 6‐(pyridine JV‐oxid‐4‐yl)‐2,4‐diamino‐l,3,5‐triazine (PyNODAT), synthesized in our laboratory, in addition to 6‐(2,5‐dichloro‐phenyl)‐2,4‐diamino‐l,3,5‐triazme (DCPDAT), or irsogladine, which is a widely used anti‐ulcer drug, were investigated for potential chemopreventive effects in a rat liver medium‐term bioassay system. A significant inhibitory influence on enzyme‐altered liver foci was found for 2CPDAT, 3CPDAT, 4CPDAT, and PyNODAT, but not for DCPDAT or PyDAT, The involvement of gap jnnctional intercellular communication in the inhibition was studied, but no change in gap Junctional intercellular communication capacity in rat liver cells in vitro or in gap junction protein (connexin 32) expression in rat liver in vivo was noted. These results indicate that, although these irsogladine analogues exert inhibitory effects on rat liver carcinogenesis, their action is independent of modification of gap Junctional intercellular communication.
Infrequent Genetic Alterations of the PTEN/MMAC1 Gene in Japanese Patients with Primary Cancers of the Breast, Lung, Pancreas, Kidney, and Ovary
Authors: Sakurada Akira , Suzuki Akihiko , Sato Masami , Yamakawa Hiromitsu , Orikasa Kazuhiko , Uyeno Shinji , Ono Tetsuya , Ohuchi Noriaki , Fujimura Shigefumi , Horii Akira
Published: August 2005

In the present study, we searched for genetic alterations of the entire coding region of PTEN/MMACl, a recently isolated candidate tumor suppressor gene, in 178 specimens from Japanese patients with various malignant tumors by the polymerase chain reaction‐single strand conformation polymorphism method. The samples consisted of 11 glioblastoma multiformes (GBMs), 14 astrocytomas, 47 breast cancers, 25 non‐small cell lung cancers, 9 small cell lung cancers, 8 pancreatic cancers, 24 renal cell carcinomas, 20 ovarian cancers, and 20 metastatic lung tumors from various organs. Only one somatic frameshift mutation at codon 319 was observed in one (9%) of eleven GBMs. Our results suggest that mutation of the PTEN/MMAC1 gene does not play a major role in carcinogenesis, at least in the tumor types from Japanese patients analyzed in this study.
Expression of Human Telomerase RNA Is an Early Event of Stomach Carcinogenesis
Authors: Kuniyasu Hiroki , Domen Tamiko , Hamamoto Takahiro , Yokozaki Hiroshi , Yasui Wataru , Tahara Hidetoshi , Tahara Eiichi
Published: August 2005

Expression of human telomerase RNA (hTR) and telomerase activity in gastric cancer and corresponding non‐cancerous mucosa were studied. Telomerase activity was detected in 23 (88%) of 26 carcinoma tissues. Although all tumor specimens and non‐cancerous mucosa expressed various levels of hTR, 21 (81%) of 26 cases expressed hTR at a higher level in the tumor than that in the corresponding mucosa. All 8 gastric carcinoma cell lines also expressed hTR at high levels. Nine (35%) of 26 non‐cancerous mucosa showed telomerase activity and all of them contained intestinal metaplasia. The incidence of telomerase‐positive mucosa in grade 2 intestinal metaplasia was significantly higher than that in grade 0 or grade 1 intestinal metaplasia, whereas hTR overexpression was found in grade 0 or grade 1 intestinal metaplasia as well as grade 2 intestinal metaplasia. The degree of Heticobacter pylori infection increased in parallel with the level of hTR expression and telomerase positivity. These results overall suggest that Helicobacter pylori infection may he a strong trigger for hTR overexpression in intestinal metaplasia, and this may lead to telomerase reactivation.
Concomitant Presence of p16/Cyclin‐dependent Kinase 4 and Cyclin D/Cyclin‐dependent Kinase 4 omplexes in LNCaP Prostatic Cancer Cell Line
Authors: Itoh Noriyuki , Kakehi Yoshiyuki , Akao Toshiya , Kinoshita Hidefumi , Okada Yusaku , Yoshida Osamu
Published: August 2005

The cyclin D/cyclin‐dependent kinase (CDK)/CDK‐inhihitory proteins/retinoblastoma protein (pRb) pathway is hypothesized to control the G1‐S check point. The role of this pathway is reported to be different depending on the status of pRb. In the present study, we examined nine human urological tumor cell lines. Cells lacking functional pRb expressed p16, instead of forming cyclin D/ CDK4 complex. In the LNCaP prostatic cancer cell line, however, both p16/CDK4 and cyclin D/ CDK4 complexes were present independently, probably because of partial loss of pRb. In view of the concomitant presence of the incompatible complexes, LNCaP should provide us with a valuable model for the study of this pathway in cancer cells.
Dose‐dependent Induction of Both Pepsinogen‐altered Pyloric Glands and Adenocarcinomas in the Glandular Stomach of C3H Mice Treated with N‐Methyl‐N‐nitrosourea
Authors: Yamamoto Masami , Furihata Chie , Fujimitsu Yasunobu , Imai Toshio , Inada Ken‐ichi , Nakanishi Hayao , Tatematsu Masae
Published: August 2005

The dose‐response relation for the appearance of pepsinogen isozyme 1 (Pg l)‐altered pyloric glands (PAPG) and the related induction of adenocarcinomas were examined in male C3H mice given N‐methyl‐N‐nitrosourea (MNU) in their drinking water at the concentration of 120 ppm (group 1), 60 ppm (group 2), 30 ppm (group 3) or 0 ppm (group 4) for 30 weeks and then normal tap water. Animals were killed at weeks 10, 30 and 42. Adenomatous hyperplasias and adenocarcinomas were noted from week 30 and their induction was dose‐dependent at week 42. Almost all cells of pyloric gland cell type in those lesions had little or no immunohistochemically demonstratable Pg 1 content, as was also the case for the cells in PAPG, whose numbers per 100 normal‐appearing pyloric glands were found to be MNU dose‐dependent at all experimental time points. The numbers of PAPG at week 10 significantly correlated with the incidences of adenomatous hyperplasias and adenocarcinomas at week 42. Investigation of proliferation by immunohistochemical detection of bromodeoxyuridine (BrdU) labeling in the PAPG at week 10 demonstrated elevation (P <0.05) as compared to normal pyloric glands. Intestinal metaplasia was not a feature in the present experiment and the results suggest that in mice, PAPG might be a preneoplastic lesion involved in gastric chemical carcinogenesis.
Decrease of Prostaglandin E2 and 5–Bromo–2′–deoxyuridine Labeling but Not Prostate Tumor Development by Indomethacin Treatment of Rats Given 3,2′–Dimethyl‐4‐aminobiphenyl and Testosterone Propionate
Authors: Kawabe Mayumi , Shibata Masa‐Aki , Sano Masashi , Takesada Yasuko , Tamano Seiko , Ito Nobuyuki , Shirai Tomoyuki
Published: August 2005

The modifying effects of indomethacin (IM) on rat prostate carcinogenesis induced by 3,2′–dimethyl–4–aminobiphenyl (DMAB) were investigated. F344 rats were given 50 mg/kg body weight of DMAB at 2–week intervals for 20 weeks and then received IM at a dose of 20 ppm in the drinking water for 37 weeks. Separate groups additionally received testosterone propionate (TP) in Silastic tubes throughout the experiment. DMAB alone induced carcinomas in situ in the ventral lobe and in combination with TP caused invasive carcinomas of the dorso–lateral and anterior lobes and seminal vesicles. No clear suppression by IM of development of in situ carcinomas or in vasive carcinomas was observed. In a short–term satellite experiment, it was revealed that prostaglandin E2 (PGE2) levels in the dorso–lateral prostate and seminal vesicles, but not the ventral prostate, were significantly reduced by IM and that TP itself also suppressed PGE2 levels. The 5–bromo–2′–deoxyuridine labeling index in the ventral prostate was significantly decreased by IM administration. These results indicate that while IM can efficiently suppress tissue PGE2 levels, it does not inhibit tumor development in the prostate or seminal vesicles of rats in the present model.
Apoptosis Induced by NS‐398, a Selective Cyclooxygenase‐2 Inhibitor, in Human Colorectal Cancer Cell Lines
Authors: Kara Akira , Yoshimi Naoki , Niwa Masayuki , Ino Natsuko , Mori Hideki
Published: August 2005

Recent studies have suggested that apoptosis is a key phenomenon in the chemopreventive action of nonsteroidal antiinflammatory drugs (NSAIDs), which exhibit cancer‐preventive and tumor‐regressive effects in the human colon. The effect of NS‐398,N‐(2‐cyclohexyloxy‐4‐nitrophenyl)methanesul‐fonamide, which is a selective inhibitor of cyclooxygenase‐2 (COX‐2), on the induction of apoptosis in two human colorectal cancer cell lines (Colo320 and THRC) was determined. The apoptotic ratios (‐fold vs. control value) of Colo320 in the presence of 100μMindomethacin and NS‐398 were 3.3 ± 1.5 and 9.0±0.94, and those of THRC were 2.3±0.46 and 7.4±0.87, respectively. The ability of NS‐398 to induce apoptosis is greater than that of indomethacin. Both indomethacin and NS‐398 reduced the cell proliferation in a concentration‐dependent manner. The IC50values of NS‐398 (54.8+3.6 and 77.2±4.9μM) were significantly lower than those of indomethacin (206.3±43.0 and 180.3±22.6/μM) at P<0.01 in Colo320 and THRC cell lines, respectively. These findings suggest that NS‐398, a selective inhibitor of COX‐2, is a possible candidate for a chemopreventive agent with a potent apoptosis‐inducing effect and low nlcerogenic activity.
Sparse Distribution of Hepatocyte Growth Factor‐producing Cells inside Hepatocellular Foci in Rats Treated with Hepatocarcinogens
Authors: Imai Toshio , Ichinose Masutaro , Yanai Tokuma , Masegi Toshiaki , Nakamura Toshikazu , Tsukamoto Tetsuya , Kitoh Kuniyoshi , Tatematsu Masae
Published: August 2005

The distribution of hepatocyte growth factor (HGF)–synthesizing cells in rat liver during development of glutathioneS–transfcrasc P form (GST–P)–positive nodules after diethylnitrosamine initiation followed hy promotion with 2–acetylaminofluorene plus partial hepatectomy (PH) was investigated using insituhybridization, HGF–produclng cells were iioii–parenchymal in nature, and were suspected to be mainly of Kupffer type. They were mostly located outside GST–P–positive lesions, in the surrounding parenchyma. In the oval cell proliferation phase 1 week after PH, they increased and they were mainly localized around the portal triads. It is concluded that HGF is directly involved in an endogenous paracrine growth pathway controlling proliferation in oval cells and in normal, hut not GST–P–positive, hepatocytes.
Mechanisms of Growth Inhibition of Human Lung Cancer Cell Line, PC‐9, by Tea Polyphenols
Authors: Okabe Sachiko , Suganuma Masami , Hayashi Moriaki , Sueoka Eisaburo , Komori Atsumasa , Fujiki Hirota
Published: August 2005

(–)‐Epigallocatechin gallate (EGCG), the main constituent of green tea, and green tea extract show growth inhibition of various cancer cell lines, such as lung, mammary, and stomach. We studied how tea polyphenols induce growth inhibition of cancer cells. Since green tea extract contains various tea polyphenols, such as EGCG, (–)‐epigallocatechin (EGC), (–)‐epicatechin gallate (ECG), and (–)‐epicatechin (EC), the inhibitory potential of each tea polyphenol on the growth of a human lung cancer cell line, PC‐9 cells, was first examined. EGC and ECG inhibited the growth of PC‐9 cells as potently as did EGCG, but EC did not show significant growth inhibition. The mechanism of growth inhibition by EGCG was studied in relation to cell cycle regulation. Flow cytometric analysis revealed that treatment with 50 μM and 100 μM EGCG increased the percentages of cells in the G2‐M phase from 13.8% to 15.6% and 24.1%, respectively. The DNA histogram after treatment with 100 μM EGCG was similar to that after treatment with genistein, suggesting that EGCG induces G2‐M arrest in PC‐9 cells. Moreover, we found by microautoradiography that [3H]EGCG was incorporated into the cytosol, as well as the nuclei. These results provide new insights into the mechanisms of action of EGCG and green tea extract as cancer‐preventive agents in humans.
Detection of Carcinoembryonic Antigen‐expressing Free Tumor Cells in Peritoneal Washes from Patients with Gastric Carcinoma by Polymerase Chain Reaction
Authors: Nakanishi Hayao , Kodera Yasuhiro , Torii Akihito , Hirai Takashi , Yamamura Yoshitaka , Kato Tomoyuki , Kito Tsuyoshi , Tatematsu Masae
Published: August 2005

Cytological examination of peritoneal washes is a useful predictor of peritoneal recurrence in gastric carcinoma patients. In the present study, even more sensitive detection of free cancer cells could be achieved through amplification of Carcinoembryonic antigen (CEA) mRNA by means of the reverse transcriptase‐polymerase chain reaction (RT‐PCR). CEA was first confirmed to be present in all the gastric cancer cell lines examined, irrespective of the differentiation degree, and absent in blood and mesothelium, indicating the specificity of this approach for detection of carcinoma cells in peritoneal lavage fluid. In sensitivity tests, CEA RT‐PCR proved to be capable of detecting 10 carcinoma cells per sample. Peritoneal washes of 15 of 48 gastric carcinoma patients, including all 10 patients with positive cytology results, proved positive for CEA mRNA. None of the 5 patients with benign disease was positive. Moreover, a close association with the depth of cancer invasion was established. The results indicate that the assay is more sensitive for detection of free carcinoma cells in the peritoneal cavity than conventional cytology. This is the first study to suggest the feasibility of the RT‐PCR method for prediction of peritoneal recurrence in gastric cancer patients.
Isolation of a Novel Gene Showing Reduced Expression in Metastatic Colorectal Carcinoma Cell Lines and Carcinomas
Authors: Fukuda Seisuke , Kuroki Tamotsu , Kohsaki Hironobu , Hayashi Seitaku , Ozaki Kouichi , Yamori Takao , Tsuruo Takashi , Nakamori Shouji , Imaoka Shingi , Nakamura Yusuke
Published: August 2005

To investigate genes involved in mctastatic stages of cancer, we analyzed expression of mRN As in three cell lines derived from murine colon adenocarcinoma 26 by means of a differential display method. Each of these lines exhibits distinct metastatic characteristics. Among many bands representing different expression patterns in the display, we confirmed by northern analysis that a gene corresponding to one amplified fragment, termed grm2 (gene related to metastasis 2), was expressed more abundantly in NL4, the derivative with the lowest metastatic potential, than in cell lines NL17, an experimentally metastatic derivative, and in NL22, a spontaneously metastatic derivative. Using thegrm.2 fragment as a probe, we isolated murine cDNA clones and subsequently human cDNA clones corresponding to the GRM2 gene. The human and mouse homologues both encode proteins of 600 amino‐acid residues, which show weak homologies to proteins belonging to the myosin family. When we examined the expression levels of this novel gene in human colon cancers and in corresponding metastatic foci, we found that in more than half of these tissues, expression was significantly reduced in association with malignant potential. Our resultsimply that in humans the GRM2 gene product may regulate the metastatic phenotype of some colorectal cancers.
Effects of Bioreductive Agents, Tirapazamine and Mitomycin C, on Quiescent Cell Populations in Solid Tumors, Evaluated by Micronucleus Assay
Authors: Masunaga Shin‐ichiro , Ono Koji , Hori Hitoshi , Shibata Toru , Suzuki Minoru , Kinashi Yuko , Takagaki Masao , Akaboshi Mitsuhiko
Published: August 2005

Mice bearing transplantable solid tumors received 10 intraperitoneal administrations of 5‐bromo‐2′‐deoxyuridine (BrdU) to label the proliferating (P) tumor cells, and were then irradiated with 60Cogamma‐rays or injected with cis‐diamminedichloroplatinum (II) (cisplatin). The tumor cells were isolated and incubated with cytochalasin‐B (a cytokinesis blocker). The micronucleus (MN) frequency in the cells without BrdU labeling, which were regarded as quiescent (Q) cells in the tumor, was determined using immunofluorescence staining for BrdU. The MN frequency in the total (P+Q) tumor cell population was determined from tumors that were not pretreated with BrdU. Pretreatment with tirapazamine, a bioreductive agent, could enhance the sensitivity of tumor cells, including Q cells, to radiation more markedly than mitomycin C pretreatment as judged from an in vivo assay immediately after irradiation. Post‐irradiation administration of tirapazamine produced a large post‐irradiation radiosensitizing effect on both the total and Q tumor cell populations in vivo. Cisplatin treatment combined with tirapazamine demonstrated that tirapazamine also has a chemosensitizing potential for both the total and Q tumor cell populations. We confirmed that the sensitivity of Q cell populations to radiation and chemotherapy using cisplatin can be enhanced by combined treatment with tirapazamine.
Effect of a very late antigen‐4 receptor antagonist on allergen‐induced airway responses and inflammation in asthma
Authors: Diamant Z. , Kuperus J. , Baan R. , Nietzmann K. , Millet S. , Mendes P. , Miller B. , Amin D. , Rohatagi S. , Sterk P. J. , Hoogsteden H. C. , Prins J.‐B.
Published: August 2005

Background
Very late antigen‐4 (VLA4) plays a key role in the recruitment of eosinophils in allergic responses in animal studies.

Objective
We investigated whether pretreatment with multiple doses of a VLA4 receptor antagonist, HMR 1031, protects against allergen‐induced airway responses and airway inflammation in humans.

Methods
Fourteen asthmatics (7F/7M), 18–49 years, PC20 forced expiratory volume in 1 s (FEV1) methacholine (M) (<8 mg/mL; FEV1 82.3–116.1% predicted) with dual responses to inhaled allergen participated in a double‐blind, placebo‐controlled, cross‐over study. Each treatment period consisted of 9 days, separated by 2 weeks. Exhaled nitric oxide (eNO), PC20FEV1(M) and hypertonic saline‐induced sputum was obtained on Days 1, 7 and 9. Subjects inhaled HMR 1031 (20 mg b.i.d.) or placebo (P) on Days 1–8. On Day 8, an allergen bronchoprovocation test was performed, the airway response was measured by FEV1, and expressed as %fall from baseline. Data from 12 evaluable subjects are presented here.

Results
Both treatments were well tolerated. There was no significant difference between HMR 1031 and P in the early asthamatic response: mean AUC (0–3 h)±SEM (%fall h): 26.01±4.26 and 17.41±4.26, respectively (P=0.18), nor in the late response: mean AUC (3–9 h)±SEM (%fall h): 97.09±8.63 and 97.61±8.63, respectively, P=0.97. This corresponded to the absence of significant allergen‐induced changes in PC20FEV1(M), eNO, sputum eosinophils and soluble inflammation markers between both treatment periods.

Conclusions
Treatment with multiple inhaled doses of the VLA4 antagonist, HMR 1031, did not result in detectable protection against allergen‐induced airway responses or airway inflammation in asthma.
Transposable element insertion location bias and the dynamics of gene drive in mosquito populations
Authors: Rasgon J. L. , Gould F.
Published: August 2005

Some vector‐borne disease control strategies using transgenic mosquitoes require transgene spread to high frequency in populations. Transposable elements (TEs) are DNA sequences that replicate and transpose within the genomes of other organisms and may therefore be represented in the next generation in higher frequencies than predicted by Mendelian segregation. This over‐representation has allowed some TEs to spread through natural populations. Transgenes incorporated within a TE sequence are expected to be driven into populations as long as there is a positive balance between fitness costs and over‐representation. Models have been used to examine parameters that affect this balance but did not take into account biased insertion of TEs to linked sites in the genome. A simulation model was created to examine the impact of insertion bias on TE spread in mosquito populations. TEs that induce no fitness costs are predicted to increase in frequency over a wide range of parameter values but spread is slower for lower levels of transposition and non‐local movement. If TEs are costly, high proportions of local movement can slow or halt spread. To function as a robust transgene drive mechanism a TE should replicate and transpose > 10%/insert/generation, induce < 1% fitness cost/insert, and move preferentially to unlinked sites in the genome.