bluuebunny/crossref_metadata_2025 huggingface dataset:
N1‐Methylnicotinamide Level in the Blood after Nicotinamide Loading as Further Evidence for Malignant Tumor Burden
Authors: Nakagawa Koji , Miyazaki Masaru , Okui Katsuji , Kato Noriko , Moriyama Yoichi , Fujimura Shinji
Published: August 2005

Nicotinamide methyltransferase (Nmd CH3transferase) activity increased in the liver of mice after i.p. transplantation of Ehrlich ascites tumor (ascitic form), but not in the liver of mice with acute inflammation induced by the i.p. administration of D‐galactosamine, and it rather showed a decrease together with necrosis after carbon tetrachloride administration. When Nmd CH3transferase activity of rat hepatocytes in primary culture was investigated with the addition of dexamethasone, epidermal growth factor, transforming growth factor‐β, tumor necrosis factor‐α and N1‐methylnicotmamide (1‐CH3Nmd), changes in activity were not correlated with DNA synthesis, suggesting that the increase of this enzyme activity in the tumor host liver was not directly related to liver cell proliferation. Thus, in order to make use of the increase of this enzyme activity as a tumor burden marker, a procedure for its estimation by measuring the blood level of l‐CH3Nmd, a metabolite of Nmd produced by Nmd CH3transfcrase, was established. The l‐CH3Nmd level in the blood of mice bearing Ehrlich ascites tumor 4 h after s.c. loading of Nmd (500 mg/kg body weight) was closely correlated with this enzyme activity in the liver (r= 0.835, P < 0.00001) from the early to the terminal stage of tumor development. Furthermore, similar correlations were seen in the animal groups bearing various other tumors, such as s.c. implanted Ehrlich ascites tumor (solid form) and i.p. implanted sarcoma S‐180, hepatoma MH‐134, Yosbida ascites sarcoma and leukemia L‐1210, but not solid tumors such as Lewis; lung carcinoma and melanoma B‐16, although almost all of the animals bearing these tumors showed a higher enzyme activity than their control normal animals.
A Case of Squamous Cell Lung Carcinoma with High Concentration of Parathyroid Hormone‐related Peptide in Serum and Pleural Effusion Presenting Hypercalcemia
Authors: Iguchi Haruo , Katakami Hideki , Ichinose Yukito , Nishi Yoshihiro , Tanaka Seiichi , Kara Nobuyuki , Ohta Mitsuo , Haji Masafumi , Nawata Hajime
Published: August 2005

A 57‐year‐old man with lung squamous cell carcinoma revealed hypercalcemia, hypophosphoremia, elevation of nephrogenous cAMP and metabolic alkalosis. Serum parathyroid hormone (PTH) and l,25(OH)2D3 concentrations were not elevated. These findings were consistent with those in humoral hypercalcemia of malignancy (HHM), PTH‐related peptide (PTHrP) concentrations were determined using N‐ and C‐terminal specific radioimmunoassays (PTHrP‐N, PTHrP‐C), and elevation of both PTHrP‐N and PTHrP‐C concentrations in the serum was noted (PTHrP‐N, 27 pmol/liter (norma<5); PTHrP‐C, 1408 pmol/liter (norma<50)). High concentration of PTHrP (946 pmol/ liter for PTHrP‐N and 5983 pmol/liter for PTHrP‐C) was also found in the pleural fluid obtained at autopsy. Immunohistochemical study, using paraffin‐embedded sections of the tumor tissue obtained at autopsy, revealed numerous PTHrP‐positive cells and expression of PTHrP gene was confirmed by Northern blot analysis. These findings indicate that PTHrP, produced in the tumor tissue, was secreted into the blood stream, which caused HHM in the patient. Gel permeation chromatography of the serum and pleural fluid revealed several peaks of both PTHrP‐N and PTHrP‐C. Molecular forms of PTHrP‐N were larger than those of PTHrP‐C in the serum as well as pleural fluid. These findings indicate that multiple forms of PTHrP molecules are present in the serum and pleural fluid. Granulocytosis was also noted in the patient. However, granulocyte‐ and granulocyte macrophage‐cotony stimulating factor were not detected in the serum, and the mechanism of the granulocytosis in the patient was unclear.
Ductectatic‐type Mucinous Cystadenoma and Cystadenocarcinoma of the Human Pancreas: A Novel Clinicopathological Entity
Authors: Yanagisawa Akio , Ohashi Kazuhiko , Hori Masaharu , Takagi Kunio , Kitagawa Tomoyuki , Sugano Haruo , Kato Yo
Published: August 2005

A new type of mucinous cystic pancreatic neoplasm is described. The lesions, which have been recognized only recently with the progress of diagnostic techniques and have not previously been described in the literature, are characterized by multilocular cysts with papillary proliferation of the lining epithelium. They occur exclusively in the head and body, predominantly in males, and coexistence of well‐differentiated adenocarcinoma as well as adenoma components is frequently encountered. These lesions are quite different from hitherto described mucinous cystic neoplasms of pancreas, not only in terms of gross features but also with regard to intrapancreatic location, and sex and age distributions. We propose to classify these lesions as a new pancreatic tumor entity: the ductectatic‐type mucinous cystadenoma/cystadenocarcinoma.
Expression of nm23‐Hl and nm23‐H2 Proteins in Prostate Carcinoma
Authors: Konishi Noboru , Nakaoka Shingo , Tsuzuki Toshihide , Matsumoto Kyoichi , Kitahori Yoshiteru , Hiasa Yoshio , Urano Takeshi , Shiku Hiroshi
Published: August 2005

The nm23 gene products/nucleoside diphosphate (NDP) kinase expression in prostate carcinomas and benign hyperplasias was evaluated immunohistochemically. Monoclonal antibodies against nm23‐H1 and nm23‐H2 proteins were prepared using the corresponding proteins fused with glutathione S‐transferase as immunogens. Of the 80 cases of nonmetastatic prostate carcinoma examined, 74% (59/80) and 60% (48/80) were immunoreactive for nm23‐H1 or nm23‐H2 protein, respectively. Negative staining for nm23‐H1 occurred in 83% of metastatic lesions, while 34% were negative for nm23‐H2. All primary tumors corresponding to the metastases examined showed positive immunostaining for nm23‐H1, indicating an inverse relationship between expression of this protein and metastatic status. nm23‐H2 protein was detected in 83% of primary tumors and its expression appeared to he significantly correlated to the degree of histological differentiation. In contrast, all cases of benign prostatic hyperplasia showed elevated levels of both nm23‐H1 and nm23‐H2 expression. These data suggest that the nm23/NDP kinase may play a role in suppressing the expression of malignant potential in prostate carcinomas.
Production, Binding and Cytotoxicity of Human/Mouse Chimeric Monoclonal Antibody‐Neocarzinostatin Conjugate
Authors: Yamaguchi Toshiharu , Tsurumi Hiroshi , Kitamura Kazuya , Otsuji Eigo , Miyagaki Takuya , Kotani Tatsuya , Takahashi Toshio
Published: August 2005

A human/mouse chimeric Fab monoclonal antibody A7 (chFabA7) was covalently coupled to neocarzinostatin (NCS) by the SPDP method at various chFabA7:NCS substitution ratios. The antigen‐binding activity of the conjugate, examined by ELISA using fixed antigen‐positive colon cancer cells, was identical to that of the parent chFabA7 when one mole of NCS was conjugated, but was reduced with 2 or 3 moles of conjugated NCS. By means of a colony‐forming assay, the cytocidal effect of the conjugate on antigen‐positive cancer cells was found to be stronger than that of free NCS, whereas in antigen‐negative cancer cells it was similar to that of free NCS. This effect was attenuated by adding an excess amount of monoclonal antibody A7. These findings indicate that the conjugate has an antigen‐specific cytocidal action, and thus chFabA7‐NCS is a promising tool for targeting cancer chemotherapy.
Influence of Aging on Multi‐organ Carcinogenesis in Rats Induced by N‐Methyl‐N‐nitrosourea
Authors: Mizoguchi Masae , Naito Hatsumi , Kurata Yasushi , Shibata Masa‐Aki , Tsuda Hiroyuki , Wild Christopher P. , Montesano Ruggero , Fukushima Shoji
Published: August 2005

The effects of aging on the multi‐organ Carcinogenesis induced in rats by N‐methyl‐N‐nitrosourea (MNU), a direct carcinogen which does not need metabolic activation to exert carcinogenicity, were examined in male F344 rats. In the first experiment, rats at 6, 52, and 98 weeks of age were treated with MNU (20 mg/kg body weight, i.p.) twice weekly for 6 weeks and then maintained without any further treatment for 24 weeks in the case of young and middle‐aged rats and for 18 weeks in the case of the old rats. In young rats, malignant lymphomas, particularly thymic types, were observed at significantly high incidence. A striking result in the middle‐aged rats was the significantly higher incidence of adenocarcinomas in the small intestine than in young or old animals. The induction of proliferative and neoplastic lesions of the large intestine also tended to be increased in middle‐aged rats. In addition, epithelial hyperplasia of the tongue, but not the forestomach, occurred at the highest incidence in the middle‐aged group. There were no differences in the induction of epithelial lesions in the urinary bladder among the groups. In a second experiment, investigation of DNA synthesis in the tongue, small and large intestines, urinary bladder and lymph nodes did reveal significant increases or tendency for increase in the MNU‐treated groups, hut without differences with age. In contrast, the thymus of young rats showed significantly increased incorporation of BrdU label after administration of MNU, whereas it was markedly reduced in middle‐aged rats. In a third experiment, O′‐methyldeoxyguanine (O6‐medG) DNA adduct formation was immunohistochemically detected in various organs including the thymus, forestomach, and small intestine without any differences with age. Thus, the results demonstrated that while the target organs of MNU are modified by the age of the animals, levels of DNA synthesis and O6‐medG DNA adduct formation in most cases can not explain the observed differences in carcinogenic susceptibility.
Alterations of Integrin Expression in Human Lung Cancer
Authors: Suzuki Susumu , Takahashi Takashi , Nakamura Shigeo , Koike Koichi , Ariyoshi Yutaka , Takahashi Toshitada , Ueda Ryuzo
Published: August 2005

Integrins are cell‐surface receptors which are involved in cell‐matrix and/or cell‐cell adhesion. They have been suggested to play a role in tumor invasion and metastasis. We examined the expression of various integrin subunits in normal and cancer cells of the lung using 33 human lung cancer cell lines as well as 6 lung cancer samples from which tumor cell lines could be established. This study clearly demonstrated that changes in the expression of certain integrins occur frequently in lung cancer, especially in small cell lung cancer. Loss of the α1 subunit of the β1 integrin family appears to be the most prominent change, although loss of other integrin subunits such as α2 or emergence of some integrin subunits such as αv can also be observed. These results suggest that changes in integrin expression may contribute to the invasive and/or metastatic behavior of lung cancer.
Multiplex Mutation Screening of the BRCA1 Gene in 1000 Japanese Breast Cancers
Authors: Emi Mitsuru , Matsushima Mieko , Katagiri Toyomasa , Yoshimoto Masataka , Kasumi Fujio , Yokota Takashi , Nakata Tomoko , Miki Yoshio , Nakamura Yusuke
Published: August 2005

To detect BRCA1 mutations in Japanese breast cancer patients, we screened 1,000 unselected primary cancers for mutations in exon 11, which accounts for 61% of the entire BRCA1 coding sequence. Using a method based on multiplex single‐strand conformational polymorphism (SSCP) analysis of multiple restriction fragments generated by restriction‐enzyme digestion of amplified DNA, we identified eight mutations. All eight were germline mutations; four of them were non‐sense mutations or small deletions resulting in premature stop codons, and the other four were missense mutations. The Japanese carriers of these mutant BRCA1 alleles had developed breast cancers at ages ranging from 45 to 62, five of them bilaterally.
Estrogen Receptor Mutations and Changes in Estrogen Receptor and Progesterone Receptor Protein Expression in Metastatic or Recurrent Breast Cancer
Authors: Umekita Yoshihisa , Sagara Yoshiatsu , Yoshida Hiroki
Published: August 2005

To investigate the frequency of estrogen receptor (ER) gene mutation in metastatic or recurrent breast cancer, metastatic lymph nodes or recurrent breast cancer tissue from 35 patients with ER‐positive primary tumors were screened for mutations in the hormone‐binding domain of the ER gene by sequence analysis. Four missense mutations, Val316Ile, Gly344Val, Ala430Val and Gly494Val, were identified in these lesions. Second, to clarify whether there is any disparity in hormone receptor status between primary and metastatic or recurrent tumors, we immunohistochemically studied 117 specimens including the above 35 specimens obtained from metastatic or recurrent breast cancer patients using monoclonal anti‐ER and progesterone receptor (PgR) antibodies. Although hormone receptor status, especially ER, was highly maintained through disease progression, negative change in PgR expression at relapse (33%) was identified more frequently than in metastatic lymph nodes (6.7%). Therefore, it was suggested that development of PgR‐negative phenotype might correlate with disease progression in some breast cancer patients. These results suggest that ER mutations in metastatic or recurrent breast cancer may be more frequent than in primary lesions, irrespective of high maintenance of ER protein expression through disease progression.
Frameshift Mutations of the hMSH6 Gene in Human Leukemia Cell Lines
Authors: Hosoya Noriko , Hangaishi Akira , Ogawa Seishi , Miyagawa Kiyoshi , Mitani Kinuko , Yazaki Yoshio , Hirai Hisamaru
Published: August 2005

Defects in DNA mismatch repair mechanisms, including frameshift mutations of the hMSH6 and hMSH3 genes at their (C)8 and (A)8 tracks, respectively, have been shown to be associated with human malignancies. To clarify the possible involvement of these mutations in hematopoietic malignancies, we screened a total of forty‐four human leukemia and lymphoma cell lines for mutations in the hMSH6 and hMSH3 genes, as well as in other genes required for DNA replication or repair, by polymerase chain reaction single‐strand conformation polymorphism analysis and sequencing analysis. Frameshift mutations at the (C)8 track of the hMSH6 gene were detected in two cell lines established from lymphoid leukemias. These two cell lines had no wild‐type alleles, and both of them showed microsatellite instability. This is the first report that describes mutations and inactivation of the hMSH6 gene in hematological malignancies, suggesting that defects of the hMSH6 gene may be associated with development of hematological malignancies.
Characterization of mRNA Expression of IκBα and NF‐κB Subfamilies in Primary Adult T‐cell Leukemia Cells
Authors: Inoue Masato , Matsuoka Masao , Yamaguchi Kazunari , Takatsuki Kiyoshi , Yoshida Mitsuaki
Published: August 2005

Tax protein of HTLV‐1 activates the transcriptional capacity of the NF‐κB family, resulting in up‐regulation of various genes, which are linked to phenotypic alterations of HTLV‐1‐infected T cells. To understand NF‐κB regulation in HTLV‐1‐infected leukemic cells in vivo, we analyzed expression of NF‐κB and IκBα in primary cells isolated from ATL patients. Using competitive polymerase chain reaction, we observed an elevated expression of IκBα mRNA in all four ATL cases tested. In contrast to the elevated mRNA levels, the levels of IκBα protein were remarkably reduced in some of these cases, suggesting destabilization of IκBα protein. On the other hand, mRNA expression of p50/p105 and p65, subfamilies of NF‐κB, was enhanced in primary cells isolated from some ATL patients. Furthermore, the expression patterns of NF‐κB subfamily were variable among patients and also different from those in T cells isolated from uninfected individuals. Although the number of cases analyzed was limited, we can conclude from these observations that activation of NF‐κB is restricted to a few subfamilies in vivo. These findings in vivo are strikingly different from those in HTLV‐1‐infected T cell lines in vitro, in which Tax is responsible for NF‐κB activation. It is therefore suggested that the elevation of NF‐κB expression in leukemic cells of ATL patients might not be supported mainly by the viral protein Tax.
Adeno‐associated Virus Vector Containing the Herpes Simplex Virus Thymidine Kinase Gene Causes Complete Regression of Intracerebrally Implanted Human Gliomas in Mice, in Conjunction with Ganciclovir Administration
Authors: Mizuno Masaaki , Yoshida Jun , Colosi Peter , Kurtzman Gary
Published: August 2005

Adeno‐associated virus (AAV) has attracted considerable interest as a potential vector for gene therapy because of its wide host range, high transduction efficiency, and lack of cytopathogenicity. In this experiment, we evaluated the efficacy of AAV vector containing the herpes simplex virus thymidine kinase (HSV‐tk) gene on human gliomas transplanted into the brain of nude mice. Complete regression of the tumors was observed after multiple AAV‐tk injections followed by intraperitoneal ganciclovir (GCV) administration, and the survival of mice treated with AAV‐tk vector and GCV administration was markedly prolonged. These results suggest that AAV‐tk vectors may be useful for gene therapy against malignant gliomas in humans.
Responses of Total and Quiescent Cell Populations in Solid Tumors to Boron and Gadolinium Neutron Capture Reaction Using Neutrons with Two Different Energy Spectra
Authors: Masunaga Shin‐ichiro , Ono Koji , Sakurai Yoshinori , Suzuki Minoru , Takagaki Masao , Kobayashi Toru , Kinashi Yuko , Akaboshi Mitsuhiko
Published: August 2005

In neutron capture therapy, whose effectiveness depends on the tumor distribution of neutron capture compound and the neutron energy distribution, controlling quiescent tumor cells with clonogenic potential is critical for therapeutic gain, as is the case in conventional radio‐ and chemotherapy. Tumor‐bearing mice were continuously given 5‐bromo‐2′‐deoxyuridine (BrdU) to label all proliferating cells. After administration of sodium borocaptate‐10B (BSH), dl‐p‐boronophenylalanine‐10B (BPA) or gadodiamide hydrate (Omniscan), the tumors were irradiated with neutrons of different cadmium (Cd) ratio, and then isolated and incubated with cytochalasin‐B (a cytokinesis blocker). The micronucleus (MN) frequency in cells without BrdU labeling (quiescent cells) was determined using immunofluorescence staining for BrdU, and that for total cells was obtained from tumors not pretreated with BrdU. Without drugs, quiescent cells showed lower MN frequencies than total cells, but neutron irradiation reduced gamma‐ray sensitivity difference between the two. Relative biological effectiveness (RBE) of neutrons compared with gamma‐rays was greater in quiescent cells than in total cells, and low Cd ratio neutrons tended to exhibit large RBE values. With neutron capture compounds, MN frequency for each cell population was increased, especially when high Cd ratio neutrons were used. BPA increased the MN frequency for total cells to a greater extent than BSH. However, the sensitivity of quiescent cells treated with BPA was lower than that in BSH‐treated quiescent cells. This tendency was clearly observed in high Cd ratio neutrons. Omniscan only slightly increased the MN frequency in both cell populations, compared with irradiation alone, without drugs. From the viewpoint of increasing the quiescent cell sensitivity, tumors should be irradiated with high Cd ratio neutrons after BSH administration.
Prevention of N‐Methylnitrosourea‐induced Colon Tumorigenesis by Ursodeoxycholic Acid in F344 Rats
Authors: Narisawa Tomio , Fukaura Yoko , Terada Kunihiko , Sekiguchi Hideaki
Published: August 2005

Bile acids are known to promote colon carcinogenesis. However, there is one study showing that ursodeoxycholic acid (UDCA) supplemented in the diet at the concentration of 0.4% prevented azoxymethane‐induced rat colon tumorigenesis. The aim of our study was to explore the inhibitory effect of a much smaller dose of UDCA on colon carcinogenesis in rats. One hundred 7‐week‐old F344 rats were given 2 mg of N‐methylnitrosourea 3 times a week for 3 weeks by intrarectal instillation, and were fed a 0% (control), 0.4% or 0.08% UDCA‐supplemented diet for the next 27 weeks. All the rats were killed and examined for tumor development at week 30. The tumor incidence and number were significantly lower and smaller, respectively, in the UDCA‐fed rats than in the control rats: 40% and 36% vs. 68%; 0.5±0.1 (mean±SEM) and 0.4±0.1 vs. 1.0±0.2. All the tumors were located in the distal half of the colon and were plaque‐shaped or polypoid, being well‐differentiated adenocarcinomas restricted to the mucosa or submucosa. Bile acids in the feces and the blood obtained at weeks 20 and 30, respectively, were analyzed by HPLC. A significant increase of UDCA was confirmed in both the faces and the blood of the UDCA‐fed rats compared with the control rats. The results suggest that the continuous feeding of a small dose of UDCA may prevent colon carcinogenesis.
Linkage Mapping of the Bra, Brb and Brg Genes for Rat Protein Phosphatase 2A 55 kDa B‐Regulatory Subunit Isotypes
Authors: Yamada Shin , Shima Hiroshi , Toyota Minoru , Ushijima Toshikazu , Kuramoto Takashi , Serikawa Tadao , Okada Kyoji , Sato Kozo , Sugimura Takashi , Nagao Minako , Nakagama Hitoshi
Published: August 2005

We previously identified the rat Bra, Brb and Brg genes, which encode α, β and γ isotypes of the 55 kDa B‐regulatory subunit of protein phosphatase 2A. Polymerase chain reaction‐single strand conformation polymorphism analysis in the present study identified polymorphisms in Bra, Brb and Brg between the ACI and BUF, ZI and TM, and BN and WTC strains, respectively. Linkage analysis using mapping panels composed of F2 or back‐crosses of these strains allowed Bra, Brb and Brg to be assigned to chromosomes 15, 18 and 14, respectively. Furthermore, it was revealed that Bra is located close to the Rb1 locus. Using polymorphism in Bra, loss of heterozygosity (LOH) was analyzed for rat mammary tumors induced in (SDxF344) F1 female rats by a food‐borne carcinogen, 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine, and a typical mammary carcinogen, 7,12‐dimethylbenz[a]anthracene. No LOH was detected at the Bra locus.
Direct Activation of Human CD8+ Cytotoxic T Lymphocytes by Interleukin‐18
Authors: Kohyama Masako , Saijyo Kaoru , Hayasida Midori , Yasugi Tomoko , Kurimoto Masashi , Ohno Tadao
Published: August 2005

Direct activation of human cytotoxic T lymphocytes (CTL) by interleukin (IL)‐18 was observed in a system in which CTL effective against autologous tumor cells were generated. Peripheral blood mononuclear cells (PBMC) from tumor‐bearing patients, after removal of natural killer (NK) cells, were cultured in a medium containing IL‐1, ‐2, ‐4, and ‐6, with or without IL‐18, and stimulated with autologous tumor cells. IL‐18 increased the activity of the CTL and the proportion of autologous CD8+ T cells present after 28 days in the induction culture. When purified CD8+ T cells were cultured in the presence of IL‐18 and IL‐2 for 7 days, the CTL showed enhanced cytotoxic activity against autologous tumor cells. Moreover, a purified CD8+ T cell population, which did not exhibit any apparent cytotoxic activity against autologous tumor cells, displayed cytotoxic activity after 7‐day incubation with IL‐18. These results suggest that IL‐18 may be useful to generate autologous CTL in humans and may thereby contribute to adoptive immunotherapy for tumors.
Telomerase Activity Correlates with Growth of Transplantable Osteosarcomas in Rats Treated with cis‐Diammine Dichloroplatinum or the Angiogenesis Inhibitor AGM‐1470
Authors: Kido Akira , Tsujiuchi Toshifumi , Morishita Toru , Tsutsumi Masahiro , Takahama Makoto , Miyauchi Yoshizumi , Mii Yoshio , Tamai Susumu , Konishi Yoichi
Published: August 2005

To determine the role of telomerase activity in the growth of tumors in rats undergoing chemotherapy, a comparison of the volumes of telomerase‐positive transplantable osteosarcomas was made in rats treated with the antineoplastic agent cis‐diammine dichloroplatinum (CDDP) or the angiogenesis inhibitor O‐(chloroacetylcarbamoyl)fumagillol (AGM‐1470). Male F344 rats, 8 weeks old, received transplants of macroscopic lung metastatic nodules into the subcutaneous back space and treatment was started on day 14 thereafter. CDDP was injected i.v. at doses of 0, 0.625, 1.25 and 2.5 mg/kg body weight (b.w.) and AGM‐1470 was administered at total doses of 0, 2.5, 5 and 10 mg/kg b.w. over 2 weeks by osmotic pumps, also implanted into the subcutaneous back space, but remote from the transplanted tumors. On day 28, all animals were killed for measurement of transplanted tumor size and determination of telomerase activities by telomeric repeat amplification protocol (TRAP) assay. The results showed telomerase activity to be highly correlated with the treated/non‐treated (T/C) tumor size ratio (r=0.96, P<0.0001). In a second experiment, CDDP at 2.5 mg/kg b.w. and AGM‐1470 at 10 mg/kg b.w., these being the most effective doses, were given as in the first experiment, and animals were serially killed on days 14, 21, 28, 35 and 42. Tumors in rats treated with CDDP and AGM‐1470 showed 18.2% and 20.5% of the control telomerase activity on days 35 and 21, respectively, when tumor growth was inhibited. However, on day 42, the activities increased to 46.5% and 92.5%, this correlating with re‐growth (r=0.73, P<0.0001). These results suggest that decline of telomerase activity may be involved in tumor growth retardation induced by chemotherapeutic agents. This possibility clearly warrants further mechanistic studies.
Generation of Metastatic Variants of Eker Renal Carcinoma Cell Lines for Experimental Investigation of Renal Cancer Metastasis
Authors: Fukuda Tomokazu , Hirayama Youko , Mitani Hiroaki , Maeda Hiroshi , Tsutsumi Masahiro , Konishi Yoichi , Hino Okio
Published: August 2005

We and others have demonstrated that a mutation in Tsc2 is the rate‐limiting step for renal carcinogenesis in the Eker rat model. Although inactivation of Tsc2 results in development of renal tumors, it is not sufficient for metastatic renal cell carcinomas (RCs) in the Eker rat. To investigate the additional genetic event(s) necessary for cancer metastasis, we have established highly metastatic S‐Lk9d‐SLM cell lines from a non‐metastatic RC cell line (Lk9dL) by co‐implantation with a foreign body (gelatin sponge). Since these cell lines were remarkably different in metastatic performance (all and none, respectively) despite having the same genetic background, they should be useful experimental tools to investigate metastasis‐promoting events in renal carcinogenesis.
Phorbol Ester‐induced G1 Arrest in BALB/MK‐2 Mouse Keratinocytes Is Mediated by δ and η Isoforms of Protein Kinase C
Authors: Ishino Keiko , Ohba Motoi , Kashiwagi Mariko , Kawabe Shoko , Chida Kazuhiro , Kuroki Toshio
Published: August 2005

We investigated the possible negative regulation of the cell cycle by protein kinase C (PKC) isoforms in synchronously grown BALB/MK‐2 mouse keratinocytes, in which PKC isoforms were overexpressed by using the adenovirus vector Ax. Cells at the G1/S boundary of the cell cycle were the most sensitive to the inhibitory effect of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), a PKC agonist, resulting in G1 arrest. TPA‐induced inhibition of DNA synthesis was augmented by overexpression of the η and δ isoforms, but rescued by the dominant‐negative and antisense η isoforms. In contrast, the α and ζ isoforms showed no effect on DNA synthesis with or without TPA treatment. Immunoblotting indicated cell cycle‐dependent expression of the η isoform, being highest in cells at the G1/S boundary. The present study provides evidence that the η and δ isoforms of PKC are involved in negative regulation of cell cycle at the G1/S boundary in mouse keratinocytes.
Mammary Fibroblast‐derived Hepatocyte Growth Factor Stimulates Growth and Morphogenesis of Mouse Mammary Tumor Cells in Primary Culture
Authors: Sasaki Masato , Nishio Masahiro , Tsukada Yasukatsu , Enami Jumpei
Published: August 2005

We have recently isolated a mammary growth factor from the conditioned medium of mouse mammary stromal fibroblasts and identified it as a mouse homologue of human HGF (hepatocyte growth factor). To elucidate the role of HGF in mouse mammary tumorigenesis, we produced recombinant mouse HGF and examined its effects on primary cultures of mouse mammary tumor cells in this study. HGF at concentrations above 20 ng/ml maximally stimulated the growth of mammary tumor cells in primary monolayer culture. HGF also stimulated the three‐dimensional growth and branching morphogenesis of mammary tumor cells cultured inside collagen gels. A comparison of the growth‐stimulating activity of HGF with that of EGF (epidermal growth factor) and KGF (keratinocyte growth factor) revealed that HGF is the most potent growth factor among the three. Immunological studies using an antibody against mouse HGF demonstrated that 74% of the growth‐stimulating activity present in the mammary fibroblast‐conditioned medium was abolished by the antibody, indicating that HGF is the major growth factor produced by the fibroblasts. These observations thus suggest a role for HGF as a mammary stromal fibroblast‐derived factor which stimulates growth and morphogenesis of adjacent mammary tumor cells in vivo.
Lithocholic Acid, a Putative Tumor Promoter, Inhibits Mammalian DNA Polymerase β
Authors: Ogawa Akio , Murate Takashi , Suzuki Motoshi , Nimura Yuji , Yoshida Shonen
Published: August 2005

Lithocholic acid (LCA), one of the major components in secondary bile acids, promotes carcinogenesis in rat colon epithelial cells induced by N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG), which methylates DNA. Base‐excision repair of DNA lesions caused by the DNA methylating agents requires DNA polymerase β (pol β). In the present study, we examined 17 kinds of bile acids with respect to inhibition of mammalian DNA polymerases in vitro. Among them, only LCA and its derivatives inhibited DNA polymerases, while other bile acids were not inhibitory. Among eukaryotic DNA polymerases α, β, δ, η, and γ, pol β was the most sensitive to inhibition by LCA. The inhibition mode of pol β was non‐competitive with respect to the DNA template‐primer and was competitive with the substrate, dTTP, with the Ki value of 10 μM. Chemical structures at the C‐7 and C‐12 positions in the sterol skeleton are important for the inhibitory activity of LCA. This inhibition could contribute to the tumor‐promoting activity of LCA.
Characterization of a Human Small‐cell Lung Cancer Cell Line Resistant to a New Water‐soluble Camptothecin Derivative, DX‐8951f
Authors: Nomoto Taisuke , Nishio Kazuto , Ishida Tomoyuki , Mori Masatomo , Saijo Nagahiro
Published: August 2005

DX‐8951f, a water‐soluble and non‐pro‐drug analogue of camptothecin, exhibits a strong inhibitory action on DNA topoisomerase I (Topo I) and in vitro cytotoxicity against various human cancer cell lines. In order to elucidate the mechanisms of its cytotoxicity, we established a DX‐8951f‐resistant cell line, SBC‐3/DXCL1, from human small cell lung cancer cells (SBC‐3) by stepwise exposure to DX‐8951f. SBC‐3/DXCL1 cells were approximately 400 times more resistant to DX‐8951f than parent cells. The SBC‐3/DXCL1 cells showed a high degree of cross‐resistance to other Topo I inhibitors such as CPT‐11, SN‐38 and camptothecin, but not to non‐Topo I targeting agents such as cisplatin, adriamycin, etoposide, and vincristine. The mechanisms of resistance of SBC‐3/DXCL1 cells to DX‐8951f were examined. Intracellular accumulation of DX‐8951f by SBC‐3 and SBC‐3/DXCL1 cells did not differ significantly. Although the Topo I activity of nuclear extracts obtained from SBC‐3/DXCL1 cells was the same as that of the parent cells, the Topo I of SBC‐3/DXCL1 cells was resistant to the inhibitory effects of DX8951f and SN‐38. Immunoblotting using anti‐Topo I antibody demonstrated similar protein levels of Topo I in SBC‐3 and SBC‐3/DXCL1 cells. The active Topo I protein of SBC‐3/DXCL1 was eluted by a high concentration of NaCl (0.4 N) compared with that of SBC‐3 (0.3 N). DX‐8951f stabilized the DNA‐Topo I cleavable complex from SBC‐3 cells, as measured by Topo I‐mediated cleavage assay. In SBC‐3/DXCL1 cells, DX‐8951f also stabilized the DNA‐Topo I complex, but with a 10‐fold lower efficiency. These results suggest that a qualitative change in Topo I contributes, at least partially, to the resistance to DX‐8951f in SBC‐3/DXCL1 cells. Therefore, SBC‐3/DXCL1 cells may have a unique mechanism of resistance to Topo I‐directed antitumor drugs.
Antitumor Effect of Diphtheria Toxin A‐Chain Gene‐containing Cationic Liposomes Conjugated with Monoclonal Antibody Directed to Tumor‐associated Antigen of Bovine Leukemia Cells
Authors: Tana  , Watarai Shinobu , Onuma Misao , Aida Yoko , Kakidani Hitoshi , Kodama Hiroshi , Yasuda Tatsuji
Published: August 2005

Monoclonal antibody c143 against tumor‐associated antigen (TAA) expressed on bovine leukemia cells was conjugated to cationic liposomes carrying a plasmid pLTR‐DT which contained a gene for diphtheria toxin A‐chain (DT‐A) under the control of the long terminal repeat (LTR) of bovine leukemia virus (BLV) in the multicloning site of pUC‐18. The specificity and antitumor effects of the conjugates were examined in vitro and in vivo using TAA‐positive bovine B‐cell lymphoma line as the target tumor. In vitro studies with the TAA‐positive cell line indicated that luciferase genecontaining cationic liposomes associated with the c143 anti‐TAA monoclonal antibody caused about 2‐fold increase in luciferase activity compared with cationic liposomes having no antibody, and also that the c143‐conjugated cationic liposomes containing pLTR‐DT exerted selective growth‐inhibitory effects on the TAA‐positive B‐cell line. Three injections of pLTR‐DT‐containing cationic liposomes coupled with c143 into tumor‐bearing nude mice resulted in significant inhibition of the tumor growth. The antitumor potency of the c143‐conjugated cationic liposomes containing pLTR‐DT was far greater than that of normal mouse IgG‐coupled cationic liposomes containing pLTR‐DT as assessed in terms of tumor size. These results suggest that cationic liposomes bearing c143 are an efficient transfection reagent for BLV‐infected B‐cell lymphoma cells, and that the delivery of the pLTR‐DT gene into BLV‐infected B‐cells by the use of such liposomes may become a useful technique for gene therapy of bovine leukosis.
The Implication of Anthracosis in the Development of Pulmonary Adenocarcinoma
Authors: Hou Mei , Morishita Yukio , Iijima Tatsuo , Mase Kentaro , Dai Yuichi , Sekine Shigeki , Noguchi Masayuki
Published: August 2005

The relationship between anthracosis, which is the deposition of black dust matter in the lung parenchyma, and the development of pulmonary adenocarcinoma has not been fully characterized. In order to clarify whether background black dust matter deposition could be implicated in the development of pulmonary adenocarcinoma, we measured the level of anthracosis at autopsy in 47 patients who had died of pulmonary adenocarcinoma. Both lungs of all 47 cadavers were examined. Twenty‐micrometer sections were cut from formalin‐fixed, paraffin‐embedded blocks of the largest cut surface of each lung. Black dust matter was extracted from the sections and blotted onto a nitrocellulose membrane. The density of the blotted black dust matter was then analyzed using an imaging densitometer. There were no significant differences in the density of black dust matter deposition between lungs affected by pulmonary adenocarcinoma and control lungs. However, well differentiated adenocarcinomas tended to develop more frequently than poorly differentiated ones in lungs showing less deposition. We found a very strong correlation between the degree of black dust matter deposition and smoking history. Patients with severe anthracosis tended to have a poorer prognosis than those with mild anthracosis.
Growth Inhibition, Enhancement of Intercellular Adhesion, and Increased Expression of Carcinoembryonic Antigen by Overexpression of Phosphoinositides‐specific Phospholipase C β1 in LS174T Human Colon Adenocarcinoma Cell Line
Authors: Nomoto Koji , Tomita Naohiro , Miyake Masami , Xhu Ding‐Bang , LoGerfo Paul R. , Weinstein I. Bernard
Published: August 2005

By using a retrovirus‐derived system we generated derivatives of the human colon adenocarcinoma cell line LS174T (ATCC CL 188) that stably overexpress a full‐length cDNA encoding the β1 isoform of bovine phosphoinositides‐specific phospholipase C (PI‐PLC). This was confirmed by the elevated levels of catalytic activity to release phosphoinositides from phosphatidylinositol (PI‐PLC) or phosphatidylinositol‐bis‐phosphate (PIP2‐PLC), and the enhanced expressions of messenger RNA and protein. PI‐PLC β1 overexpresser clones grew to form cell clumps floating in liquid medium, whereas the pMV7‐introduced control clones displayed morphologic characteristics that were very similar to those of the parent LS174T cell line. Three individual PI‐PLC β1 overexpresser cell lines displayed increased doubling time (18.0 h, 21.5 h, and 23.8 h) when compared with 4 individual pMV7‐introduced control cell lines (13.1 h, 10.7 h, 12.9 h, and 9.3 h). Anchorage‐independent growth ability in soft agar medium was dramatically suppressed by overexpression of PLC β1, and the ability of PLC‐overproducer clones to form aggregates when cultured in liquid medium was dramatically enhanced when compared with that of pMV7‐introduced control clones. Tumorigenicity of PLC β1‐overproducers was much weaker than that of vector‐transduced control clones. The spontaneous release of carcinoembryonic antigen from PLC β1‐overproducer clones was much higher than that from pMV7 control clones. The ability of PLC β1‐overproducer clones to form aggregates during suspension culture was much stronger than that of the control clones. These results provide the first evidence that elevated levels of endogenous PI‐PLC β1 suppress tumor cell growth, but enhance the ability to form cell aggregates and to release carcinoembryonic antigen, an intercellular adhesion molecule.
Expression of Mucin‐associated Sulfo‐Lea Carbohydrate Epitopes on Human Colon Carcinoma Cells
Authors: Tsuiji Hitomi , Hayashi Makiko , Wynn Dianna M. , Irimura Tatsuro
Published: August 2005

The level of sulfo‐Lea (SO3‐3Galβ1‐3(Fucα1‐4)GlcNAc) epitope recognized by monoclonal antibody (mAb) 91.9H in hepatic metastasis of colon carcinoma is known to be lower than at the primary sites. We examined 19 human colon carcinoma cell lines for their production of this epitope. Sixteen cell lines were found to produce high Mr components that metabolically incorporated [35S]sulfate and were resistant to heparitinase I and chondroitinase ABC, and 8 of them were reactive with mAb 91.9H as shown by western blotting analysis. These were all of the 4 cell lines derived from well differentiated primary tumors (HCCP‐2998, LS174T, GEO, and CBS), 2 of 10 cell lines (DLD‐1 and HCT116) from moderately to poorly differentiated primary tumors, and 2 of 5 cell lines (SW480 and HCC‐M1544) from metastases. Incubation of LS174T cells with benzyl‐N‐acetyl‐α‐d‐galactosaminide abrogated the incorporation of [35S]sulfate and the reactivity of mAb 91.9H with high Mr components in the cell lysates. Sodium chlorate, which inhibits the formation of 3′‐phosphoadenosine 5′‐phosphosulfate, also inhibited the [35S]sulfate incorporation and reactivity with mAb 91.9H. These treatments did not change the incorporation of [14C]threonine into high Mr components. These results indicated that sulfo‐Lea epitopes were expressed on O‐linked carbohydrate chains in sulfomucins. Immunohistochemical studies of tumor tissues in nude mice indicated that sulfo‐Lea was expressed at the site of orthotopic transplantation in the cecum. The expression appeared to be suppressed in liver metastatic foci in nude mice.
Role of Sialylglycoconjugate(s) in the Initial Phase of Metastasis of Liver‐metastatic RAW117 Lymphoma Cells
Authors: Kikkawa Hironori , Miyamoto Daisei , Imafuku Hidetoshi , Koike Chieko , Suzuki Yasuo , Okada Shoji , Tsukada Hideo , Irimura Tatsuro , Oku Naoto
Published: August 2005

To elucidate the early events of blood‐borne metastasis under actual blood flow, real‐time trafficking of RAW117 large cell lymphoma cells, namely parental RAW117‐P and liver‐metastatic RAW117‐H10 cells, was investigated using positron emission tomography (PET). Both types of cells accumulated in the liver immediately after injection via the portal vein, and were eliminated from the liver time‐dependently. The elimination rate of RAW117‐H10 cells, however, was slower than that of RAW117‐P cells, suggesting that RAW117‐H10 cells interact more strongly with hepatic sinusoidal endothelium than the parental cells. This result correlated with the metastatic potential of these cells: RAW117‐H10 cells metastasized in the liver to a greater extent than RAW117‐P cells after injection via this route. To investigate the role of sialylglycoconjugates in the interaction of RAW117‐H10 cells with the hepatic endothelium after injection via the portal vein, the trafficking of RAW117‐H10 cells was examined after the cells had been treated with sialidase. The elimination rate of RAW117‐H10 cells from liver was observed to be greatly accelerated by sialidase treatment. To elucidate what kind of sialylglycoconjugates is related to this phenomenon, we analyzed the distribution of sialyl Lewis A and sialyl Lewis X antigens of both sublines of RAW117 by using flow cytometry. RAW117‐H10 cells were found to express a much higher level of sialyl Lewis A than RAW117‐P cells, whereas the amount of sialyl Lewis X did not differ significantly. These findings suggest that some sialylglycoconjugates, perhaps sialyl Lewis A in particular, play an important role in the initial interaction of RAW117‐H10 cells with the hepatic endothelium, leading to metastasis.
FK317, a Novel Substituted Dihydrobenzoxazine, Exhibits Potent Antitumor Activity against Human Tumor Xenografts in Nude Mice
Authors: Naoe Yoshinori , Inami Masamichi , Matsumoto Sanae , Takagaki Shoji , Fujiwara Tomoichi , Yamazaki Sachiko , Kawamura Ikuo , Nishigaki Fusako , Tsujimoto Susumu , Manda Toshitaka , Shimomura Kyoichi
Published: August 2005

The antitumor effects of FK317, a novel substituted dihydrobenzoxazine, were evaluated using human tumor xenografts (small cell lung cancer, non‐small cell lung cancer, stomach cancer, colon cancer, pancreatic cancer, breast cancer, cervical cancer and ovarian cancer). Tumor growth‐inhibitory effects and the effective dose‐range of FK317 were much stronger and broader, respectively, than those of reference drugs such as mitomycin C, adriamycin, cisplatin, taxol and irinotecan. Furthermore, the body weight decrease and myelosuppression in FK317‐treated mice were less than in the animals given any of the reference drugs. To explain this tumor selectivity, the distribution of FK317 was investigated after dosing tumor‐bearing mice with the 14C‐labelled compound. The concentration of FK317 in tumor tissues was relatively low, and long tumor retention was not observed. However, thin‐layer chromatographic separation revealed that the radioactivity in the tumor resided mainly in strongly cytotoxic metabolites, while that in other tissues resided mainly in non‐cytotoxic metabolites. These results suggest that FK317 shows strong antitumor activity without side effects, and one reason for this is its specific metabolite pattern. FK317 is now undergoing phase I clinical trials.
Anti‐cachectic Effect of FK317, a Novel Anti‐cancer Agent, in Colon26 and LX‐1 Models in Mice
Authors: Naoe Yoshinori , Kawamura Ikuo , Inami Masamichi , Matsumoto Sanae , Nishigaki Fusako , Tsujimoto Susumu , Manda Toshitaka , Shimomura Kyoichi
Published: August 2005

The effects of FK317 (11‐acetyl‐8‐carbamoyloxymethyl‐4‐formyl‐6‐methoxy‐14‐oxa‐1,11‐diazatetra‐cyclo[7.4.1.02,7.010,2]tetradeca‐2,4,6‐trien‐9‐yl acetate), a novel anti‐cancer agent, on murine adenocarcinoma colon26‐ and human lung carcinoma LX‐1‐induced cachexia were investigated in mice. Mice bearing colon26 or LX‐1 s.c. lost weight and became cachectic, associated with tumor growth. FK317 and mitomycin C (MMC) inhibited the growth of both tumors. FK317 ameliorated the weight loss induced by the presence of colon26 or LX‐1, while MMC enhanced it. An attenuation of the reduction in the weights of epididymal fat, gastrocnemius muscle and carcass was observed in FK317‐treated tumor‐bearing mice in both cachexia models, but not in MMC‐treated mice. The decreases in the circulating levels of triglyceride, glucose and non‐esterified fatty acid, which were induced by the presence of colon26, was partially inhibited by treatment with FK317. Overall, this study revealed that FK317 is a potent anti‐cancer drug with anti‐cachectic activity, suggesting that FK317 has potential utility for the treatment of cancer.
Calcium, Magnesium, and Nitrate in Drinking Water and Gastric Cancer Mortality
Authors: Yang Chun‐Yuh , Cheng Ming‐Fen , Tsai Shang‐Shyue , Hsieh Ya‐Lun
Published: August 2005

The possible association between the risk of gastric cancer and the levels of calcium, magnesium, and nitrate in drinking water from municipal supplies was investigated in a matched case‐control study in Taiwan. Records of gastric cancer deaths among eligible residents in Taiwan from 1987 through 1991 were obtained from the Bureau of Vital Statistics of the Taiwan Provincial Department of Health. Controls were deaths from other causes and were pair‐matched to the cases by sex, year‐of‐birth, and year‐of‐death. Each matched control was selected randomly from the set of possible controls for each case. Data on calcium, magnesium, and nitrate levels in drinking water throughout Taiwan were obtained from the Taiwan Water Supply Corporation. The municipality of residence of the cases and controls was assumed to be the source of the subject's calcium, magnesium, and nitrate exposure via drinking water. The subjects were divided into tertiles according to the levels of calcium, magnesium, and nitrate in their drinking water. The results of the present study show that there is a significant positive association between drinking water nitrate exposure and gastric cancer mortality. The present study also suggests that there was a significant protective effect of calcium intake from drinking water on the risk of gastric cancer. Magnesium also exerts a protective effect against gastric cancer, but only for the group with the highest levels.
Suppressive Effect of the Angiogenesis Inhibitor TNP‐470 on the Development of Carcinogen‐induced Hepatic Nodules in Rats
Authors: Ikebe Takashi , Yamamoto Takatsugu , Kubo Shoji , Hirohashi Kazuhiro , Kinoshita Hiroaki , Kaneda Kenji , Sakurai Masami
Published: August 2005

Tumor metastasis can be prevented by inhibiting angiogenesis. In the present study, we have demonstrated that the angiogenesis inhibitor TNP‐470 also suppresses the development of primary hepatic nodules. Hepatocarcinogenesis was performed by the feeding of 2‐acetylaminofluorene to hepatectomized rats during 8–14 weeks of age. Predominantly arterial‐to‐portal circulation and sinusoidal capillarization were determined by the staining of nodules with arterially infused ink and immunostaining for factor VIII‐related antigen, respectively. Intraperitoneal administration of 30 mg/kg b.w. of TNP‐470 twice a week significantly reduced the number of hepatic nodules. Among the nodules, hyperplastic nodules stained with ink, atypical hyperplastic nodules and hepatocellular carcinoma, all of which possess structurally altered sinusoidal endothelial cells or capillary‐type endothelial cells, were dramatically decreased in number. Suppression was observed equally in nodules of all sizes. TNP‐470 was more effective when administered during 8–20 weeks than during 14–26 weeks. In contrast, ink‐non‐stained hyperplastic nodules, which have normal sinusoidal endothelial cells, were not affected at all. The present results indicate that TNP‐470 suppresses the development of primary hepatic nodules whose microvessels are capillaries or transitional forms from sinusoids to capillaries.