bluuebunny/crossref_metadata_2025 huggingface dataset:
Metabolic and Morphologic Characteristics of Adipose Tissue Associated with the Growth of Malignant Tumors
Authors: Ishiko Osamu , Nishimura Sadako , Yasui Tomoyo , Sumi Toshiyuki , Hirai Kouzo , Honda Ken‐ichi , Ogita Sachio
Published: August 2005

Changes in total body fat and the metabolic and morphologic characteristics of adipose tissue were sequentially investigated in individual rabbits implanted with VX2 tumors to elucidate the pathology of the fat reduction in animals with malignant tumors as compared with that of diet‐restricted rabbits. Lipogenesis in normal, VX2–implanted, and diet‐restricted rabbit groups on day 40 after the start of the experiments was 19.1±2.9, 13.3±3.5, and 41.7±6.0×l05 cpm/g/h, respectively, and glycerol liberation by their adipose tissue was 199±21, 528±94, and 301±45 nmol/g/h, respectively. In addition, apoptotic cells were noted in the adipose tissue of VX2–implanted rabbits on days 20–30 after implantation, but not in diet‐restricted rabbits. The results showed clear differences between the total body fat reduction profiles of VX2–implanted rabbits and diet‐restricted rabbits, suggesting a characteristic lipid metabolism with enhanced lipolysis and diminished lipogenesis in VX2–implanted rabbits. The results strongly suggest that adipocyte apoptosis might be involved in these phenomena.
Improvement of Survival Over Time for Colon Cancer Patients by Anatomical Sub‐sites
Authors: Kawazuma Yoshikazu , Tanaka Hideo , Tsukuma Hideaki , Ajiki Wakiko , Oshima Akira
Published: August 2005

The aims of this study are to describe and to evaluate improvement of survival over time for colon cancer patients by anatomical sub‐sites. Data on 661 patients newly diagnosed as having colon cancer at Osaka Medical Center for Cancer and Cardiovascular Diseases from 1978 to 1991 were examined in this study. Corrected survival was calculated with the Kaplan‐Meier method according to the period of diagnosis: early period (1978‐84) and later period (1985‐91). Factors concerning the difference in survival between the two periods were examined with the Cox proportional hazards regression model according to sub‐site. Five‐year corrected survival of the patients with left colon cancer improved significantly (60 to 72%; P <0.01), probably due to advances in treatment, while that of patients with transverse colon cancer also improved significantly (39 to 67%; P<0.01), mainly because of progress in diagnosis. The five‐year corrected survival of those with right colon cancer did not increase (57 to 46%; P=0.14), owing to lack of improvement in stage at diagnosis. Among the three sub‐sites, the right showed the worst five‐year survival in the later period. We concluded that survival of patients with right colon cancer, differing from the other anatomical sub‐sites, did not improve, possibly because of lack of symptoms. The screening programs for colon cancer introduced in Japan in 1992 may be expected to improve the survival of patients with colon cancer, including that of the right colon.
Development and Distribution of 2‐Amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]‐pyridine (PhIP)‐induced Aberrant Crypt Foci in the Rat Large Intestine
Authors: Tsukamoto Tetsuya , Kozaki Ken‐ichi , Nishikawa Yoko , Yamamoto Masami , Fukami Hiroko , Inoue Manami , Wakabayashi Keiji , Tatematsu Masae
Published: August 2005

Aberrant crypt foci (ACF) are generally considered to be preneoplastic lesions for colon cancer. To assess their induction by 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP), a colon carcinogen, we performed a sequential study of ACF morphology and localization. F344 male rats were given PhIP, and methylene blue‐stained colon epithelium and isolated crypts were analyzed at weeks 12, 25, 50, and 75. Each crypt was classified into 2 groups, “single” with round bottoms and “bifurcating” displaying V‐shaped clefts (indicating proliferation). In combination with the number of crypts in an ACF, this classification was a good indicator for the generation of ACF in line with the fission mechanism of growth. Increasing numbers of crypts in ACF through weeks 12 to 75 and decreased percentages of ACF with bifurcating crypts at the late time points indicated that proliferation of crypts occurs predominantly during the early stages. The distribution pattern showed a significant shift (P < 0.000005) from the distal to the proximal part of the large intestine between weeks 25 and 50. Adenocarcinomas were first found to develop at week 50 in the ascending colon and cecum where bifurcating crypts were generally lacking at weeks 12 and 25. These data suggest the existence of (1) proliferating ACF which contains bifurcating crypt(s) and (2) quiescent or senescent ACF which consists of only single crypts.
Mxi1 Mutations in Human Neurofibrosarcomas
Authors: Li Xiao‐Jin , Wang Dong‐Yu , Zhu Yue , Guo Rong‐Jun , Wang Xing‐Duo , Lubomir Kaco , Mukai Kiyoshi , Sasaki Hiroki , Yoshida Haruhiko , Oka Teruaki , Machinami Rikuo , Shinmura Kazuya , Tanaka Masamitsu , Sugimura Haruhiko
Published: August 2005

Mxi1 is thought to negatively regulate Myc function and may therefore be a potential tumor suppressor gene. Little effort has yet been made to find alterations involving this gene in human solid tumors. We screened 31 human gastric cancers, 7 esophageal cancers, 85 bone and soft tissue tumors of various types, including 4 neurofibrosarcomas. We also examined 29 human tumor celllines consisting of 12 esophageal cancers, 7 glioma/glioblastomas and 10 others for Mxi1 mutations in exons 1, 2, 4 (HLH domain), 5 and 6. Polymerase chain reaction‐single‐strand conformation polymorphism (PCR‐SSCP) and subsequent sequencing revealed three distinct polymorphisms in the intron‐exon boundary upstream from exon 6. We discovered a missense mutation, GCA to GTA (Ala 54 Val), in exon 2 in a neurofibrosarcoma patient (case 1), two missense mutations, AAA to CAA (Lys 118 Gln) and GAA to GGA (Glu 154 Gly) in exon 5 of another neurofibrosarcoma patient (case 2), and 3 amino acid substitutions, GTG to GCG (Val 179 Ala), GTT to GCT (Val 181 Ala) and TTC to CTC (Phe 186 Leu), in a third neurofibrosarcoma patient (case 3). In case 3, loss of heterozygosity was also demonstrated by informative (TTC)3/(TTC)2 polymorphism. Our data demonstrate that mutations occur in the Mxi1 gene in neurofibrosarcoma. Missense mutations in the functional domain of Mxi1 in these cases may be involved in the pathogenesis of neurofibrosarcoma.
Membrane Transport and Antitumor Activity of Pirarubicin, and Comparison with Those of Doxorubicin
Authors: Sugiyama Tomomi , Sadzuka Yasuyuki , Nagasawa Kazuki , Ohnishi Noriaki , Yokoyama Teruyoshi , Sonobe Takashi
Published: August 2005

We have compared the membrane transport and antitumor activity of pirarubicin with those of doxorubicin in M5076 ovarian sarcoma, which exhibits low sensitivity to doxorubicin. Pirarubicin was rapidly taken up by M5076 cells and the intracellular concentration of pirarubicin reached more than 2.5‐fold that of doxorubicin. In terms of the 50% cell growth‐inhibitory concentration in vitro, pirarubicin was more effective than doxorubicin. Thus, the intracellular concentration influenced the cytotoxicity of these anthracycline agents. On comparison of the nuclear uptake of pirarubicin and doxorubicin, the nucleus/cell ratio of pirarubicin was found to be about 40%, whereas that of doxorubicin reached more than 80%. As the intranuclear concentration of pirarubicin is dependent on nuclear transport, the increases in not only cell membrane transport, but also nuclear membrane transport contributed to the enhancement of the efficacy of pirarubicin. In M5076 solid tumor‐bearing mice, pirarubicin reduced the tumor weight to 60% of the control level, although doxorubicin had no effect. These results were supported by the intracellular uptake of pirarubicin. Moreover, theanine, which inhibited the pirarubicin efflux from M5076 cells, increased by 1.3‐fold the pirarubicin concentration in the tumor and enhanced the therapeutic efficacy of pirarubicin 1.7‐fold. In conclusion, our results suggest that an increase in the concentration of an anthracycline derivative in tumor cells due to alteration of cell membrane transport results in enhancement of the antitumor activity.
Multidrug Resistance Phenotype in the RMS‐GR Human Rhabdomyosarcoma Cell Line Obtained after Polychemotherapy
Authors: Prados Jose , Melguizo Consolación , Marchal Juan Antonio , Vélez Celia , Alvarez Luis , Aránega Antonia
Published: August 2005

Classical cytotoxic treatment of rhabdomyosarcoma (RMS), the most common soft tissue malignancy in children, is often accompanied by significant morbidity and poor response. Chemotherapy may induce multidrug resistance (MDR) associated with the expression of P‐glycoprotein, a drug efflux pump which modifies the sensitivity of tumoral cells to drugs. To analyze MDR in RMS we used the RMS‐GR cell line, obtained from an embryonal rhabdomyosarcoma treated in vivo with polychemotherapy. The RMS‐GR cells showed cross‐resistance to vincristine, doxorubicin and actinomycin D, the drugs of choice in the conventional treatment of RMS. Polymerase chain reaction (PCR) analysis showed that these RMS cells overexpressed mdr1/P‐glycoprotein. The pattern of resistance and the level of P‐glycoprotein expression were similar to those found in the resistant RMS TE.32.7.DAC cell line obtained in vitro. Southern blot analysis showed that mdr1 overexpression was not due to amplification of the gene. Our results showed that the in vivo treatment of embryonal RMS may induce an MDR phenotype mediated by mdr1/P‐glycoprotein in RMS cells.
Tumorigenic Conversion of a Rat Urothelial Cell Line by Human Polymorphonuclear Leukocytes Activated by Lipopolysaccharide
Authors: Tamatani Tetsuya , Turk Patrick , Weitzman Sigmund , Oyasu Ryoichi
Published: August 2005

Chronic inflammation is a significant risk factor for the development of urinary bladder cancer. We have shown that inflammation induced by killed Escherichia coli and also by its lipopolysaccharide (LPS) strikingly enhances N‐methyl‐N‐nitrosourea (MNU)‐initiated rat bladder carcinogenesis. Aspirates from the bladder lumen contained a large quantity of hydrogen peroxide (H2O2) and several cytokines. In this study, we tested the hypothesis that reactive oxygen intermediates (ROI) released from activated polymorphonuclear leukocytes (PMN) are involved in inflammation‐associated bladder carcinogenesis. Using an immortalized nontumorigenic rat urothelial cell line, MYP3, we examined the effect of LPS‐activated PMN on malignant transformation. MYP3 cells pretreated with or without MNU were exposed daily to LPS‐activated PMN for one week and were then tested for growth in soft agar. In contrast to no colony formation by the parental cells, a varying number of colonies developed from cells treated with LPS‐activated PMN. Although combined treatment with MNU and PMN was most effective (P < 0.01), cells treated with LPS‐activated PMN alone also formed a small number of colonies. Addition of catalase, which decomposes H2O2, and/or an antioxidant, α‐tocopherol, reduced the number of colonies induced by LPS‐activated PMN (P<0.05). Cells derived from colonies were tumorigenic in athymic nude mice. However, tumorigenicity in mice was greater with cells treated with both MNU and PMN than with cells treated with PMN alone. Our results suggest that ROI released from LPS‐activated PMN may be one of the mechanisms involved in the carcinogenesis associated with active urinary tract infection.
Retention of Cell Adhesion and Growth Capability in Human Cervical Cancer Cells Deprived of Cell Anchorage
Authors: Kikuchi Keiji , Yasumoto Shigeru
Published: August 2005

Cell adhesion is linked to various regulatory processes of growth as well as apoptotic cell death in normal and transformed epithelial cells. We investigated changes of cellular responses to the deprivation of cell anchorage associated with immortalization or malignant transformation. Normal human ectocervical keratinocytes (NCE cells) deprived of cell anchorage become susceptible to apoptosis, and in parallel they lose their adhesion to the culture substratum. The loss of cell adhesion is not directly due to apoptosis. NCE16 cells, an immortalized but not malignantly transformed subline of NCE, underwent apoptosis and lost cell adhesion in suspension, as the NCE cells did. By contrast, apoptosis was not inducible in human cervical cancer‐derived C33A cells in suspension. Of other cell lines derived from human cervical cancer, SiHa cells showed a weak apoptotic response and Caski cells were highly sensitive to apoptosis in the absence of cell anchorage. Unlike NCE or NCE16 cells, all these cancer cells retained cell adhesion as well as growth capability in suspension cultures. These results indicate that retention of cell adhesion and growth capability in the absence of cell anchorage is more closely associated with cancer cell lines than resistance to apoptosis upon the deprivation of cell anchorage.
Wakame Seaweed Suppresses the Proliferation of 7,12‐Dimethylbenz(a)‐anthracene‐induced Mammary Tumors in Rats
Authors: Funahashi Hiroomi , Imai Tsuneo , Tanaka Yuji , Tsukamura Kyousuke , Hayakawa Yasusyuki , Kikumori Toyone , Mase Takahiro , Itoh Takahiro , Nishikawa Mikiko , Hayashi Hiromiti , Shibata Arihiro , Hibi Yatsuka , Takahashi Masahide , Narita Tatsuhiko
Published: August 2005

We examined the anti‐tumor proliferation effects of wakame seaweed on 7,12‐dimethylbenz(a)‐anthracene (DMBA)‐induced rat mammary tumor. DMBA was administered to 8‐week‐old female Sprague‐Dawley rats, and rats which developed mammary tumors were assigned randomly to three groups. Commercial rat feed was used in a control group (group I‐A), and two feed mixtures were prepared, which contained commercial rat feed blended with wakame at 1.0% (group I‐B) and 5.0% (group I‐C) by weight. The respective feeds were given to each group for 8 weeks, and changes in mammary tumor size were compared. At the end of the experiment, mammary tumors and thyroid glands were resected to compare their weights. Serum total iodine and thyroxin (T4) levels were measured. Immunohistochemical studies for bromodeoxyuridine (BrdU) labeling, transforming growth factor (TGF)‐β, and apoptosis were carried out in the resected tumor. Significant suppression of tumor growth was observed in groups I‐B and I‐C compared with I‐A. In groups I‐B and I‐C, the weights of resected mammary tumors were significantly lower and serum total iodine concentration was significantly higher than in I‐A. BrdU indices were significantly lower in groups I‐B and I‐C, compared with I‐A. TGF‐β and apoptotic index were inversely related to BrdU. These results suggest that iodine is transported from the serum into mammary tissues and induces apoptosis through the expression of TGF‐β. In conclusion, wakame suppressed the proliferation of DMBA‐induced mammary tumors.
Shedding of Membrane Type 1 Matrix Metalloproteinase in a Human Breast Carcinoma Cell Line
Authors: Harayama Takeshi , Ohuchi Eiko , Aoki Takanori , Sato Hiroshi , Seiki Motoharu , Okada Yasunori
Published: August 2005

Membrane type 1 matrix metalloproteinase (MT1‐MMP) with a transmembrane domain is a new member of the MMP gene family and is expressed on the cell surfaces of many carcinoma cells to activate the zymogen of MMP‐2 (gelatinase A). We have previously reported that MT1‐MMP is released into culture media in a complex form with tissue inhibitor of metalloproteinases 2 (TIMP‐2) from a human breast carcinoma cell line, MDA‐MB‐231, treated with concanavalin A (Con A). In the present study, we further studied the release mechanism of MT1‐MMP. Immunoblot analysis indicated that the amounts of MT1‐MMP in culture media increase with the time of exposure and the concentration of Con A, and those in cell lysates conversely decrease in a similar way. Time‐ and dose‐dependent release of MT1‐MMP into the media was confirmed by a sandwich enzyme immunoassay specific to MT1‐MMP. The molecular weight of the immunoreactive MT1‐MMP in the media was Mr 56,000, which was 4,000‐Mr smaller than that in the cell lysates. Northern blot analysis demonstrated that the mRNA expression level of MT1‐MMP is about 3‐fold enhanced after a 24 h‐exposure to Con A and this is maintained up to 72‐h exposure. The release of MT1‐MMP from the Con A‐treated cells was inhibited by metalloproteinase inhibitors such as EDTA and o‐phenanthroline, but not by MMP inhibitors including TIMP‐1, TIMP‐2 and BB94 or other proteinase inhibitors of serine, cysteine and aspartic proteinases. During the Con A treatment of the cells, cell viability decreased time‐ and dose‐dependently and dead cells reacted positively in the TdT‐mediated dUTP Nick‐End Labeling (TUNEL) method. Con A‐treated MDA cells showed apoptotic morphology when stained with Hoechst dye and hematoxylin and eosin. DNA ladder formation was detected by electrophoresis of the DNA from Con A‐treated MDA cells. These results suggest that MT1‐MMP release from Con A‐treated cells is due to shedding mediated by metalloproteinase(s) other than MMPs, and is associated with apoptosis.
Analysis of APCL, a Brain‐specific Adenomatous Polyposis Coli Homologue, for Mutations and Expression in Brain Tumors
Authors: Nakagawa Hidewaki , Koyama Kumiko , Monden Morito , Nakamura Yusuke
Published: August 2005

We recently identified a novel homologue of the adenomatous polyposis coli (APC) tumor suppressor gene, APCL, whose abundant and specific expression in the central nervous system indicated an important role in neuronal proliferation and differentiation. To investigate possible involvement of APCL alterations in brain tumors, we first analyzed the expression of APCL mRNA in seven glioma tissues by semi‐quantitative reverse transcription polymerase chain reaction (RT‐PCR) analysis, and in nine glioma cell lines by northern blotting. APCL expression was reduced significantly in most of the glioma tissues and all nine cell lines in comparison with normal brain tissue. However, single‐strand conformation polymorphism (SSCP) analysis and DNA sequencing of the entire coding region of APCL detected no mutations in any of the glioma cell lines, or in any of the 35 astrocytic gliomas and five medulloblastomas examined. Our results suggested that some epigenetic mechanism is responsible for the decrease in APCL expression in our panel of brain tumors.
Receptor for Hyaluronan‐mediated Motility and CD44 Expressions in Colon Cancer Assessed by Quantitative Analysis Using Real‐time Reverse Transcriptase‐Polymerase Chain Reaction
Authors: Yamada Yoichi , Itano Naoki , Narimatsu Hisashi , Kudo Takashi , Hirohashi Setsuo , Ochiai Atsushi , Niimi Atsushi , Ueda Minoru , Kimata Koji
Published: August 2005

Receptor for hyaluronan (HA)‐mediated motility (RHAMM) is a receptor for HA‐mediated motility and its expression is correlated with malignancy of ras‐transformed cells in that binding of HA to this receptor activates their migratory ability. CD44, a cell surface receptor for HA is also implicated in metastatic behavior of some cancer cells. In this study we examined the relationships of cancer progression with mRNA levels of RHAMM, CD44 (all forms), and exon 6 of CD44 using the real‐time reverse transcriptase‐polymerase chain reaction method in specimens of colon cancers at different diagnostic stages from 30 patients. Increased mRNA levels of RHAMM were observed in 29 specimens (97%), CD44s (all forms) in 21 specimens (70%), and its exon 6 in 19 specimens (63%) in comparison with those in the corresponding noncancerous tissue specimens. A statistically significant correlation between RHAMM expression and cancerous specimens at any of Dukes' stages A, B, and C was found, and the overexpression of CD44 mRNAs was confirmed in specimens at Dukes' stage C. Thus, our present study for the first time suggests that RHAMM expression may be a clinically useful indicator of colon cancer.
Disruption of E‐Cadherin‐mediated Cell Adhesion Systems in Gastric Cancers in Young Patients
Authors: Saito Atsushi , Kanai Yae , Maesawa Chihaya , Ochiai Atsushi , Torii Akira , Hirohashi Setsuo
Published: August 2005

The aim of this study was to the pathogenetic backgrounds of early‐onset gastric cancers. Mutations of the E‐cadherin and β‐catenin genes were analyzed by subjecting microdissected cancer cells and corresponding non‐cancerous epithelial cells obtained from 9 gastric cancer patients under 35 years old to polymerase chain reaction‐single strand conformation polymorphism analysis. Somatic, but no germline, E‐cadherin gene mutations were detected in 6 (67%) of the patients. The cancer cells of 2 patients were exon 9‐deleted E‐cadherin molecule‐immunoreactive. Neither somatic nor germline mutations in exon 3 of the β‐catenin gene were observed in any patient. One patient lacked β‐catenin immunoreactivity and the cancer cells of 6 others showed cytoplasmic β‐catenin immunoreactivity. The E‐cadherin‐mediated cell adhesion system in the cancer cells of all the patients examined appeared to be disrupted, indicating that somatically acquired dysfunction of this system plays an important role in early‐onset diffuse‐type gastric cancers. Helicobacter pylori infection was observed in 6 (67%) of our 9 patients, an incidence higher than the average in young Japanese individuals. Thus, early‐onset gastric cancers may be attributable to environmental factors such as Helicobacter pylori infection.
Antitumor Effects of Interleukin‐2 Gene‐modified Fibroblasts in an Orthotopic Colon Cancer Model
Authors: Terasawa Hiroshi , Tanimura Hiroshi , Nakamori Mikihito , Tsunoda Takuya , Iwahashi Makoto , Tani Masaji , Yamaue Hiroki
Published: August 2005

We transduced the interleukin‐2 (IL‐2) gene into murine fibroblasts BALBCL7 or murine colon cancer CT26 using a retroviral vector. BALBCL7 transduced with IL‐2 gene secreted 748 pg/ml of IL‐2, whereas IL‐2 gene‐modified CT26 secreted 1,167 pg/ml of IL‐2 (48 h incubation, 1×106/ml). Then, we inoculated gene‐modified BALBCL7 and/or CT26 cells into BALB/c female mice, and observed the tumor growth. The tumor growth was inhibited in mice inoculated with parental CT26 plus IL‐2 gene‐modified BALBCL7, compared with that in mice given parental CT26 alone (P < 0.01). Moreover, we investigated the cytotoxic activity of spleen cells derived from mice treated with gene‐modified cells, and performed phenotypic analysis of the effector cells. The killer cells derived from mice inoculated with IL‐2 gene‐modified BALBCL7 plus parental CT26 showed higher cytotoxic activity than those from mice inoculated with CT26 alone. The cytotoxic activity was almost completely blocked by anti‐CD8 antibody (Ab), and partially blocked by anti‐asialo GM1 Ab. Next, we inoculated CT26 tumor tissue into murine cecum orthotopically, and treated the animals with gene‐modified BALBCL7 plus parental CT26. The tumor size in the cecum was significantly decreased, compared with parental CT26 alone (P < 0.01).
Cytokines Required for Induction of Histocompatibility Leukocyte Antigen‐class I‐restricted and Tumor‐specific Cytotoxic T Lymphocytes by a SART1‐derived Peptide
Authors: Matsunaga Kazuko , Nakao Masanobu , Masuoka Kazuhiro , Inoue Yoshiko , Gouhara Rumi , Imaizumi Toshihiro , Nishizaka Shinya , Itoh Kyogo
Published: August 2005

Although there have been several reports on peptides of human tumor‐rejection antigens capable of inducing histocompatibility leukocyte antigen (HLA)‐class I‐restricted and tumor‐specific cytotoxic T lymphocytes (CTLs), it is not yet clear which cytokines are required for CTL induction. This study has investigated the cytokine combinations required for optimal induction of CTLs by SARTI690–698 peptide, which is capable of inducing HLA‐A24‐restricted and tumor‐specific CTLs in peripheral blood mononuclear cells (PBMCs). Pretreatment of PBMCs as a source of antigen‐presenting cells (APCs) with interferon (IFN)‐γ, or to some extent with IFN‐α, but not with any of the other cytokines tested, augmented the peptide‐induced CTL activity in HLA‐A24 heterozygotes, but not in HLA‐A24 homozygotes. This IFN‐γ ‐mediated augmentation was inhibited by either interleukin (IL)‐4 or IL‐10. IL‐2 alone in culture, along with weekly stimulation by peptide‐pulsed APCs, was sufficient for the differentiation and proliferation of CTLs for the initial several weeks of culture. This IL‐2‐mediated activation of CTLs was inhibited by the addition of IFN‐γ, IL‐4, or IL‐10 to the IL‐2 culture. For further expansion of the CTLs, dendritic cells (DCs) induced from PBMCs with IL‐4 and granulocyte macrophage colony‐stimulating factor (GM‐CSF) were required as APCs. These results indicate that IFN‐γ and IL‐2 are important in the activation of APCs and CTLs, respectively, while GM‐CSF and IL‐4 are needed for the induction of DCs, which in turn are required for further expansion of mature CTLs. These results are important in allowing for a better understanding of the cellular and molecular basis of tumor‐specific immunity, and also for the development of peptide‐based specific immunotherapy.
Chemopreventive Effect of Bovine Lactoferrin on 4‐Nitroquinoline 1‐Oxide‐induced Tongue Carcinogenesis in Male F344 Rats
Authors: Tanaka Takuji , Kawabata Kunihiro , Kohno Hiroyuki , Honjo Shiro , Murakami Manabu , Ota Takahide , Tsuda Hiroyuki
Published: August 2005

The modifying effects of dietary feeding of bovine lactoferrin (bLF) on tongue carcinogenesis initiated with 4‐nitroquinoline 1‐oxide (4‐NQO) were investigated in male F344 rats. The activities of phase II detoxifying enzymes, glutathione S‐transferase (GST) and quinone reductase (QR), polyamine content and ornithine decarboxylase (ODC) activity in the tongue were also examined for mechanistic analysis of possible modifying effects of bLF on carcinogenesis. At 7 weeks of age, all animals except those treated with bLF alone and untreated rats were given 20 ppm 4‐NQO in drinking water for 8 weeks to induce tongue neoplasms. Starting 7 days before 4‐NQO exposure, experimental groups were fed experimental diets containing bLF (0.2% and 2%) for 10 weeks (“initiation feeding”). Starting 1 week after the cessation of exposure to 4‐NQO, the other experimental groups given 4‐NQO and a basal diet were fed the experimental diets for 22 weeks (“postinitiation feeding”). At week 32, the incidence and multiplicity of tongue neoplasms in the “initiation feeding’ groups of 0.2% and 2% bLF and the “post‐initiation feeding” group of 0.2% bLF were lower than those of the 4‐NQO alone group, but without statistical significance. However, “post‐initiation feeding” of 2% bLF caused a significant reduction in the incidence (20% vs. 55%, P=0.02418) and multiplicity (0.25±0.54 vs. 0.70±0.71, P< 0.05) of tongue squamous cell carcinoma (by 64%, P=0.02418). bLF treatment elevated liver and tongue GST activities and liver QR activity. The “post‐initiation feeding' with 2% bLF significantly decreased QR activity, proliferating cell nulcear antigen‐positive index and ODC activity in the tongue. In addition, feeding with bLF decreased tongue polyamine content. These results suggest that bLF, when given at the 2% dose level during the post‐initiation phase, exerts chemopreventive action against tongue tumorigenesis through modification of cell proliferation activity and/or the activities of detoxifying enzymes.
Adenovirus‐mediated Gene Transduction of IkB or IkB Plus Bax Gene Drastically Enhances Tumor Necrosis Factor (TNF)‐induced Apoptosis in Human Gliomas
Authors: Shinoura Nobusada , Yamamoto Naoki , Yoshida Yoko , Fujita Takashi , Saito Nobuto , Asai Akio , Kirino Takaaki , Hamada Hirofumi
Published: August 2005

Tumor necrosis factor‐α (TNF), which was initially supposed to be a promising cancer therapeutic reagent, does not kill most types of cancer cells partly due to the activation of an anti‐apoptotic gene, NF‐kB. NF‐kB forms an inactive complex with the inhibitor kappa B alpha (IkBα), which is rapidly phosphorylated and degraded in response to various extracellular signals. To disrupt this protective mechanism, we introduced an inhibitor kappa B alpha (IkBdN) gene, a deletion mutant gene lacking the nucleotides for the N‐terminal 36 amino acids of IkBα, into human glioma cells (U251, T‐98G, and U‐373MG) via an adenoviral (Adv) vector in addition to treatment of the glioma cells with recombinant TNF. Immunohistochemical analysis revealed that NF‐kB was translocated to nuclei by TNF treatment in U251 and T‐98G cells, but not in U‐373MG cells. Neither transduction of IkBdN nor treatment with TNF protein alone induced apoptosis in U251 and T‐98G cells, whereas both cell lines underwent drastic TNF‐induced apoptosis after transduction of IkBdN. On the other hand, U‐373MG cells were refractory to TNF‐induced apoptosis even when they were transduced with the IkBdN gene. U‐373MG cells underwent drastically increased apoptosis when co‐transduced with the IkBdN and Bax gene in the presence of TNF. Adv‐mediated transfer of IkBdN or IkBdN plus Bax may be a promising therapeutic approach to treat gliomas through TNF‐mediated apoptosis.
Elevated Expression of Interleukins in Lung Adenocarcinomas Induced by N‐Nitrosobis(2‐hydroxypropyl)amine in Rats
Authors: Tsujiuchi Toshifumi , Sasaki Yasutaka , Tsutsumi Masahiro , Konishi Yoichi
Published: August 2005

The expression of interleukins (ILs) in lung adenocarcinomas induced by N‐nitrosobis(2‐hydroxypropyl)amine (BHP) in rats was investigated using a multiprobe RNase protection assay (RPA) followed by densitometric quantification. Male Wistar rats, 6 weeks old, were given 2000 ppm BHP in their drinking water for 12 weeks and maintained without further treatment until they were killed at week 25. Total RNAs were extracted from 14 individual adenocarcinomas and 2 specimens of normal lung tissue of untreated rats. In adenocarcinomas, elevated expression of IL‐1α (6/14), IL‐1β (14/14), IL‐3 (7/14), IL‐4 (11/14), IL‐5 (9/14), IL‐6 (11/14) and IL‐10 (8/14) was observed, compared with normal lung tissues. In contrast, no expression of IL‐2 was detected in any case. The results suggest that preferential expression of these ILs and their complex networks may contribute to the development and progression of lung adenocarcinomas induced by BHP in rats.
Birth Cohort Effects on Incidence of Lung Cancers: A Population‐based Study in Nagasaki, Japan
Authors: Soda Hiroshi , Oka Mikio , Soda Midori , Nakatomi Katsumi , Kawabata Shigeru , Suenaga Mitsuhiro , Kasai Takashi , Yamada Yasuaki , Kamihira Shimeru , Kohno Shigeru
Published: August 2005

Smoking prevalence remains high (around 60%) among Japanese males, but smoking initiation among males born in the 1930s decreased by approximately 10% due to economic difficulties following World War II. The present study was designed to examine whether this temporary decline in smoking initiation influenced the subsequent incidence of lung cancers, especially adenocarcinoma. Trends of lung cancer incidence by histological type in both sexes were investigated using data from the population‐based cancer registry in Nagasaki, Japan, from 1986 through 1995. During this period, 5668 males and 2309 females were diagnosed as having lung cancer, and the overall incidence of lung cancers among both sexes remained stable. However, males aged 55–59 years showed a decrease in the age‐specific incidence of adenocarcinoma and squamous‐cell carcinoma (P < 0.05 and P < 0.01, respectively). In birth cohort analyses, the incidence of adenocarcinoma and squamous‐cell carcinoma was lower in the 1935–1939 birth male cohort than in the successive cohorts. The incidence of lung cancers among females with low smoking prevalence did not change with birth cohort. The low smoking initiation among the 1935–1939 birth male cohort appeared to have resulted in a decreased incidence of adenocarcinoma and squamous cell carcinoma among middle‐aged Japanese males. The present study suggests that smoking prevention has an effect in reducing the incidence of lung adenocarcinoma, as well as squamous‐cell carcinoma, among smokers.
Increase in 8‐Hydroxyguanine and Its Repair Activity in the Esophagi of Rats Given Long‐term Ethanol and Nutrition‐deficient Diet
Authors: Asami Shinya , Hirano Takeshi , Yamaguchi Raizo , Tsurudome Yosuke , Itoh Hideaki , Kasai Hiroshi
Published: August 2005

Epidemiological studies have shown that an increased risk of esophageal cancer is associated with the chronic consumption of alcoholic beverages, although alcohol itself is not a carcinogen in animal models. Reactive oxygen species produced by the metabolism of ethanol or by chronic inflammation may play an important role in the carcinogenic process. In this study, we analyzed one type of oxidative DNA damage, 8‐hydroxyguanine (8‐OH‐Gua), and its repair activity in the esophagus as indicators of cellular oxidative stress in rats given long‐term ethanol and an autoclaved diet (nutrition‐deficient diet). Three‐week‐old male Sprague‐Dawley rats were fed an ethanol beverage whose concentration was increased from 12 to 70% over 20 weeks. When the concentration reached 50%, the diet of one group was changed from the regular diet to an autoclaved diet. At the feeding periods of 20, 25, 30, and 35 weeks, the rats were sacrificed and the 8‐OH‐Gua levels and repair activities within the esophagi were measured. After 30 weeks of ethanol‐ and autoclaved diet‐feeding, significant increases of 8‐OH‐Gua and its repair activity were observed in the esophagi, but not in those of the ethanol‐ and normal diet‐fed rats. This result indicates that the combined effects of long‐term ethanol consumption and nutritional deficiency may be involved in inducing oxidative stress in the rat esophagus.
Human Tissue Distribution of TA02, Which is Homologous with a New Type of Aspartic Proteinase, Napsin A
Authors: Hirano Takashi , Auer Gert , Maeda Masahiro , Hagiwara Yoshiaki , Okada Shinya , Ohira Tatsuo , Okuzawa Ken , Fujioka Kaoru , Franzén Bo , Hibi Nozomu , Seito Tsutomu , Ebihara Yoshiro , Kato Harubumi
Published: August 2005

The N‐terminal amino acid sequence of TA02 (molecular weight 35.0 kDa, isoelectric point 5.29), which is associated with primary lung adenocarcinoma, was determined and a fragment peptide was used to generate mouse monoclonal antibodies (mAbs) against TA02. The amino acid sequence suggested that TA02 might be homologous with napsin A, a new type of aspartic proteinase. In this context, we confirmed the expression of napsin A in primary lung adenocarcinoma using reversetranscription polymerare chain reaction (RT‐PCR) and showed that the TA02 mAbs reacted with glutathione‐S‐transferase (GST)‐napsin A fusion protein. We concluded that TA02 is the same molecule as napsin A, and showed immunohistochemically that it is distributed mainly in type II pneumocytes, alveolar macrophages, renal tubules and exocrine glands and ducts in the pancreas. In particular, type II pneumocytes and alveolar macrophages showed high expression of TA02 among human normal tissues. In primary lung adenocarcinoma, 47 out of 58 (81.0%) primary lesions were positive. All well‐differentiated adenocarcinomas except those of goblet cell type showed high expression of TA02. In addition, two out of seven (28.6%) large cell carcinomas showed low expression of TA02. The other histopathological types of primary lung cancer did not express TA02 at all. A few cases of renal cell cancer, pancreatic cancer, breast cancer, thyroid cancer, colon cancer and ovarian cancer showed low expression, but the staining patterns were completely different from that of primary lung adenocarcinoma, which showed a granular staining pattern. Our novel mAbs should be valuable for immunochemical detection of TA02/napsin A.
Trans‐regulated Silencing and Reactivation of TP53 Tumor Suppressor Gene in Malignant Transformation and Its Reversion
Authors: Yang Lichun , Sasaki Masao S.
Published: August 2005

Despite growing interest in the methylation‐mediated silencing of tumor suppressor genes in the neoplastic process, its signaling mechanism remains largely unknown. Here we show in a cultured murine cell line system that the silencing and reactivation of tumor suppressor gene TP53 were reversibly controlled by a trans‐acting regulatory mechanism. The gene product p53, which was constitutively expressed and activated upon X‐ray irradiation in non‐malignant parental cell line, was undetectable in its X‐ray‐induced malignant transformants, while they retained a wild‐type TP53. The silencing was cancelled by transferring a human chromosome 11 and the expression of p53 was restored. The non‐malignant revertants thus obtained were again susceptible to transformation by X‐irradiation, giving rise to re‐transformants, in which p53 was again repressed while the human chromosome 11 retained the ability to turn on TP53 when it was transferred into other malignant clone. The silent TP53 could be reactivated by treatment with the demethylating agent 5‐azadeoxycytidine. These observations indicate the presence of a trans‐acting signaling mechanism in the methylation‐mediated regulation of TP53 expression which is associated with the acquisition of malignancy.
Regulation of Dihydropyrimidine Dehydrogenase and Pyrimidine Nucleoside Phosphorylase Activities by Growth Factors and Subsequent Effects on 5‐Fluorouracil Sensitivity in Tumor Cells
Authors: Ueda Masatsugu , Kitaura Kozo , Kusada Osamu , Mochizuki Yoshino , Yamada Naomi , Terai Yoshito , Kumagai Koji , Ueki Ken , Ueki Minoru
Published: August 2005

Dihydropyrimidine dehydrogenase (DPD) and pyrimidine nucleoside phosphorylase (PyNPase) are the first and rate‐limiting enzymes that regulate 5‐fluorouracil (5‐FU) metabolism, and tumoral DPD activity appears to be a promising predictor of 5‐FU sensitivity. However, the regulatory mechanisms determining these enzyme activities have not been fully understood. We investigated the biological effects of epidermal growth factor (EGF) and transforming growth factor (TGF)α on cell growth and tumoral DPD and PyNPase activities, and the subsequent effects on 5‐FU sensitivity in uterine cervical carcinoma SKG‐IIIb cells. The treatment of tumor cells with EGF or TGF‐α resulted in a concentration‐dependent increase in tumor cell growth and PyNPase activity, whereas tumoral DPD activity was inhibited. Their stimulatory effects on tumor cell growth correlated well with PyNPase activity, but were inversely related to DPD activity (P<0.01). 5‐FU sensitivity of tumor cells increased in the presence of EGF or TGF‐α. These growth factors were shown to stimulate the first, rate‐limiting enzyme activity in 5‐FU anabolism and to inhibit that in 5‐FU catabolism, leading to enhancement of the antiproliferative action of 5‐FU at achievable therapeutic levels. The tumor environmental factors, EGF and TGF‐α, may act as intrinsic regulators of DPD and PyNPase activities that affect the 5‐FU sensitivity of individual tumors.
UCN‐01 (7‐Hydroxystaurosporine) Enhances 5‐Fluorouracil Cytotoxicity through Down‐regulation of Thymidylate Synthetase Messenger RNA
Authors: Abe Sadanori , Kubota Tetsuro , Otani Yoshihide , Furukawa Toshiharu , Watanabe Masahiko , Kumai Koichiro , Kitajima Masaki
Published: August 2005

UCN‐01 (7‐hydroxystaurosporine) is a newly developed cell cycle inhibitor known to have several modes of action, including inhibition of cyclin‐dependent kinase, induction of p21 and suppression of pRb phosphorylation. In order to test a combination therapy of UCN‐01 and 5‐fluorouracil (5‐FU), growth inhibition of CRL 1420 (MIA PaCa‐2; undifferentiated pancreatic carcinoma) by four different treatments was measured using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) assay. The treatments used were UCN‐01 alone, 5‐FU alone, 5‐FU followed by UCN‐01 (5‐FU/UCN‐01) and UCN‐01 followed by 5‐FU (UCN‐01/5‐FU). We also assessed changes in thymidylate synthetase (TS) mRNA levels, TS activity, and 5‐FU incorporation by RNA (FRNA) for each treatment. Although treatment with UCN‐01 alone, 5‐FU alone, and 5‐FU/UCN‐01 inhibited CRL 1420 growth in a concentration‐dependent manner, treatment with UCN‐01/5‐FU inhibited the growth of CRL 1420 synergistically at less than 1 μg/ml drug concentration. The down‐regulation of TS mRNA by UCN‐01 resulted in stable total TS and decreased free TS, and UCN‐01/5‐FU resulted in enhanced thymidylate synthetase inhibition rate (TSIR) compared to UCN‐01 alone and 5‐FU/UCN‐01. This increased TSIR due to UCN‐01 pretreatment was accompanied by elevated F‐RNA concentrations in the UCN‐01/5‐FU treatment. The suppression of TS mRNA and TS activity by UCN‐01 may lead to higher sensitivity of tumor cells to 5‐FU and may explain the synergistic antitumor effect of UCN‐01/5‐FU. In conclusion, low concentrations of UCN‐01 (from 0.01 to 1 μg/ml) may be clinically useful, affording low cytotoxicity of UCN‐01, while enhancing the antitumor effect of 5‐FU.
An Experimental Model of Tumor Dormancy Therapy for Advanced Head and Neck Carcinoma
Authors: Nishimura Goshi , Yanoma Shunsuke , Satake Kenichi , Ikeda Yoichi , Taguchi Takahide , Nakamura Yoshiyasu , Hirose Fumikazu , Tsukuda Mamoru
Published: August 2005

An experimental model of tumor dormancy therapy for advanced head and neck carcinoma was developed. After transplantation of KB cells into nude mice, the mice were given tiracoxib, a selective cyclooxygenase (COX)‐2 inhibitor, probucol, an antioxidant, and S‐1, an oral pro‐drug of 5‐fluorouracil (5‐FU), or combinations of two of them. The combined administration of tiracoxib with probucol significantly inhibited the tumor growth. The angiogenesis in this group was markedly reduced. Tiracoxib and probucol did not affect the intratumoral concentration of 5‐FU when coadministered with S‐1. The combined use of tiracoxib and probucol is thus a candidate for use in maintenance therapy after the primary therapy for patients with advanced head and neck carcinoma.
Enhanced Cell Killing by Overexpression of Dominant‐negative Phosphatidylinositol 3‐Kinase Subunit, Δp85, Following Genotoxic Stresses
Authors: Tachiiri Seiji , Sasai Keisuke , Oya Natsuo , Hiraoka Masahiro
Published: August 2005

Phosphatidylinositol 3‐kinase (PI3‐K) is a heterodimer of a regulatory subunit, p85, and a catalytic subunit, p110. A number of previous reports showed that PI3‐K functions in diverse cellular phenomena such as cell proliferation, glucose catabolism, cell adhesion, and vesicle transport. It is also well known that a survival signal from the receptor tyrosine kinases utilizes Akt via PI3‐K to protect cells from apoptosis. To examine the role of PI3‐K in cellular sensitivity to genotoxic stresses such as cisplatin and ultraviolet (UV), we introduced deletion type p85 (Δp85) into two human glioblastoma cell lines (T98G and A172) and one melanoma cell line (G361). The Δp85 works in a dominant‐negative fashion on PI3‐K activity by disrupting its p85/p110 interaction. In all three transfected cell lines, the overexpression of Δp85 rendered the cells markedly more sensitive to these DNA‐damaging stresses than the cells transfected with the vector alone. Apoptosis was vigorously induced in cells overexpressing Δp85 following the treatment. The present results imply that PI3‐K plays a critical role in determining cellular sensitivity to genotoxic stresses in human cancer cells, and that disruption of the p85/p110 interaction of PI3‐K may be a potential molecular target for developing a novel strategy for cancer treatment.
Molecular Cytogenetic Analysis of 17 Renal Cancer Cell Lines: Increased Copy Number at 5q31‐33 in Cell Lines from Nonpapillary Carcinomas
Authors: Yang Zeng‐Quan , Yoshida Mitsuaki A. , Fukuda Yoji , Kurihara Naoki , Nakamura Yusuke , Inazawa Johji
Published: August 2005

Comparative genomic hybridization (CGH) was used to screen for genomic imbalances in cell lines derived from 13 nonpapillary renal‐cell carcinomas (RCCs), two papillary RCCs, one renal squamous‐cell carcinoma, and one transitional‐cell carcinoma of the renal pelvis. Aberrations were found in all 17 lines. The most frequent changes in nonpapillary RCC cell lines were gains of 5q (85%), 7q (69%), 8q (69%) and 1q (54%) and losses of 3p (92%), 8p (77%), 4q (62%) and 14q (54%). High‐level gains (HLGs) were detected at 4q12, 5p, 5q23‐33, 7q22‐qter, 8q23‐24, 10q21‐qter, 12p and 12q13‐22. By means of fluorescence in situ hybridization (FISH) we narrowed the smallest common region involving 5q gains to the genomic segment between D5S642 and D5S673, and found that the HLG at 4q12 possibly involved amplifications of c‐kit and PDGFRA. Two papillary RCC cell lines showed gains of entire chromosomes 7, 12 and 17. The CGH data reported here should help to facilitate the choice of individual renal‐tumor cell lines for exploring target genes in regions of interest.
Characterization of High‐grade Neuroendocrine Tumors of the Lung in Relation to menin Mutations
Authors: Haruki Nobuhiro , Yatabe Yasushi , Travis William D. , Nomoto Shuji , Osada Hirotaka , Nakamura Shigeo , Nakao Akimasa , Fujii Yoshitaka , Takahashi Takashi
Published: August 2005

It has been suggested that mutations in the menin gene play a role in the development of multiple endocrine neoplasia type 1 (MEN1)‐associated and of sporadic forms of low‐ and intermediategrade neuroendocrine tumors of the lung. In the present study, eight tumor specimens of large cell neuroendocrine carcinoma (LCNEC) and 13 of small cell lung cancer (SCLC), which represent a high‐grade category of neuroendocrine tumors, were examined for the potential involvement of menin alterations as well as for the expression of various neuroendocrine markers and p53 and Rb abnormalities. All specimens expressed multiple neuroendocrine markers as expected and almost invariably carried p53 and Rb alterations. Unexpectedly, however, mutations in the menin gene were not detected in any of the high‐grade neuroendocrine tumors examined. We thus conclude that menin mutations do not play a crucial role in the pathogenesis of high‐grade subsets, in contrast to their suggested significant role in the development of low‐ and intermediate‐grade subsets. Interestingly, loss of heterozygosity (LOH) in the menin gene appeared to be more prevalent in LCNEC (50%) than in SCLC (22%), suggesting a possible distinction between SCLC and LCNEC.
Clinicopathological Significance of Expression of CD44 Variants in Head and Neck Squamous Cell Carcinoma
Authors: Kanke Minoru , Fujii Masato , Kameyama Kaori , Kanzaki Jin , Tokumaru Yutaka , Imanishi Yorihisa , Tomita Toshiki , Matsumura Yasuhiro
Published: August 2005

Splice variants of the cell surface glycoprotein CD44 have been reported to be associated with the progression of various human tumors. The aim of this study is to determine the correlation between the expression of CD44 isoforms, especially CD44 variant 2 (CD44v2), and the clinicopathological features of head and neck squamous cell carcinomas (HNSCCs). The expression of CD44 isoforms was evaluated immunohistochemically in paraffin‐embedded tissues from 89 primary lesions, using monoclonal antibodies against CD44 standard (CD44st), CD44 variant 6 (CD44v6) and CD44v2. Cancer tissues from 89 (100%), 85 (95.5%) and 59 (66.3%) patients showed positive immunoreactivity for CD44st, CD44v6 and CD44v2, respectively. A significant correlation was observed between the down‐regulation of CD44v2 and poorer differentiation of the tumor cells (P=0.02). We could not find any significant correlation between the expression of CD44v2 and T stage or N stage (lymph node status). However, the rate of positive cervical lymph node metastasis tended to increase with reduced expression of CD44v2 (P=0.08). Down‐regulation of CD44v2 expression was correlated with shorter overall survival (P=0.01). Furthermore, Cox's multivariate analysis revealed that only CD44v2 expression and lymph node status were independent prognostic factors. These findings suggest that down‐regulation of CD44v2 expression may be one of the biological markers for the degree of malignancy in HNSCCs.