bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of p53 Gene Mutations in Aspiration Biopsy Specimens from Suspected Breast Cancers by Polymerase Chain Reaction‐Single Strand Conformation Polymorphism Analysis
Authors: Sato Takashi , Okazaki Akira , Okazaki Minoru , Takahashi Shuji , Hirata Koichi
Published: August 2005

Genomic DNA was extracted from aspiration biopsy specimens taken from 15 suspected cases of breast cancer, including 7 known cases of breast cancer, and the p53 gene was studied for evidence of mutation by using a polymerase chain reaction‐single strand conformation polymorphism (PCR‐SSCP) analysis. In 5 of the 15’cases (33%), p53 gene mutation was identified and these tumors were subsequently histologically diagnosed as malignant. Further, DNA flow cytometry of the 15 tumors demonstrated that 6 (40%) were aneuploid and malignant, whereas 9 (60%) were diploid and benign. It was also found that the tumor cells in 5 aspirated cases that showed p53 gene mutations were all aneuploid, the p53 protein expression was positive, and the tumors were proved to be histologically malignant. It was thus concluded that the detection of p53 gene mutation by PCR‐SSCP analysis of aspirated biopsy specimens from suspected breast cancers is a helpful method for achieving a more accurate diagnosis.
A Possible Role of 92 kDa Type IV Collagenase in the Extramedullary Tumor Formation in Leukemia
Authors: Kobayashi Masanobu , Hamada Jun‐ichi , Li Young‐Quing , Shinobu Noriaki , Imamura Masahiro , Okada Futoshi , Takeichi Noritoshi , Hosokawa Masuo
Published: August 2005

Production of metalloproteinases such as collagenases has been reported to be involved in the metastasis of cancer cells. Granulocytic sarcoma in extramedullary sites can be formed by similar steps to other cancers. In this study, we have examined the secretion of type IV collagenases and a tissue inhibitor of metalloproteinase‐1 (TIMP‐1) in several human leukemia cell lines, including a granulocytic sarcoma‐derived cell line established from a patient with granulocytic sarcomas in dermal tissues. We have also examined the invasive capacity of these leukemia cell lines into reconstituted basement membrane, Matrigel, which was used for in vitro invasion assay. Among the human leukemia cell lines used in this study, only the granulocytic sarcoma cell line was found to secrete type IV collagenase constitutively. Other myeloid leukemia cell lines such as HL‐60 and U‐937 produced type IV collagenase only after treatment with 12‐O‐tetradecanoylphorbol‐13‐acetate. All the cell lines secreted similar amounts of the tissue inhibitor of metalloproteinases. In vitro invasion assay revealed that the granulocytic sarcoma cell line showed higher invasive capacity than the other cell lines. These results suggest that the secretion of 92 kDa type IV collagenase plays a role in the leukemia cells’ invasion of extramedullary tissues.
Augmentation of Cancer Chemotherapy by Preinjection of Human Macrophage Colony‐stimulating Factor in L1210 Leukemic Cell‐inoculated Mice
Authors: Douzono Miyuki , Suzu Shinya , Yamada Muneo , Yanai Nobuya , Kawashima Takuji , Hatake Kiyohiko , Motoyoshi Kazuo
Published: August 2005

Human macrophage colony‐stimulating factor (hM‐CSF) is a potent stimulator of the effector functions of monocytes/macrophages. We investigated the antitumor effects of this factor in CDF1 male mice inoculated with L1210 cells, a mouse B‐cell leukemia line. Mice preinoculated with various numbers of L1210 cells on day 0 were given intravenous injections of vehicle (human serum albumin; HSA) (100μg/kg/day) or hM‐CSF (20μg/kg/day) for 3 days from day 1. In mice preinoculated with 102 L1210 cells but not with 103 or more L1210 cells, a marked increment in survival rate was observed with hM‐CSF treatment. We next examined the effect of hM‐CSF treatment combined with chemotherapy on the survival of mice that had been preinoculated with 105 L1210 cells. In our system, the administration of 4.9 mg/kg adriamycin (ADM) alone slightly prolonged survival of the tumorbearing mice, but all of the mice died within 20 days. When hM‐CSF was injected for 3 days before this ADM treatment, the invasion and proliferation of tumor cells in the liver and spleen were markedly inhibited and 50% of the mice were still alive at day 50. We detected inhibitory activity toward L1210 growth in serum of mice administered with hM‐CSF, and the degree of the inhibitory activity was correlated with the level of nitrite (NO2) in the serum. When L1210 cells were co‐cultured with peritoneal macrophages from mice intraperitoneally injected with hM‐CSF, the uptake of [3H]thymidine in L1210 cells was inhibited. The inhibition was abolished by the addition of NG‐monomethyI‐L‐arginine, an inhibitor of NO2− synthesis, suggesting that the reactive nitrogen oxide intermediate is involved in hM‐CSF‐induced inhibition of L1210 growth.
Detection of Homing, Proliferation, and Infiltration Sites of Adult T Cell Leukemia Cells in Severe Combined Immunodeficiency Mice Using Radiometric Techniques
Authors: Takaori‐Kondo Akifumi , Hosono Makoto , Imada Kazunori , Yao Zheng‐Sheng , Sakahara Harumi , Yamabe Hirohiko , Konishi Junji , Okuma Minoru , Uchiyama Takashi
Published: August 2005

To clarify the mechanism of in vivo proliferation of adult T cell leukemia (ATL) cells, we examined the organ distribution of ATL‐43T cell line cells derived from original leukemic cells in severe combined immunodeficiency (SCID) mice using radiometric techniques. First, we injected 111In‐oxine‐labeled ATL‐43T cells into SCID and CB17 mice. On day 6, significant accumulation of radioactivity was found in the spleen and thymus of SCID mice (33.3±9.4 and 10.0±3.6 % injected dose/g of tissue [%ID/g], respectively) in comparison with that in CB17 mice (19.1±2.5 and 3.7±0.9 %ID/g, respectively). Next, we injected radiolabeled anti‐Tac monoclonal antibody (MoAb) recognizing human interleukin‐2 receptor (IL‐2R) α chain or isotype‐matched control MoAb RPC5 in SCID mice bearing ATL‐43T cells 4 weeks after cell inoculation. The amounts of radioactivity found in the spleen and thymus of SCID mice injected with 125I‐labeled anti‐Tac MoAb (22.5±6.9 and 22.8±9.6 %ID/g, respectively) were significantly higher than those in the corresponding organs of SCID mice injected with 125I‐labeled RPC5 MoAb (12.0±5.1 and 7.5±4.6 %ID/g, respectively). Similar results were obtained with 111In‐labeled anti‐Tac MoAb. These results were consistent with the histological findings of SCID mice bearing ATL‐43T cells, indicating that ATL‐43T cells infiltrated preferentially into the lymphoid organs, such as the spleen and thymus, and proliferated there. Thus, the radiometric techniques employed in this study were very useful to evaluate the proliferation sites of ATL‐43T cells in SCID mice. Furthermore, this murine model could give us an opportunity to test the feasibility of therapeutic application of radiolabeled anti‐Tac MoAb.
Frequent Genetic Instability in Small Intestinal Carcinomas
Authors: Hibi Kenji , Kondo Ken , Akiyama Seiji , Ito Katsuki , Takagi Hiroshi
Published: August 2005

To determine whether genetic instability plays a part in the development of digestive tract carcinomas, we analyzed 3 microsatellite loci isolated from tumors and surrounding normal tissue samples obtained during surgery. The polymerase chain reaction (PCR) technique was used to assess differences between tumor and matched normal DNAs. Replication errors (RERs) were observed in 3 of the 29 cases (10%) of gastric carcinoma and in 11 of the 72 cases (15%) of colorectal carcinoma. None of the 13 (0%) esophageal carcinoma cases showed any RER, but 5 of the 11 cases of small intestinal carcinoma (45%) had RERs, a significantly frequent finding. These results suggest that genetic instability plays an important role in the pathogenesis of small intestinal carcinomas.
Ubenimex Activates the E‐Cadherin‐mediated Adhesion of a Breast Cancer Cell Line YMB‐S
Authors: Fujioka Seiji , Kohno Nobuoki , Hiwada Kunio
Published: August 2005

It has been reported that ubenimex, a biological response modifier, has a direct anti‐tumor effect. To clarify the mechanism involved, we examined the effects of ubenimex on the growth and adhesive property of a breast cancer cell line YMB‐S. The cells proliferate in a floating manner without aggregation in normal complete medium. Ubenimex induced cell‐cell and cell‐surface adhesion of the cells accompanied with growth suppression. E‐Cadherin localized at cell‐cell contact sites of adhered cells, and anti‐E‐cadherin antibody inhibited the adhesion. Both Western blot analysis and binding assay disclosed that there was no apparent difference between E‐cadherin levels of the cells before and after the treatment with ubenimex. These results indicate that ubenimex inhibits the proliferation of YMB‐S cells and augments cell‐to‐cell adhesion through the induction of E‐cadherin ‐mediated adhesion resulting from the functional activation of pre‐expressed but inefficient E‐cadherin.
Pharmacokinetics of All‐trans Retinoic Acid in Pediatric Patients with Leukemia
Authors: Takitani Kimitaka , Tamai Hiroshi , Morinobu Takao , Kawamura Naohisa , Miyake Munenori , Fujimoto Takeo , Mino Makoto
Published: August 2005

Since all‐trans retinoic acid (ATRA) induces complete remission in a high proportion of patients with acute promyelocytic leukemia (APL), and its effectiveness appears to be related to the plasma or serum level, a pharmacokinetic study of ATRA was undertaken in nine patients with various leukemias. After oral administration at a dose of 30 mg/m2, the time required to reach the peak plasma level of ATRA (20–1198 ng/ml) was between 120 and 240 min and the apparent plasma elimination half life was 21–51 min. In addition, 13‐cis retinoic acid was detected in the plasma of seven patients, indicating the occurrence of ATRA isomerization in vivo. ATRA therapy did not induce complete remission in all patients, even when high plasma levels were achieved. Among the six APL patients given ATRA therapy, one who failed to respond had a very low plasma ATRA level. These findings suggest that it may be useful to monitor plasma levels during oral ATRA therapy in order to achieve an appropriate treatment regimen.
Subchromosomal Mapping of a Putative Transformation Suppressor Gene on Human Chromosome 1
Authors: Horikawa Izumi , Yamada Hideto , Kugoh Hiroyuki , Yuasa Yoshihiro , Suzuki Mikio , Oshimura Mitsuo
Published: August 2005

We previously reported that the introduction of a normal human chromosome 1 via microcell‐mediated chromosome transfer suppressed the transformed phenotypes, including anchorage‐independent growth, of Kirsten murine sarcoma virus‐transformed NIH3T3 (DT) cells. Soft‐agar clones derived from DT‐#1 cells (DT cells with an intact transferred human chromosome 1) exclusively failed to retain an intact form of this chromosome. Thus, a gene(s) with a suppressive activity on this chromosome had probably been lost. We therefore attempted to identify a commonly deleted region on human chromosome 1 in these soft‐agar clones. Although eight of the 9 soft‐agar clones examined still contained regions on this chromosome, to a greater or lesser degree, four loci on 1q21 and 1q23‐q24 were commonly lost in all of them. Furthermore, the soft‐agar clones had growth properties similar to those of DT cells. Thus, chromosome and DNA analyses suggested that human 1q21 and/or 1q23‐q24 carries a transformation suppressor gene(s) which controls the transformed phenotypes of DT cells.
Direct Transplantation of Chronic Myelogenous Leukemia Cells into Nude Mice and Establishment of a Leukemic Stem Cell (Ph1+, CD34+) Line Dependent on Mouse Bone Marrow Stromal Cells in vitro
Authors: Kubonishi Ichiro , Takeuchi Seisho , Uemura Yoshiki , Kamioka Mikio , Asahi Yasutomo , Ohtsuki Yuji , Miyoshi Isao
Published: August 2005

Peripheral blood cells from a female patient with Ph1‐positive chronic myelogenous leukemia (CML) in blast crisis were serially transplanted in BALB/c nude mice for 16 passages. This in vivo cell line, designated CML‐N‐1, had Ph1chromosome abnormality and BCR gene rearrangement. The cells expressed CD11b, CD13, CD33, CD34, CD38, and HLA‐DR antigens until the 11th passage and subcutaneous tumors produced by these passages were composed of admixtures of immature and maturing cells that differentiated to basophils when cultured in vitro. From the 12th passage on, the tumors became composed mainly of immature cells expressing CD13, CD34, and HLA‐DR, and no longer differentiated to basophils even upon in vitro culture. In contrast to the vigorous proliferation in vivo, CML‐N‐1 cells from any passage failed to proliferate in vitro under standard liquid culture conditions with or without growth factors, such as granulocyte‐macrophage colony‐stimulating factor, granulocyte colony‐stimulating factor, monocyte colony‐stimulating factor, interleukin 3, interleukin 6 and stem cell factor. However, a continuously growing cell line, designated CML‐C‐1, was established by culturing CML‐N‐1 cells on feeder layers of mouse bone marrow stromal cells. This mouse bone marrow stromal cell‐dependent cell line showed immature cell morphology and expressed early myeloid phenotype positive for CD13, CD34, and HLA‐DR. These results indicate that mouse bone marrow stromal cells provide a certain growth factor(s) active on human leukemia cells.
Establishment and Characterization of Non‐small Cell Lung Cancer Cell Lines Resistant to Mitomycin C under Aerobic Conditions
Authors: Shibata Kazuhiko , Kasahara Kazuo , Bando Takuma , Nakatsumi Yasuto , Fujimura Masaki , Tsuruo Takashi , Matsuda Tamotsu
Published: August 2005

To elucidate the mechanisms of acquired resistance to mitomycin C (MMC) in non‐small cell lung cancer (NSCLC), we established two MMC‐resistant NSCLC sublines by continuous exposure to MMC, using PC‐9 as a parent cell line. The sublines, PC‐9/MC2 and PC‐9/MC4, were 6.4‐ and 10‐fold more resistant to MMC than their parent cell line, respectively, at the IC50 value as determined by MTT assay. They exhibited cross‐resistance to EO9, but were not resistant to cisplatin, vindesine, etoposide, carboquone, or KW‐2149, a novel MMC derivative. They were collaterally sensitive to adriamycin and menadione. Accumulation of the drug was decreased in the resistant sublines to about 60% of that in the parent cells. Cytosolic DT‐diaphorase (DTD) activities were decreased to 13.5±3.2 in PC9/MC2 and 1.3±0.6 in PC‐9/MC4 from 261.5±92.7 nmol/min/mg protein in the parent PC‐9. NADH:cytochrome b5 reductase activities in both of the resistant cell lines were significantly decreased as compared to that in the parent cell line. Addition of dicumarol resulted in a two‐fold increase in IC50 value in PC‐9, whereas the IC50 value showed no change in PC‐9/MC4. Moreover, dicumarol did not affect the sensitivities to KW‐2149 but decreased the sensitivities to EO9 in both the parent and the resistant cell lines. Formation of an alkylating metabolite was significantly decreased in the resistant cells, in parallel to the degree of resistance. We concluded that deficient drug activation due to decreased DTD activity was important as a mechanism of resistance to MMC in PC‐9, a relatively DTD‐rich NSCLC cell line.
Functional and T Cell Receptor Gene Usage Analysis of Cytotoxic T Lymphocytes in Fresh Tumor‐infiltrating Lymphocytes from Human Head and Neck Cancer
Authors: Chikamatsu Kazuaki , Eura Masao , Nakano Koji , Masuyama Keisuke , Ishikawa Takeru
Published: August 2005

Twenty‐one cytotoxic T lymphocyte (CTL) clones or lines that killed autologous tumor cells, but not allogeneic tumor, K562, or Daudi cells, were established from fresh tumor‐infiltrating lymphocytes of two individuals (HP‐1 and HP‐2) with head and neck cancer by limiting dilution in the presence of recombinant interleukin‐2. Sixteen (76%) of these 21 clones or lines comprised CD4+ CTLs and the other five comprised CD8+ CTLs. These observations suggest that autologous tumor cell‐specific CD4+ CD8− and CD4− CD8+ CTLs are present in vivo at the tumor site in head and neck cancer. Analysis of T cell receptor (TCR) gene arrangements in 20 of the 21 CTL isolates with reverse transcriptase and the polymerase chain reaction revealed that five of 12 and five of eight isolates from HP‐1 and HP‐2, respectively, were clones, the other isolates being lines comprised of two or more clones. Each CTL clone showed a different combination of Vα and Vβ gene expression, suggesting that more than five different tumor‐associated antigens may be expressed on head and neck cancer cells. In spite of the diversity of TCR αβ combinations, TCR Vα1, Vα3, Vα8, Vα10, Vβ8, and Vβ9, and Vβ17 were also frequently expressed in both patients. These data suggest that specific CTLs proliferate oligoclonally and contribute to the specific immune response against head and neck cancer in vivo.
Phase I Study and Clinical Pharmacological Evaluation of Daily Oral Etoposide Combined with Carboplatin in Patients with Lung Cancer
Authors: Ohune Terumasa , Fujiwara Yasuhiro , Sumiyoshi Hidetaka , Yamaoka Naoki , Yamakido Michio
Published: August 2005

Twenty‐eight patients with inoperable or relapsed lung cancer were given a combination of oral etoposide, administered once a day at doses ranging from 40 to 60 mg/m2/day (d) for 21 consecutive days, and carboplatin, administered intravenously over 1 h at doses ranging from 300 to 400 mg/m2 on day 1 to determine the appropriate doses of this combination. In addition, pharmacokinetic and pharmacodynamic analyses were performed. All the patients had a performance status of 0 to 1. Serum etoposide and free platinum (Pt) concentrations were measured using high‐performance liquid chromatography and atomic absorption, respectively. Myelosuppression, nausea and vomiting were the dose‐limiting toxicities of this schedule. The maximum tolerated dose (MTD) was 50 mg/m2/d oral etoposide for 21 days and 400 mg/m2 i.v. carboplatin on day 1. For heavily pretreated patients, the MTD was 40 mg/m2/d oral etoposide for 21 days and 350 mg/m2 i.v. carboplatin on day 1. No cumulative increase in the area under the concentration‐time curve (AUC) for oral etoposide over time was observed. There were significant correlations between the free Pt serum level (6, 8, 12, 24 h post‐dose) and etoposide AUC level (days 1, 10 and 21) for graded hematological toxicity, and the percentage decreases and nadir counts of hemoglobin, leukocytes, neutrophils and platelets. Several pharmacodynamic models were developed to predict the hematological toxicity. In order to facilitate pharmacodynamic evaluations in future studies, a limited sampling model for oral etoposide was also developed and validated.
Anti‐tumor Activity of Arginine Deiminase from Mycoplasma arginini and Its Growth‐inhibitory Mechanism
Authors: Takaku Haruo , Matsumoto Mitsuhiro , Misawa Satoru , Miyazaki Kaoru
Published: August 2005

Two kinds of arginine deiminase (AD, EC 3.5.3.6) were purified from cell extracts of Mycoplasma arginini (a‐AD) and Mycoplasma hominis (h‐AD), and their enzymic properties and anti‐tumor activities were compared. The a‐AD enzyme strongly inhibited the growth of mouse hepatoma cell line MH134 in vitro, and its concentration required for 50% growth inhibition (IC50) was estimated to be about 10 ng/ml. The IC50 value of h‐AD against the same cell line was estimated to be ahout 100 ng/ml, due to its low enzyme activity under the physiological pH condition, i.e., pH 7.4. These results show that the reaction pH profile of the a‐AD was superior to that of the h‐AD as an anti‐tumor enzyme. Moreover, the effects of l‐arginine metabolism‐related substances on the anti‐tumor activity of the a‐AD were examined to study the growth‐inhibitory mechanism of this enzyme. The addition of 2 or 4 mMl‐arginine restored, in a dose‐dependent manner, the growth of mouse MH134 hepatoma and Meth A fibrosarcoma cell lines that had been inhibited by 20 ng/ml of the a‐AD. The addition of 2 or 4 mMl‐ornithine, which is biosynthesized from l‐arginine in the urea cycle and is the starting material in the polyamine‐biosynthesis pathway, also partially restored it in a dose‐dependent manner. These results indicate that the tumor cell growth inhibition caused by a‐AD originates from the depletion of the essential nutrient l‐arginine, and that the resulting block of the polyamine‐biosynthesis pathway is involved in part in the inhibitory mechanism.
Double Determinant Immuno‐Polymerase Chain Reaction: A Sensitive Method for Detecting Circulating Antigens in Human Sera
Authors: Suzuki Asako , Itoh Fumio , Hinoda Yuji , Imai Kohzoh
Published: August 2005

A sensitive method for the detection of antigens in sera, termed double determinant immunopolymerase chain reaction (double determinant immuno‐PCR) was developed, using two monoclonal antibodies (MoAbs), in which the antigens are sandwiched, and a specific DNA molecule is used as a marker. Instead of the antigen itself, the first MoAb to bind the circulating antigens was immobilized. After the biotinylated second MoAb was bound to the antigen, free streptavidin was used to attach a biotinylated DNA to the biotinylated second MoAb. The biotinylated DNA complexed with antigen‐antibody‐streptavidin was amplified by PCR, and the PCR products were analyzed by Southern blot hybridization after agarose gel electrophoresis. Compared with the conventional enzyme linked immunosorbent assay (ELISA) using soluble intercellular adhesion molecule‐1 (sICAM‐1) in the supernatant of cultured Panc‐1 cells as an antigen, our double determinant immuno‐PCR was 103 times more sensitive in terms of the detection limit. Not only in culture medium, but also in sera from gastric cancer patients of high sICAM‐1 titer, an approximately 103‐fold enhancement in detection sensitivity was obtained compared with ELISA. In addition, this system can detect the antigen in sera at a level below the detection limit of traditional ELISA methods with high sensitivity. Thus, double determinant immuno‐PCR has the significant advantage that it can be readily applied to any antigen‐antibody system for which two MoAbs are available.
The Relationship between Aryl Hydrocarbon Hydroxylase and Polymorphisms of the CYP1A1 Gene
Authors: Kiyohara Chikako , Hirohata Tomio , Inutsuka Satoru
Published: August 2005

We examined the relationship between aryl hydrocarbon hydroxylase (AHH) and the frequency of a Msp I mutation in the 3′‐flanking region of cytochrome P450 (CYP) 1A1 (Mspl polymorphism) and another mutation in exon 7 (Ile‐Val polymorphism) in 84 healthy male subjects in Fukuoka, Japan. AHH inducibility (3‐methylcholanthrene (MC)‐induced AHH activity/non‐induced AHH activity) was correlated with the MspI polymorphism (P<0.0001) and age class (P=0.015), whereas no correlation was found for the Ile‐Val polymorphism (P=0.509). Age‐adjusted AHH inducibility (mean±SE) of the predominant homozygote (genotype A), the heterozygote (genotype B) and a homozygote rare allele (genotype C) genotypes was 4.89±0.36, 4.82±0.29 and 13.61±1.44, respectively. The genotype C showed much higher AHH inducibility than genotypes A and B (P<0.001), while no significant difference was observed between genotypes A and B. Non‐induced AHH activity was also correlated with these polymorphisms. The AHH activity of a homozygous mutant Val/Val genotype (0.076±0.010 pmol/min/106 cells) was significantly higher (P<0.05) than that of the wild‐type homozygous Ile/Ile (0.044±0.004 pmol/min/106 cells) and heterozygous Ile/Val (0.047±0.007 pmol/min/106 cells) genotypes. Our study suggests that the genotypes C and Val/Val, which are more frequent in smoking‐related lung cancer, are closely related with high AHH inducibility and high non‐induced AHH activity, respectively. Thus, the positive relationship between AHH inducibility and lung cancer is supported by our study. If our results are confirmed and the assessment of genotype becomes feasible on a population basis, identification of smokers who have genetically high susceptibility to lung cancer (genotype C or Val/Val) may become important for the prevention of lung cancer.
Hepatocellular Carcinoma Induction in LEC Rats by a Low Dose of 2‐Amino‐3,8‐dimethylimidazo[4,5‐f]quinoxaline
Authors: Sone Hideko , Wakabayashi Keiji , Kushida Hiromi , Enomoto Katsuhiko , Mori Michio , Takeichi Noritoshi , Tsuda Hiroyuki , Sugimura Takashi , Nagao Minako
Published: August 2005

A food‐borne heterocyclic amine, 2‐amino‐3,8‐dimethylimidazo[4,5‐f]qninoxaline (MeIQx), induces hepatocellular carcinomas (HCCs) in F344 male rats at an incidence of 95%, when fed in the diet at 400 ppm for 61 weeks. In this study, the effect of a low dose of MeIQx was examined in Long‐Evans with cinnamon‐like coat color (LEC) rats, which have a mutation in Atp7b and suffer from hereditary hepatitis and HCCs, with high levels of copper accumulation in the liver. Rats of the LEC and Long‐Evans with agouti coat color (LEA) sibling lines were given a diet containing 40 ppm MeIQx from the age of 23 weeks to 63 weeks, for a total administration period of 40 weeks. In LEC rats, HCCs were observed in 8/8 animals administered MeIQx, and 2/8 rats receiving a normal diet. The number of HCCs per rat (mean±SD) was 2.8±2.0 and 0.3±0.5, respectively. In the LEA rats, however, no tumors were induced by administration of MeIQx. These results indicate that damaged liver associated with compensatory cell proliferation is much more susceptible to chemical hepatocarcinogens, including MeIQx, than the normal liver.
Heparin‐binding Epidermal Growth Factor‐like Growth Factor: p91 Activation, Induction of Plasminogen Activator/Plasminogen Activator Inhibitor, and Tubular Morphogenesis in Human Microvascular Endothelial Cells
Authors: Ushiro Shin , Ono Mayumi , Izumi Hiroto , Kohno Kimitoshi , Taniguchi Naoyuki , Higashiyama Shigeki , Kuwano Michihiko
Published: August 2005

Epidermal growth factor (EGF) or transforming growth factor‐α (TGF‐α) stimulates cell migration, proliferation and the formation of tube‐like structures of human microvascular endothelial cells in culture. Heparin‐binding EGF‐like growth factor (HB‐EGF), which shows 35% homology with EGF/ TGF‐α, is a member of the EGF family, and it is ubiquitous in many tissues and organs. We examined whether or not HB‐EGF induced angiogenic responses in human microvascular endothelial cells. HB‐EGF inhibited the binding of 125I‐EGF to the EGF receptor and induced autophosphorylation of the receptor on endothelial cells. Exogenous HB‐EGF induced the loss of more than 70% of the EGF receptor from the cell surface within 30 min, with similar kinetics to that of EGF. The level of c‐fos mRNA markedly increased at 30 min in response to HB‐EGF as well as EGF. A gel shift assay demonstrated the activation of the transcription factor p91 by HB‐EGF and EGF. This factor directly interacts with the EGF receptor and mediates the activation of c‐fos gene promoter. HB‐EGF enhanced the mRNA expression of tissue‐type plasminogen activator (t‐PA) and plasminogen activator inhlbitor‐1 (PAI‐1) mRNA. However, the enhancement of t‐PA and PAI‐1 by HB‐EGF was less than that by EGF. Heparitinase/chlorate, which digests the heparan sulfate proteoglycan of the endothelial cell surface, restored both t‐PA and PAI‐1 mRNA levels in response to HB‐EGF in a manner similar to that by EGF. HB‐EGF at 10 ng/ml developed tube‐like structures in type I collagen gel at similar levels to that of EGF at 10 ng/ml, suggesting that HB‐EGF is also a potent angiogenic factor in the model system for angiogenesis. The tubulogenesis activity of HB‐EGF is discussed in relation to the expression of the t‐PA and PAI‐1 genes.
Leukocyte Function‐associated Antigen‐1‐dependent Adhesion of Rat Ascites Hepatoma AH66F to Mesentery‐derived Mesothelial Cells
Authors: Nomura Masaaki , Yamamoto Hideo , Sugiura Norihiko , Kuroda Kazunari , Kawaguchi Hiroki , Miyamoto Ken‐ichi
Published: August 2005

Rat ascites hepatoma AH66F cells adhered better than AH130 cells to mesentery‐derived mesothelial cells (M‐cells), though both cells secreted M, 92,000 matrix metalloproteinase on a gelatin zymogram with similar activity. AH66F cells expressed leukocyte function‐associated antigen‐1 (LFA‐1) and intercellular adhesion molecule‐1 (ICAM‐1), a ligand of LFA‐1, on the cell surface, while AH130 cells had ICAM‐1 alone. The adhesion to M‐cells of AH66F cells was inhibited to the adhesion level of AH130 cells by anti‐rat LFA‐1 α‐ and/or β‐chain monoclonal antibody (mAb) and also by anti‐rat ICAM‐1 mAb. This is the first report to show the LFA‐1‐dependent adhesion of cells other than leukocytes, because AH66F cells did not express CD45, T cell‐αβ receptor or CD11b/c (Mac‐1/p150,95). These results indicate that a part of the adhesion of AH66F cells to M‐cells is due to LFA‐1/ICAM‐1 interaction, and we suggest that this characteristic feature of AH66F cells may be related to the malignant properties.
Association between Family History and Gastric Carcinoma among Young Adults
Authors: Kikuchi Shogo , Nakajima Toshifusa , Nishi Tsunehiro , Kobayashi Osamu , Konishi Toshiro , Inaba Yutaka , Wada Osamu , Satou Hiroshi , Ishibashi Teruo , Ichikawa Shoichi , Okamoto Naoyuki , Hirata Toru , Kubo Takuji , Sato Nobuhiro , Miki Kazumasa , Myoga Akio
Published: August 2005

The relationship between family history of gastric carcinoma and gastric carcinoma in Japanese under 40 years of age was analyzed. The subjects were 108 gastric carcinoma patients (86% were diffuse type) at 9 hospitals in the Kanto area of Japan. Firstly, incidence of gastric carcinoma among the parents of the subjects were compared with that in the general population. Observed/expected (O/E) ratios (P‐value) were 1.8 (0.06) for all subjects, 1.3 (0.62) for male subjects, 2.1 (0.04) for female subjects, 0.5 (0.41) for early carcinoma, 2.6 (P<0.01) for advanced carcinoma, 2.3 (0.22) for intestinal‐type carcinoma and 1.7 (0.13) for diffuse‐type carcinoma. Association between gastric carcinoma and parents’ history of gastric carcinoma was strong among women and regarding advanced carcinoma, and the difference in O/E ratios between early and advanced carcinoma was remarkable. Secondly, factors related to advanced‐stage gastric carcinoma were analyzed. Histological type (diffuse and intestinal types) was not related, but family history of gastric carcinoma among parents and grandparents was related to advanced stage, and the relationship was independent of other factors. The odds ratio (95% confidence interval) was 3.3 (1.1–9.9). Family history may be related to stage of gastric carcinoma through its relationship to the manner or speed of the tumor's progression. We hypothesis that some genetic factor exists which is involved both in progression from early to advanced stage and in occurrence of gastric carcinoma.
Inhibition of in vitro Tumor Cell Invasion by Ginsenoside Rg3
Authors: Shinkai Kiyoko , Akedo Hitoshi , Mukai Mutsuko , Imamura Fumio , Isoai Atsushi , Kobayashi Motomasa , Kitagawa Isao
Published: August 2005

The effect of plant glycosides on tumor cell invasion was examined. Among the glycosides tested, ginsenoside Rgs was found to be a potent inhibitor of invasion by rat ascites hepatoma cells (MM1), B16FE7 melanoma cells, human small cell lung carcinoma (OC10), and human pancreatic adenocarcinoma (PSN‐1) cells, when examined in a cell monolayer invasion model. Structurally analogous ginsenosides, Rb2, 20(R)‐ginsenoside Rg2 and 20(S)‐ginsenoside Rg3 (a stereoisomer of Rg3), showed little inhibitory activity. Neither Rh1, Rh2, 20(R)‐ginsenosides Rh1 Rb1, Rc nor Re had any effect. The effective ginsenoside, Rg3, tended to inhibit experimental pulmonary metastasis by highly metastatic mouse melanoma B16FE7 cells as well. Taking account of our previous finding that 1‐oleoyl‐lysophosphatidic acid (LPA) induced invasion by MM1 cells in the monolayer invasion model, the effect of Rg3 on molecular events associated with the invasion induced by LPA was analyzed in order to understand the mechanism of the inhibition. Rg3, which suppressed the invasion induced by LPA, dose‐dependently inhibited the LPA‐triggered rise of intracellular Ca2+. Protein tyrosine phosphorylation triggered by LPA was not inhibited by Rg3.
A Novel Monoclonal Antibody Specific for Sialylated MUC1 Mucin
Authors: Yamamoto Masaya , Bhavanandan V. P. , Nakamori Shoji , Irimura Tatsuro
Published: August 2005

Development of a new monoclonal antibody (mAb) MY.1E12 which reacts with sialylated MUC1 mucins is described. The mAb did not react with any component in the lysates of COS‐1 cells, whereas it bound to sialylated MUC1 mucins produced by COS‐1 cells transiently transacted with MUC1 mucin cDNA, strongly suggesting that the expression of the epitope of mAb MY.1E12 depends on the presence of the MUC1 mucin core peptide. The requirement of sialyl residues for antibody recognition was established by Western blotting analysis of extracts of various carcinoma cells and in situ desialylation. In all cases, the mAb binding of electrophoretically separated MUC1 mucin diminished after desialylation by mild acid hydrolysis. When Capan‐1 pancreatic carcinoma cells were pretreated with benzyl‐JV‐acetylgalactosaminide in culture, the MUC1 mucins produced under these conditions, which were detected by core peptide‐specific mAbs, did not react with mAb MY.1E12. These results suggest that 0‐linked carbohydrate chains are important for the mAb binding.
Production of Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases in Human Breast Carcinomas
Authors: Iwata Hiroji , Kobayashi Shunzo , Iwase Hirotaka , Masaoka Akira , Fujimoto Noboru , Okada Yasunori
Published: August 2005

We examined production and tissue localization of matrix metalloproteinase (MMP)‐1 (tissue collagenase), MMP‐2 (gelatinase A), MMP‐3 (stromelysin‐1), MMP‐9 (gelatinase B), tissue inhibitors of metalloproteinase (TIMP)‐1 and TIMP‐2 in human breast carcinomas. In more than half of the cases, MMP‐1, MMP‐2, MMP‐9, TIMP‐1 and TIMP‐2 were immunolocalized in carcinoma cells and MMP‐2 was on the carcinoma cell membranes as well, whereas MMP‐3 was positively stained in less than 15% of the cases. MMP‐1 staining in carcinoma cells was significantly higher in scirrhous carcinoma than in other types of carcinoma. MMP‐9 expression was remarkably higher in the carcinoma cases with lymphnode metastasis than in the non‐metastatic cases. MMP‐3 was mainly expressed in T‐lymphocytes infiltrated in the tumor stroma. Stromal fibroblasts were positive for all these MMPs except for MMP‐3. The TIMP‐1 levels released into the culture media by carcinoma tissues were significantly lower than those by fibroadenoma tissues, although there were no significant differences in the levels of MMP‐1, MMP‐2, MMP‐9 and TIMP‐2. Gelatin zymographical analyses showed that the activation rate of the zymogen of MMP‐2 (proMMP‐2) is significantly higher in the more advanced carcinoma group with lymphnode metastasis than in the metastasis‐negative and fibroadenoma groups. These data indicate that MMP‐1, MMP‐2 and MMP‐9 are highly expressed in human breast carcinoma tissue and suggest that activation of proMMP‐2 may be an indicator of lymphnode metastasis of the breast carcinoma.
Leuchtenberger Bodies in Flat Adenomas of the Colorectal Mucosa: A Comparison between Japanese and Swedish Patients
Authors: Rubio Carlos A. , Kumagai Jiro , Nakamura Kyoichi , Yanagisawa Akio , Kato Yo
Published: August 2005

The dysplastic epithelium of flat colorectal adenomas was found to contain DNA inclusion granules, known as Leuchtenberger bodies (LB). Hematoxylin and cosin‐stained sections of 263 colorectal flat neoplastic lesions (i.e., flat adenomas and flat adenocarcinomas) collected in Japan and Sweden were scrutinized for the presence of such intraepithelial inclusions. LB were recorded in 87.1% of the 263 flat colorectal neoplastic lesions. The frequency of flat colorectal lesions having LB was similar in Japan (160 of 178 or 89.8%) to that in Sweden (69 of 85 flat lesions or 81.2%), suggesting that the occurrence of LB may not be influenced by race or environment. Direct immunoperoxidase detection of nuclear DNA fragmentation and transmission electron microscopy studies indicated that the inclusions contained apoptotic material. The semiquantitative assessment of histochemically labeled apoptotic granules indicated that the number of lesions having moderate to large numbers of apoptotic granules (++/+++) was higher in flat adenomas with high‐grade dysplasia, suggesting that the occurrence of these granules may be related to the degree of severity of the dysplastic process. A better knowledge of LB may improve our understanding of the possible relationship between apoptosis, host immune response and carcinogenesis in flat colorectal adenomas.
Inhibition of Natural Killer Cell Cytotoxicity by Cell Growth‐related Molecules
Authors: Tamura Yasuaki , Takashima Satoru , Cho Joong‐Moon , Qi Weimin , Kamiguchi Kenjiro , Torigoe Toshihiko , Takahashi Shuji , Hirai Itaru , Sato Noriyuki , Kikuchi Kokichi
Published: August 2005

Certain MHC class I molecules on target cells are known to inhibit the cytotoxic action of NK cells. By using monoclonal antibody (mAb) Cho‐1, we have found inhibitory non‐MHC class I cell surface molecules that are noncovalently‐associated with 200 kDa and 40 kDa antigens. Poly I‐C‐induced rat NK cells were not cytotoxic to rat fetus‐derived fibroblast WFB cell line. In contrast, NK cells were cytotoxic to H‐ras oncogene‐induced transformants of WFB, W14 and W31. FACS analysis indicated that mAb Cho‐1 reacts with WFB, but not with W14 and W31 cells. Thus, this antigen may disappear concomitantly with cell growth and transformation. Cho‐1 antigens were also expressed on other NK‐resistant lines, such as mouse BALB3T3 fibroblast, EL‐4 lymphoma and human fibroblast HEPM. However, they were not expressed on NK‐sensitive mouse YAC‐1 and H‐ras transformant (Brash) of BALB3T3 cells. Furthermore, treatment of target cells with IFN‐γ clearly induced the cell surface expression of Cho‐1 antigens, and conferred a resistance to NK cytolysis on target cells. These data strongly suggest that Cho‐I antigen expression may correlate with target cell susceptibility to NK cells. Indeed, treatment of NK‐resistant WFB as well as HEPM cells with F(ab')2 fragments of mAb Cho‐1 resulted in the acquisition of susceptibility to NK cytolysis. Cho‐1 antigens may be novel molecules that regulate the NK resistance of cells.
Immunohistochemical Localization of P‐Glycoprotein and Expression of the Multidrug Resistance‐1 Gene in Human Pancreatic Cancer: Relevance to Indicator of Better Prognosis
Authors: Suwa Hirofumi , Ohshio Gakuji , Arao Shinji , Imamura Takashi , Yamaki Kenichirou , Manabe Tadao , Imamura Masayuki , Hiai Hiroshi , Fukumoto Manabu
Published: August 2005

We investigated immunohistochemical localization of P‐glycoprotein (F‐gp) on paraffin‐embedded sections from 103 cases of previously untreated pancreatic tumors and also analyzed multidrug resistance‐1 (MDR1) gene expression by polymerase chain reaction after reverse transcription in 35 cases. High positive staining for P‐gp was observed in 72.8% of pancreatic tumors and in 73.2% of ductal adenocarcinoma. In ductal adenocarcinoma, immunoreactivity of P‐gp was inversely correlated with biological aggressiveness of tumors determined by histologic grading (P<0.01), tumor size (P<0.01), retroperitoneal invasion (P<0.01) and portal invasion (P<0.05). Expression of the MDR1 gene was detected in all the pancreatic tumors examined and was significantly higher than that in normal pancreas (P<0.05). The levels of MDR1 mRNA showed a moderate correlation with those of P‐gp (r=0.62, P<0.0001). Higher expression levels of MDR1/P‐gp significantly correlated with better prognosis of patients with ductal carcinoma (P<0.05). Among patients with ductal carcinoma, the high staining group for P‐gp revealed a 3.5‐fold better prognosis compared with the low staining group (HR=3.47, 95% CI=1.62, 7.45; P=0.0016). In conclusion, MDR1 gene/P‐gp expression in pancreatic cancer without chemotherapy inversely correlates with biological aggressiveness and is an independent indicator of favorable prognosis.
Equivalent Parental Distribution of Frequently Lost Alleles and Biallelic Expression of the H19 Gene in Human Testicular Germ Cell Tumors
Authors: Mishina Mutsuki , Ogawa Osamu , Kinoshita Hidefumi , Oka Hiroya , Okumura Kazuhiro , Mitsumori Kenji , Kakehi Yoshiyuki , Reeve Anthony E. , Yoshida Osamu
Published: August 2005

Epigenetic alterations such as genomic imprinting might play an important role in human tumorigenesis, in addition to specific genetic alterations. To clarify the role of genetic and/or epigenetic alterations in the tumorigenesis of testicular germ cell tumors (GCTs), we analyzed 40 primary and 3 metastatic testicular GCTs with regard to specific chromosomal losses and their parental origin. A high incidence of loss of heterozygosity (LOH) was demonstrated on chromosomes 1p, 3p, 11p, and 17p: 9/19 (47%), 18/39 (46%), 13/40 (33%) and 20/36 (56%), respectively. However, there was no correlation between the frequency of LOH on any chromosome and clinicopathological features. Regarding the parental origin of the lost allele at these chromosomes, preferential loss was not demonstrated in this study. To clarify the imprinting status in GCTs, we analyzed the allele‐specific expression of the H19 gene, which is paternally imprinted on chromosome 11p. All of 11 tumors without LOH at this locus showed biallelic expression of H19. Based on previous work demonstrating the biallelic expression of H19 in primordial germ cells and spermatogonia in the mouse germ line, these results suggest that the biallelic expression of H19 in testicular GCTs reflects the characteristics of the original germ cells in which the imprinting marking has been erased and not established, rather than loss of imprinting during tumorigenesis. It is also possible that a failure to re‐establish the imprinting might be an initial event which leads to testicular GCTs.
Screening for Colorectal Cancer by Immunochemical Fecal Occult Blood Testing
Authors: Saito Hiroshi
Published: August 2005

Screening for colorectal cancer using the conventional Hemoccult test has been shown to reduce mortality associated with cancer by 33% through a randomized controlled trial. However, the magnitude of effectiveness is small in terms of cost‐effectiveness. The recently developed immunochemical fecal occult blood test (IFOBT) provides a potential replacement for the Hemoccult test as a screening test, due to its superior performance characteristics such as higher sensitivity shown in preliminary studies and the fact that it does not require any dietary restriction. The IFOBT method is reviewed, especially in relation to its specificity. In known colorectal cancer subjects, IFOBTs have shown both higher sensitivity and specificity than the Hemoccult test. Similarly, IFOBT has demonstrated a higher sensitivity than Hemoccult for colorectal cancer in an asymptomatic population. A nationwide screening program in Japan has demonstrated the feasibility of this approach for large population screening. However, the positivity rate varied according to the conditions at each screening facility. Therefore, technical factors that influence the positivity rate of IFOBTs in the screening program are discussed. Case‐control studies have strongly suggested that screening using IFOBT would reduce mortality from colorectal cancer by 60% or more. Several observational studies have provided support for this estimate. The feasibility and effectiveness of population‐based screening by IFOBT are discussed.
Inhibitory Effect of Chlorophyllin on Diethylnitrosamine and Phenobarbital‐induccd Hepatocarcinogenesis in Male F344 Rats
Authors: Sugie Shigeyuki , Okamoto Kiyohisa , Makita Hiroki , Ohnishi Masami , Kawamori Toshihiko , Watanabe Tomoyuki , Tanaka Takuji , Mori Hideki
Published: August 2005

Modifying effects of chlorophyllin (CHL) on the diethylnitrosamine (DEN)‐phenobarbital (PB) hepatocarcinogenesis model were examined in rats. Five‐week‐old male F344 rats were divided into 8 groups. Groups 1 through 5 were given i.p. injections of DEN (100 mg/kg body weight) once a week for 3 weeks beginning one week after the start of the experiment, while groups 6 through 8 received vehicle treatment. Groups 1, 2, 3 and 7 received drinking water with 500 ppm PB from one week after the end of carcinogen or vehicle treatment. CHL‐containing diet (2000 ppm) was given to group 2 during the initiation phase and to groups 3 and 5 during the promotion and the post‐initiation phase, respectively. Group 6 was given the experimental diet alone throughout the experiment (24 weeks). Liver neoplasms were present in DEN‐treated groups and PB treatment promoted liver tumorigenesis. The incidences of adenoma in groups 2 and 3 were significantly smaller than in group 1 (P<0.05 and P<0.02), although the reductions in the incidences of liver cell cancer were not significant. The average numbers of liver neoplasms/rat in group 2 were significantly smaller than in group 1 (P<0.05‐P<0.005), Glutathione S‐transferase placental form‐positive foci were also significantly decreased by CHL treatment (P<0.05 and P<0.001). DEN and PB exposure increased liver ornithine decarboxylase activity and this increase was significantly inhibited by feeding of CHL during the initiation phase (P<0.001). These results suggest that CHL is a chemopreventive agent for liver neoplasia.
Expression of c‐met/HGF Receptor in Human Non‐small Cell Lung Carcinomas in vitro and in vivo and Its Prognostic Significance
Authors: Ichimura Eiji , Maeshima Arafumi , Nakajima Takashi , Nakamura Toshikazu
Published: August 2005

The expression of c‐met/HGF receptor was evaluated in non‐small cell lung cancers (NSCLC) by western blot analysis of 11 established cell lines and 104 surgically resected tissues. All cancer cell lines (eight adenocarcinomas, two squamous cell carcinomas and a large cell carcinoma) showed strong c‐met protein bands of 145 kDa and 170 kDa. Moreover, c‐met protein was demonstrated in 34 (72.3%) of 47 surgically resected adenocarcinomas, 20 (38.5%) of 52 squamous cell carcinomas and 3 of 5 others, and the results were mostly confirmed immunohistochemically in formalin‐fixed and paraffin‐embedded tumors of the same case. Although squamous cell carcinomas showed relatively high c‐met protein expression in established cell lines, more adenocarcinomas than squamous cell carcinomas showed c‐met protein expression in the original cancers. Furthermore, two cell lines used in this study originated from primary cancers negative for c‐met protein expression, suggesting that c‐met protein expression might be influenced by cultivation. Furthermore, clinicopathological study revealed that NSCLC with c‐met protein expression tended to be in a higher pathological tumor stage and to have a worse outcome than those without such expression. In conclusion, c‐met protein is expressed in cell lines and primary tumors of NSCLC, and this phenomenon is probably closely related to the aggressive behavior or progression of NSCLC, especially of adenocarcinomas.
In vivo Anti‐tumor Efficacy of Polyethylene Glycol‐modified Tumor Necrosis Factor‐α against Tumor Necrosis Factor‐resistant Tumors
Authors: Tsutsumi Yasuo , Tsunoda Shinichi , Kaneda Yoshihisa , Kamada Haruhiko , Kihira Tetsunari , Nakagawa Shinsaku , Yamamoto Yoko , Horisawa Yoshifumi , Mayumi Tadanori
Published: August 2005

We previously reported that the optimally PEGylated tumor necrosis factor‐α (MPEG‐TNF‐α), in which 56% of the TNF‐α‐lysine amino groups were coupled with polyethylene glycol (PEG), had about 100‐fold greater anti‐tumor effect than native TNF‐α. Here, we assessed the usefulness of MPEG‐TNF‐α as a systemic anti‐tumor therapeutic drug, using B16‐BL6 melanoma and colon‐26 adenocarcinoma, which have been reported to be resistant to TNF‐αin vivo, as compared with Meth‐A fibrosarcoma. MPEG‐TNF‐α markedly inhibited the growth of both tumors without causing any TNF‐α‐mediated side‐effects, whereas native TNF‐α had no anti‐tumor effects and caused adverse side‐effects. In addition, MPEG‐TNF‐α drastically inhibited the metastatic colony formation of B16‐BL6 melanoma. MPEG‐TNF‐α may, thus, be a potential systemic anti‐tumor therapeutic agent.