bluuebunny/crossref_metadata_2025 huggingface dataset:
Interrelationships between Cellular Nucleotide Excision Repair, Cisplatin Cytotoxicity, HER‐2/neu Gene Expression, and Epidermal Growth Factor Receptor Level in Non‐small Cell Lung Cancer Cells
Authors: Tsai Chun‐Ming , Chang Kuo‐Ting , Li Lan , Perng Reury‐Perng , Yang Li‐Ying
Published: August 2005

Nucleotide excision repair (NER) is a major repair mechanism for DNA lesions induced by cisplatin. Overexpressions of epidermal growth factor receptor (EGFR) and HER‐2/neu have been reported to affect the sensitivity of certain human cancer cells to cisplatin, presumably by modification of DNA repair activity through interference with NER. Using an in vitro repair assay, we investigated NER activity of cisplatin‐induced DNA lesions in a panel of 16 non‐small cell lung cancer (NSCLC) cell lines. The interrelationships between NER activity, cisplatin sensitivity, HER‐2/neu expression and EGFR level, were also analyzed. The results showed that high NER activity was closely correlated with cisplatin resistance and high levels of HER‐2/neu expression (P < 0.05). Analysis of the relationships between EGFR level and each of the other three parameters revealed no statistically significant correlations (all P values were > 0.05 by Spearman rank correlation), but a trend of association (all the values of proportion of accordance were ≥62.5% by using a 2x2 contingency table). These results suggest that NER activity may play an important role in the cisplatin resistance of NSCLC cells and there may be an association between enhanced NER activity and high levels of p185neu and probably EGFR in NSCLC cells. The finding that high levels of EGFR showed very little influence on the relationship between p185neu and cisplatin resistance suggests that EGFR may be a less crucial factor in modulating the chemoresistance of NSCLC cells when compared with HER‐2/neu.
Transforming Growth Factor‐α Induces the Differentiation of Sarcomatoid Cholangiocarcinoma Cells
Authors: Enjoji Munechika , Nakashima Manabu , Nakamuta Makoto , Nawata Hajime
Published: August 2005

A sarcomatoid cholangiocarcinoma cell line, ETK‐1, was established from a patient. Phenotypically, the cells corresponded to immature biliary epithelial cells. Because a small number of ETK‐1 cells appeared to differentiate spontaneously along a biliary epithelial lineage in continuous culture, we examined the factors that initiate and/or promote the differentiation of the cells. Transforming growth factor‐α (TGFα) induced significant changes in ETK‐1 cells. After stimulation with the factor, ETK‐1 cells displayed morphologic transformation at a much higher frequency, with the appearance of many large cells with intracytoplasmic vacuoles, and the production of mucinous substances. These morphologically transformed cells were phenotypically similar to welldifferentiated adenocarcinoma cells. The expression pattern of integrins after TGFα treatment also supported the maturation of the ETK‐1 cells. The antibody against the receptor of TGFα inhibited these changes by TGFα. Moreover, the proliferation rate of ETK‐1 cells was suppressed by TGFα. Our data suggest that TGFα can act as a differentiation factor along a biliary epithelial lineage.
Practical Utility of the Revised European‐American Classification of Lymphoid Neoplasms for Japanese Non‐Hodgkin's Lymphomas
Authors: Izumo Toshiyuki , Maseki Nobuo , Mori Shigeo , Tsuchiya Eiju
Published: August 2005

A clinicopathological study of 515 non‐Hodgkin's lymphoma (NHL) cases was performed using the revised European‐American classification of lymphoid neoplasms (REAL classification) in an HTLV1‐nonendemic area of Japan. The following characteristics were revealed: 1) frequency of extranodal lymphomas was high (59%) with 79% B‐cell lymphomas in this series, while the overall ratio of B:T/NK lineage was 3.7:1; 2) the most common type was the diffuse large B‐cell lymphoma (46%), follicle center lymphomas occurred at an incidence lower (15%) than that in European and American populations, and marginal zone B‐cell lymphomas accounted for as much as 12%; 3) peripheral T‐cell lymphomas were common (19%), with the unspecified type predominant (11%), while adult T‐cell lymphomas were present at a level equivalent to that among European and American patients (1%). Clear segregation of survival curves was rated according to cell lineage and B‐cell lymphomas had a better prognosis than T/NK‐cell lymphomas. Furthermore, new subtypes in the REAL classification, such as marginal zone B‐cell and mantle cell lymphomas, exhibited distinct curves. Taken altogether, the REAL classification demonstrated advantages for assessment of Japanese NHL cases.
Cationic Liposome Conjugation to Recombinant Adenoviral Vector Reduces Viral Antigenicity
Authors: Natsume Atsushi , Mizuno Masaaki , Ryuke Yasushi , Yoshida Jun
Published: August 2005

Adenoviral (Ad) vectors are commonly used in gene therapy trials because of their efficiency in gene transfer. However, their use is limited by immune responses that reduce transgene expression and decrease the efficacy of repeated vector administration. In this study, we demonstrated that conjugation of Ad vector with our novel cationic liposomes could reduce viral antigenicity in vivo. Mice subcutaneously injected with liposome‐conjugated Ad vector showed a 6.5‐fold reduction of anti‐Ad antibodies with neutralizing activity, compared to those with unconjugated Ad vector. Interestingly, we also found that the conjugated vector is less susceptible to inactivation by neutralizing antibodies in vitro and in vivo. Our results suggest that liposome conjugation reduces viral antigenicity, shields vectors from neutralizing antibody, and may allow repeated Ad vector administration.
Polymerase Chain Reaction with Confronting Two‐pair Primers for Polymorphism Genotyping
Authors: Hamajima Nobuyuki , Saito Toshiko , Matsuo Keitaro , Kozaki Ken‐ichi , Takahashi Takashi , Tajima Kazuo
Published: August 2005

A novel PCR method using confronting two‐pair primers, named PCR‐CTPP, is introduced to detect a single nucleotide polymorphism (base X or Y). One primer for the X allele is set to include X'at the 3′end (antisense), where X'is the antisense of X, with the counterpart sense primer upstream. For the Y allele, a sense primer including Y at the 3′end is set, with the antisense primer downstream. One common band and one specific band for each allele are amplified, which allows genotyping directly by electrophoresis. This method is exemplified by application to the polymorphisms of beta‐adrenoceptor 2 and interleukin 1B. It is simpler than PCR‐RFLP (restriction fragment length polymorphism), which requires incubation with a restriction enzyme, and is suitable for genotyping in studies of genetic epidemiology involving hundreds of samples.
Prognostic Significance of Occult Bone Marrow Micrometastases of Breast Cancer Detected by Quantitative Polymerase Chain Reaction for Cytokeratin 19 mRNA
Authors: Ikeda Noriko , Miyoshi Yasuo , Motomura Kazuyoshi , Inaji Hideo , Koyama Hiroki , Noguchi Shinzaburo
Published: August 2005

Amplification of cytokeratin 19 (CK19) transcripts by reverse transcriptase‐polymerase chain reaction (RT‐PCR) has been shown to be a highly sensitive assay for the detection of bone marrow micrometastases (BMM) of breast cancer, but recent studies have demonstrated the occurrence of false‐positive results due to low‐level, illegitimately transcribed CK19 in normal bone marrow tissue. One approach to solve this problem is to develop a quantitative CK19 RT‐PCR assay and to introduce a cut‐off value which can distinguish between illegitimate expression and cancer‐specific expression levels. In the present paper, we describe a quantitative CK19 RT‐PCR assay using a real‐time automated PCR system. The number of CK19 transcripts was normalized to that of GAPDH transcripts as an internal control for quality and quantity of cDNA. The cut‐off value for the ratio of CK19 to GAPDH transcripts was set at 10−4 since the ratio never exceeded this value in the control bone marrow samples (n=12). In total, 117 bone marrow aspirates from stage I‐III patients with invasive breast cancers were subjected to CK19 RT‐PCR assay and immunocytological examination. Forty (34.2%) were found to be BMM‐positive by CK19 RT‐PCR assay whereas only three (2.6%) were found to be BMM‐positive by immunocytology. Multivariate analysis has shown that occult BMM detected by CK19 RT‐PCR is a significant risk factor for relapse, being independent of axillary lymph node metastases.
Relations of Insulin Resistance and Serum Concentrations of Estradiol and Sex Hormone‐binding Globulin to Potential Breast Cancer Risk Factors
Authors: Nagata Chisato , Shimizu Hiroyuki , Takami Rieko , Hayashi Makoto , Takeda Noriyuki , Yasuda Keigo
Published: August 2005

There is a hypothesis that hyperinsulinemia or insulin resistance may be a mediator for breast cancer risk factors. On the other hand, some, but not all, of the well‐known risk factors of breast cancer have been associated with serum estrogen concentrations. We assessed the relationships of potential breast cancer risk factors to indicators of insulin resistance, fasting plasma insulin concentration and homeostasis model assessment insulin resistance (HOMA‐R), in 88 postmenopausal Japanese women. We also examined whether insulin resistance would explain the association of breast cancer risk factors with serum estradiol and sex hormone‐binding globulin (SHBG). Information on potential breast cancer risk factors, such as demographic characteristics, smoking and drinking habits, diet, exercise, menstrual and reproductive factors, was obtained by self‐administered health questionnaire including a validated semiquantitative food frequency questionnaire. Body mass index (BMI) was significantly correlated with the ratio of estradiol to SHBG (Spearman r=0.30, P=0.0004), fasting plasma insulin (r=0.45) and HOMA‐R (r=0.43, P=0.0001) after controlling for age. The correlations were still significant between BMI and estradiol/SHBG ratio (r=0.21, P=0.047) after controlling for fasting plasma insulin and between BMI and fasting plasma insulin (r=0.40, P=0.0001) as well as HOMA‐R (r=0.38, P=0.0003) after controlling for estradiol/SHBG ratio. There is a possibility that effect of BMI on breast cancer risk is mediated by both insulin resistance and estrogen metabolism.
Life Style and Urinary 8‐Hydroxydeoxyguanosine, a Marker of Oxidative DNA Damage: Effects of Exercise, Working Conditions, Meat Intake, Body Mass Index, and Smoking
Authors: Kasai Hiroshi , Iwamoto‐Tanaka Naoko , Miyamoto Toshiaki , Kawanami Kiyoshi , Kawanami Shoko , Kido Rie , Ikeda Masato
Published: August 2005

The urinary levels of 8‐hydroxydeoxyguanosine (8‐OH‐dG), a marker of oxidative DNA damage, of 318 healthy men aged 18‐58 were measured with high resolution by a newly developed automated high‐pressure liquid chromatography (HPLC) system coupled to an electrochemical detector (ECD). The mean 8‐OH‐dG level (μMg/g creatinine) was 4.12±1.73 (SD). An eleven‐fold inter‐individual variation was observed. The accuracy of the measurement estimated from the recovery of an added 8‐OH‐dG standard was 90‐98%. By univariate analysis, it was found that moderate physical exercise (P=0.0023) and high body mass index (BMI) (P=0.0032) reduced the 8‐OH‐dG level, while physical labor (P=0.0097), smoking (P=0.032), and low meat intake (less than once/ week) (P=0.041) increased its level. Based on a multi‐regression analysis of the log‐transformed values, moderate physical exercise (P=0.0039), high BMI (P=0.0099), and age (P=0.021) showed significant reducing effects on the 8‐OH‐dG level, while low meat intake (P=0.010), smoking (P=0.013), and day‐night shift work (P=0.044) increased its level. These results suggest that many types of life‐style factors that either generate or scavenge oxygen radicals may affect the level of oxidative DNA damage of each individual.
Identification and Characterisation of a Platelet GPIb/V/IX‐like Complex on Human Breast Cancers: Implications for the Metastatic Process
Authors: Suter Catherine M. , Hogg Philip J. , Price John T. , Chong Beng H. , Ward Robyn L.
Published: August 2005

The glycoprotein (GP) Ib/V/IX receptor complex is an important adhesion molecule, originally thought to be unique to the megakaryocytic lineage. Recent evidence now indicates that GPIb/V/IX may be more widely expressed. In this study we report the presence of all subunits of the complex on four breast cancer cell lines, and 51/80 primary breast tumours. The surface expression of GPIb/V/IX was confirmed by flow cytometry, and by immunoprecipitation of biotin surface‐labelled tumour cells. Western blotting of cell lysates under reducing conditions revealed that tumour cell‐GPIba had a relative molecular weight of 95 kDa as compared to 135 kDa on platelets. Despite the discrepant protein size, molecular analyses on the tumour cell‐GPIba subunit using RT‐PCR and DNA sequencing revealed 100% sequence homology to platelet GPIba. Tumour cell‐GPIb/V/IX was capable of binding human von Willebrand factor (vWf), and this binding caused aggregation of tumour cells in suspension. Tumour cells bound to immobilised vWf in the presence of EDTA and demonstrated prominent filapodial extensions indicative of cytoskeletal reorganisation. Furthermore, in a modified Boyden chamber assay, prior exposure to vWf or a GPIba monoclonal antibody, AK2, enhanced cell migration. The presence of a functional GPIb/V/IX‐like complex in tumour cells suggests that this complex may participate in the process of haematogenous breast cancer metastasis
Molecular Significance of Excess Body Weight in Postmenopausal Breast Cancer Patients, in Relation to Expression of Insulin‐like Growth Factor I Receptor and Insulin‐like Growth Factor II Genes
Authors: Suga Kenji , Imai Kazue , Eguchi Hidetaka , Hayashi Shin‐ichi , Higashi Yasuhiro , Nakachi Kei
Published: August 2005

A number of epidemiological and clinical studies have revealed that excess body weight increases the risk of postmenopausal breast cancer and also adversely affects subsequent malignant progression. To elucidate the molecular mechanisms underlying these observations, we examined mRNA expression of various genes in normal (non‐cancerous) mammary gland and cancer tissue of Japanese patients with primary breast cancer, in association with their body mass index (BMI). On the basis of analysis of 106 breast cancer patients, we found that mRNA expression of insulin‐like growth factor I receptor (IGF‐IR) and insulin‐like growth factor II (IGF‐II) in the normal mammary gland showed a significant and positive association with increased BMI among postmenopausal patients. Furthermore, the positive association of increased BMI with IGF‐IR mRNA expression was also found in postmenopausal breast cancer tissue, while this association was not observed among premenopausal patients. In addition, increased mRNA expression of cyclin Dl and bcl‐2 was observed in association with increased mRNA levels of IGF‐IR among the patients regardless of menopausal status. These findings suggest that the molecular consequence of the increased BMI is the increased expression of IGF‐II and IGF‐IR, resulting in development of postmenopausal breast cancer and its progression mediated through modulation of the cell cycle and apoptosis.
Progesterone Receptor A and B Isoforms in the Human Breast and Its Disorders
Authors: Ariga Naohiro , Suzuki Takashi , Moriya Takuya , Kimura Michio , Inoue Tsukasa , Ohuchi Noriaki , Sasano Hironobu
Published: August 2005

Two different isoforms of progesterone receptor (PR), PRA and PRB, are expressed in target tissues at comparable levels. In this study, we first examined PRA and PRB immunoreactivity in human breast cancer and various intraductal proliferative epithelial lesions, and correlated these findings with clinicopathologic parameters. We then examined mRNA expression of PRA and PRB in six cases of invasive ductal carcinoma using RT‐PCR. Immunoreactivity for both PRA and PRB was positive in the great majority of proliferative disease without atypia (PDWA) (85% for PRA and 96% for PRB) and atypical ductal hyperplasia (ADH) (100% for PRA and 100% for PRB), but the ratio of immunopositive cases and immunohistochemical (IHC) scores was significantly smaller in ductal carcinoma in situ (DCIS) (65% for PRA and 75% for PRB) and invasive ductal carcinoma (IDC) (66% for PRA and 55% for PRB) than in PDWA and ADH. There was a significant positive correlation between IHC scores for PRA and estrogen receptor α (ERα) in IDC, DCIS and ADH but not between PRB and ERα. In IDC, both PRA and PRB IHC scores were significantly associated with histological grade, but there was no association between PRA or PRB status and lymph node involvement, tumor size, or prognosis of the patients. The expression of mRNAs for both PRA and PRB was detected in all six cases of IDC examined. These results suggest that both PRA and PRB are strongly associated with ERa in human breast and this relation may be disturbed in breast cancer.
Troglitazone, a Ligand for Peroxisome Proliferator‐activated Receptor γ Inhibits Chemically‐induced Aberrant Crypt Foci in Rats
Authors: Kohno Hiroyuki , Yoshitani Shin‐ichiro , Takashima Shigeki , Okumura Ataru , Hosokawa Masashi , Yamaguchi Nobuo , Tanaka Takuji
Published: August 2005

The biological roles of peroxisome proliferator‐activated receptors (PPARs) in various diseases, including inflammation and cancer, have been highlighted recently. Although PPARγ ligand is suspected to play an important role in carcinogenesis, its effects on colon tumorigenesis remain undetermined. The present tune‐course study was conducted to investigate possible modifying effects of a PPARγ ligand, troglitazone, on the development and growth of aberrant crypt foci (ACF), putative precursor lesions for colon carcinoma, induced by azoxymethane (AOM) or dextran sodium sulfate (DSS) in male F344 rats. Oral troglitazone (10 or 30 mg/kg body weight (b.w.)) significantly reduced AOM (two weekly subcutaneous injections, 20 mg/kg b.w.)‐induced ACF. Treatment with troglitazone increased apoptosis and decreased polyamine content and ornithine decarboxylase (ODC) activity in the colonic mucosa of rats treated with AOM. Gastric gavage of troglitazone also inhibited colitis and ACF induced by DSS (1% in drinking water), in conjunction with increased apoptosis and reduced colonic mucosal polyamine level and ODC activity. Our results suggest that troglitazone, a synthetic PPARγ ligand, can inhibit the early stage of colon tumorigenesis with or without colitis.
Cell‐free Entry of Human T‐Cell Leukemia Virus Type 1 to Mouse Cells
Authors: Feng Renqing , Kabayama Ayako , Uchida Kazuhiko , Hoshino Hiroo , Miwa Masanao
Published: August 2005

Human T‐cell leukemia virus type 1 (HTLV‐1) is the etiologic agent for adult T‐cell leukemia and HTLV‐1‐associated myelopathy/tropical spastic paraparesis. Recently we infected newborn mice by inoculating HTLV‐1‐producing human cells, and found that T‐cells, B‐cells and granulocytes were infected in vivo. To understand the mechanism of viral‐cell interaction and the pathogenesis of HTLV‐1 using the mouse model, it is important to clarify the cellular tropism using a cell‐free HTLV‐1 transmission system. We employed a highly transmissible cell‐free HTLV‐1 produced by a feline kidney cell line, c77, and studied the susceptibility of 9 kinds of mouse cell lines, EL4, RLml, CTLL‐2, J774.1, DA‐1, Ba/F3, WEHI‐3, NIH3T3 and Bl, and two kinds of human cell lines, Molt‐4 and Hut78. HTLV‐1 proviral sequence was found by PCR in all 9 mouse cell lines as well as in 2 human cell lines and viral entry was blocked with sera from an HTLV‐1 carrier and an adult T‐cell leukemia patient. Unexpectedly, mouse cell lines EL4 and RLml and human cell lines Molt‐4 and Hut78 showed similar efficiency for viral entry. These results suggest a wide distribution of HTLV‐1 receptor in mouse cells.
A Novel Amplicon at 9p23‐24 in Squamous Cell Carcinoma of the Esophagus That Lies Proximal to GASC1 and Harbors NFIB
Authors: Yang Zeng‐Quan , Imoto Issei , Pimkhaokham Atiphan , Shimada Yutaka , Sasaki Kohsuke , Oka Masaaki , Inazawa Johji
Published: August 2005

The non‐random amplification of DNA at 9p23‐24 observed in various types of human cancers, including esophageal squamous cell carcinomas (ESCs), may reflect the locations of important tumor‐associated genes. Our previous studies using ESC cell lines defined an amplicon in this region and identified a novel gene, GASC1, as a target of the amplification. Since different regions within the same chromosome arm are often involved in amplification in a syntenic or non‐syntenic manner, we characterized the amplicon at 9p23‐24 in 35 ESC cell lines (29 KYSE series and 6 YES series), and examined possible involvement of non‐syntenic amplifications at 9p23‐24 in 32 primary ESCs. Our results clearly indicated that two target regions for DNA amplification exist at 9p23‐24; the major amplicon contains GASC1, and the minor one harbors a transcription factor, NFIB, centromeric to the GASC1 locus.
Seaweed Prevents Breast Cancer?
Authors: Funahashi Hiroomi , Imai Tsuneo , Mase Takahiro , Sekiya Masanori , Yokoi Kazuki , Hayashi Hiromichi , Shibata Arihiro , Hayashi Takako , Nishikawa Mikiko , Suda Namiko , Hibi Yatsuka , Mizuno Yutaka , Tsukamura Kyosuke , Hayakawa Akemi , Tanuma Seiichi
Published: August 2005

To investigate the chemopreventive effects of seaweed on breast cancer, we have been studying the relationship between iodine and breast cancer. We found earlier that the seaweed, wakame, showed a suppressive effect on the proliferation of DMBA (dimethylbenz(a)anthracene)‐induced rat mammary tumors, possibly via apoptosis induction. In the present study, powdered mekabu was placed in distilled water, and left to stand for 24 h at 4°C. The filtered supernatant was used as mekabu solution. It showed an extremely strong suppressive effect on rat mammary carcinogenesis when used in daily drinking water, without toxicity. In vitro, mekabu solution strongly induced apoptosis in 3 kinds of human breast cancer cells. These effects were stronger than those of a chemothera‐peutic agent widely used to treat human breast cancer. Furthermore, no apoptosis induction was observed in normal human mammary cells. In Japan, mekabu is widely consumed as a safe, inexpensive food. Our results suggest that mekabu has potential for chemoprevention of human breast
Low Expression Myeloperoxidase Genotype Negatively Associated with Helicobacter pylori Infection
Authors: Hamajima Nobuyuki , Matsuo Keitaro , Suzuki Takashi , Nakamura Tsuneya , Matsuura Akira , Tajima Kazuo , Tominaga Suketami
Published: August 2005

Our previous study revealed that a polymorphism of the interleukin (IL) 1B gene, encoding the pro‐inflammatory cytokine IL‐lβ, influenced the prevalence of persistent Helicobacter pylori (HP) infection. In this paper, a polymorphism of another inflammation‐related enzyme, myeloperoxidase (MPO), was examined with respect to association with the HP infection. The polymorphism is due to a G‐to‐A transition at ‐463 in the promoter region of MPO. The G allele is the wild type with normal expression, while the A allele is a low expression allele. The subjects were 241 non‐cancer outpatients (118 males and 123 females) aged 39 to 69 who participated in an HP eradication program at Aichi Cancer Center Hospital. High‐molecular weight Campylobacter‐Associated‐Protein (HM‐CAP) ELISA (Enteric Products Ins., Westbury, NY) was used for the identification of HP‐infected participants. The frequency was 79.7% (192/241) for the GG genotype, 19.5% (47/241) for the GA genotype, and 0.8% (2/241) for the AA genotype. The sex‐age‐adjusted odds ratio (OR) relative to GG was 0.69 (95% confidence interval (CI), 0.35–1.35) for individuals with the A allele, but among male participants the OR was 0.31 (0.11–0.84). Subgroup analysis revealed significantly reduced ORs with the GA/AA genotypes for current smokers (0.19, 0.04–0.96), and for those who were occasional/no milk drinkers (0.25, 0.09–0.72). These findings are consistent with the results for IL‐1B in our earlier study, suggesting that inflammatory responses in the gastric mucosa may influence persistent HP infection, and that smoking and milk intake may be effect ‐modifiers.
Suicide Gene Therapy for Chemically Induced Rat Bladder Tumor Entailing Instillation of Adenoviral Vectors
Authors: Akasaka Shuji , Suzuki Satoru , Shimizu Hiroyuki , Igarashi Takehito , Akimoto Masao , Shimada Takashi
Published: August 2005

The efficacy of an in vivo gene therapy protocol making use of an adenoviral vector in the treatment of bladder cancer was examined. Bladder tumors were induced in rats by oral administration of BBN (N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine). Histologically, such tumors resemble those seen in human bladder cancer, and the cells can be selectively transduced using adenoviral vectors. The therapeutic protocol thus entailed instillation of an adenoviral vector containing the HSV‐tk suicide gene into rat bladder followed by a regimen of intraperitoneal ganciclovir (GCV) injections. Histological examination after a short‐term GCV regimen (3 days) revealed marked vacuolization of the tumor cells. Moreover, TUNEL assays showed that the cytotoxic reaction was mediated by apopto‐sis. Following a long‐term GCV regimen (14 days), tumor growth was significantly inhibited and glandular metaplasia was observed. This is the first report demonstrating the efficacy of in vivo suicide gene therapy in a chemically induced transitional cell carcinoma like that seen in most human bladder cancer. Intravesical instillation is already a well established clinical technique. Our findings indicate that now there is a strong potential for its incorporation into new and useful gene therapies aimed at the treatment of human bladder cancer.
Screening of Differentially Expressed Genes in 5‐Fluorouracil‐resistant Human Gastrointestinal Tumor Cells
Authors: Takechi Teiji , Koizumi Katsuhisa , Tsujimoto Hiroaki , Fukushima Masakazu
Published: August 2005

To identify genes differentially expressed in association with resistance to 5‐fluorouracil (5FU), an niRNA differential display (DD) analysis was used to compare transcripts from the NUGC‐3 human gastric tumor cell line and the NUGC‐3/5FU/L line, which had acquired 208‐fold resistance as a consequence of repeated exposure to escalating concentrations of 5FU. The 110 cDNA fragments differentially expressed in the DD analysis of either the NUGC‐3 or NUGC‐3/5FU/L cells were sequenced and subjected to a homology search, and 29 overexpressed and 22 underexpressed genes were identified in NUGC‐3/5FU/L as a result. To confirm whether the changes in the gene expression levels in the NUGC‐3/5FU/L cells were shared by other 5FU‐resistant cells, 35 genes were analyzed by northern hybridization in 3 pairs of parent/5FU‐resistant human gastrointestinal tumor cell lines. The analysis revealed 20 overexpressed and 10 underexpressed genes in at least one of the three 5FU‐resistant cells as compared with those in the parent cells. Among them, P‐glycoprotein, equilibrative nucleoside transporter 1, and methylenetetrahydrofolate dehydrogenase were highly expressed in two of the three 5FU‐resistant cells and cytidine deaminase and integrin o3 were underexpressed. The acquisition of resistance to 5FU by tumor cells may result from multiple changes in cellular functions.
Geranylgeraniol, an Intermediate Product in Mevalonate Pathway, Induces Apoptotic Cell Death in Human Hepatoma Cells: Death Receptor‐independent Activation of Caspase‐8 with Down‐regulation of Bcl‐xL Expression
Authors: Takeda Yoshio , Nakao Kazuhiko , Nakata Keisuke , Kawakami Atsushi , Ida Hiroaki , Ichikawa Tatsuki , Shigeno Masaya , Kajiya Yuji , Hamasaki Keisuke , Kato Yuji , Eguchi Katsumi
Published: August 2005

Geranylgeraniol (GGOH), an intermediate of mevalonate metabolism, is known to induce apoptosis in various lines of cancer cells. The present study was undertaken to clarify the signaling pathways of apoptosis induced by GGOH in human hepatoma cells. HuH‐7 human hepatoma cells were incubated in the absence or presence of GGOH. Activation of caspase‐8/‐9/‐3 in HuH‐7 cells was found after 8 h treatment with GGOH, at which tune DNA fragmentation and loss of mitochondrial transmembrane potential (ΔΨm) occurred. HuH‐7 cells do not express Bcl‐2; however, down‐regulation of Bcl‐xL expression preceded activation of the caspase cascade in GGOH‐treated HuH‐7 cells, while Bax expression was not changed by GGOH treatment. Addition of caspase inhibitors restored the decreased cell viability of HuH‐7 cells by GGOH, including ΔΨm, to the baseline level, which indicated that caspase triggers mitochondria‐dependent apoptotic pathways in GGOH‐treated HuH‐7 cells. Similarly, GGOH‐mediated apoptosis of HuH‐7 cells was clearly prevented by coadministration of ursodeoxycholic acid (UDCA), which led to restoration of the level of Bcl‐xL expression. Activation of caspase‐8/‐9/‐3, as well as ΔΨm, by GGOH treatment was suppressed by addition of UDCA. Our results indicate that activation of the caspase cascade initiating from caspase‐8, which could be accelerated by down‐regulation of Bcl‐xL expression, plays a key role in an apoptotic process induced by GGOH in human hepatoma cells.
A Novel Renal Carcinoma Predisposing Gene of the Nihon Rat Maps on Chromosome 10
Authors: Hino Okio , Okimoto Kazuo , Kouchi Mami , Sakurai Junko
Published: August 2005

A novel rat model of hereditary renal cell carcinoma (RC) was found in a rat colony of the Spra‐gue‐Dawley (SD) strain in Japan, and named the “Nihon” rat in 2000. This study was designed to map the RC susceptibility gene in the Nihon rat using 113 backcross annuals. Our present data clearly show that the Nihon gene is genetically linked to interleukin‐3 (IL3) gene (χ2=93.6, Lod score=25.16), lethal (2) giant larvae (LLGL1) locus (χ2=109.0, Lod score=31.56) and myosin heavy chain, embryonic skeletal muscle (MYHSE) gene (χ2=90.6, Lod score=23.87), which are located on the distal part of rat chromosome 10. The order of the genes is the Eker (Tsc2) gene (located on the proximal part of rat chromosome 10; human chromosome 16p 13.3)–21.3 cM–IL3 gene (human 5q23‐31)–4.4 cM–Nihon gene–0.9 cM–LLGL1 locus (human 17p11.2)‐4.4 cM–MYHSE gene (human 17pl3.1). We also detected loss of the wild‐type allele at the MYHSE locus, fitting Knudson's “two hit” model. Thus, the Nihon rat should have a mutation of a novel tumor suppressor gene related to renal carcinogenesis.
Hydroxyurea Induces Site‐specific DNA Damage via Formation of Hydrogen Peroxide and Nitric Oxide
Authors: Sakano Katsuhisa , Oikawa Shinji , Hasegawa Keishi , Kawanishi Shosuke
Published: August 2005

Hydroxyurea is a chemotherapeutic agent used for the treatment of myeloproliferative disorders (MPD) and solid tumors. The mutagenic and carcinogenic potential of hydroxyurea has not been established, although hydroxyurea has been associated with an increased risk of leukemia in MPD patients. To clarify whether hydroxyurea has potential carcinogenicity, we examined site‐specific DNA damage induced by hydroxyurea using 32P‐5′‐end‐labeled DNA fragments obtained from the human p53 and p16 tumor suppressor genes and the c‐Ha‐ras‐1 protooncogene. Hydroxyurea caused Cu(II)‐mediated DNA damage especially at thymine and cytosine residues. NADH efficiently enhanced hydroxyurea‐induced DNA damage. The DNA damage was almost entirely inhibited by catalase and bathocuproine, a Cu(I)‐specific chelator, suggesting the involvement of hydrogen peroxide (H2O2) and Cu(I). Typical free hydroxyl radical scavengers did not inhibit DNA damage by hydroxyurea, but methional did. These results suggest that crypto‐hydroxyl radicals such as Cu(I)‐hydroperoxo complex (Cu(I)‐OOH) cause DNA damage. Formation of 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) was induced by hydroxyurea in the presence of Cu(H). An electron spin resonance spectroscopic study using N‐(dithiocarboxy)sarcosine as a nitric oxide (NO)‐trapping reagent demonstrated that NO was generated from hydroxyurea in the presence and absence of catalase. In addition, the generation of formamide was detected by both gas chromatography‐mass spectrometry (GC‐MS) and time‐of‐flight‐mass spectrometry (TOF‐MS). A high concentration of hydroxyurea induced depurination at DNA bases in an H2O2‐independent manner, and endonu‐clease IV treatment led to chain cleavages. These results suggest that hydroxyurea could induce base oxidation as the major pathway of DNA modification and depurination as a minor pathway. Therefore, it is considered that DNA damage by hydroxyurea participates in not only anti‐cancer activity, but also carcinogenesis.
Introduction of Clusterin Gene into Human Renal Cell Carcinoma Cells Enhances Their Resistance to Cytotoxic Chemotherapy through Inhibition of Apoptosis both in vitro and in vivo
Authors: Hara Isao , Miyake Hideaki , Gleave Martin E. , Kamidono Sadao
Published: August 2005

Recent studies have revealed the powerful antiapoptotic activity of clusterin in various malignant tumors; however, the significance of clusterin expression in the acquisition of a resistant phenotype against several kinds of treatment in human renal cell carcinoma (RCC) has not been well characterized. We, therefore, transfected the clusterin cDNA into RCC ACHN cells, that scarcely express clusterin protein, to examine whether overexpression of clusterin inhibits chemotherapy‐induced apoptosis both in vitro and in vivo. Although no significant differences were observed in the in vitro growth rates between clusterin‐transfected ACHN (ACHN/CL) and the vector only‐transfected cell line (ACHN/Co), ACHN/CL exhibited high resistance to cisplatin treatment compared with ACHN/Co, with a greater than 5‐fold higher IC50 through the inhibition of apoptotic cell death, which was demonstrated by DNA fragmentation analysis and western blotting of PARP protein. Moreover, intravenous administration of cisplatin into athymic nude mice bearing ACHN/CL tumors resulted in 2‐ to 3‐times faster tumor growth compared with ACHN/Co tumors. These findings suggest that clusterin overexpression helps confer a chemoresistant phenotype through inhibition of apoptosis in human RCC cells.
Paclitaxel and SN‐38 Overcome Cisplatin Resistance of Ovarian Cancer Cell Lines by Down‐regulating the Influx and Efflux System of Cisplatin
Authors: Komuro Yuuki , Udagawa Yasuhiro , Susumu Nobuyuki , Aoki Daisuke , Kubota Tetsuro , Nozawa Shiro
Published: August 2005

Cisplatin (DDP) is one of the key drugs used to treat patients with ovarian cancer, although resistance to DDP can occur. Paclitaxel and SN‐38 (an active metabolite of irinotecan (CPT‐11)) are two drugs that are effective in patients with DDP‐resistant ovarian cancer. To study how these drugs may overcome the intrinsic and/or acquired resistance of cancer cells to DDP, we investigated the effect of a combination of DDP with paclitaxel and a combination of DDP with SN‐38 on three ovarian cancer cell lines, RTSG (intrinsically resistant cell line), KF (DDP‐sensitive cell line), and KFra (acquired resistant cell line obtained from KF). We found that these combinations showed additive to synergistic antitumor activity. A time‐dependent platinum (Pt) accumulation was observed in the DDP‐sensitive KF cell line, while a decrease occurred in the KFra cell line. Little accumulation was observed in RTSG. Intracellular Pt accumulation was increased in all three cell lines by exposure to paclitaxel or SN‐38. Ouabain, a Na+,K+‐ATPase inhibitor, decreased Pt accumulation in KF and KFra cell lines and inhibited the paclitaxel‐ and SN‐38‐induced increases in Pt accumulation in these cell lines. When we assessed the mRNA levels of the multidrug resistance‐associated protein (MRP), which may be an efflux pump for DDP, the combination of paclitaxel or SN‐38 with DDP down‐regulated these levels, which are up‐regulated by DDP alone. These results suggest that paclitaxel and SN‐38 overcome DDP resistance of ovarian cell lines by controlling intracellular accumulation of DDP via both the influx and efflux systems
A Radicicol Derivative, KF58333, Inhibits Expression of Hypoxia‐inducible Factor‐1α and Vascular Endothelial Growth Factor, Angiogenesis and Growth of Human Breast Cancer Xenografts
Authors: Kurebayashi Junichi , Otsuki Takemi , Kurosumi Masafumi , Soga Shiro , Akinaga Shiro , Sonoo Hiroshi
Published: August 2005

A novel oxime derivative of radicicol, KF58333, binds to the heat shock protein 90 (Hsp90) and destabilizes its associated signaling molecules. These effects play a critical role in the growth inhibition of tumor cells. To further investigate the effects of this agent, it was administered to two human breast cancer cell lines, KPL‐1 and KPL‐4, both in vitro and in vivo. KF58333 dose‐dependently inhibited the growth and vascular endothelial growth factor (VEGF) secretion, concomitantly with a decrease in VEGF mRNA expression, in each cell line. This agent also suppressed the increase of VEGF secretion and expression induced by hypoxia (1% O2). Intravenous injections of this agent into nude mice bearing either KPL‐1 or KPL‐4 xenografts significantly inhibited the tumor growth associated with a decrease in the Ki67 labeling index and microvascular area and an increase in apoptosis and the necrotic area. These findings indicate that the antitumor activity of this radicicol derivative may be partly mediated by decreasing VEGF secretion from tumor cells and inhibiting tumor angiogenesis. To explore the action mechanisms of the anti‐angiogenic effect, the expression level of hypoxia‐inducible factor (HIF)‐lα was investigated. KF58333 provided a significant decrease in the HTF‐lα protein expression under both normoxic and hypoxic conditions. In contrast, the mRNA expression of HIF‐lα was not decreased by this agent. It is suggested that the post‐transcriptional down‐regulation of HIF‐lα expression by this agent may result in a decrease of VEGF expression and tumor angiogenesis.
Establishment of Cell Lines with High and Low Metastatic Potential from A549 Human Lung Adenocarcinoma
Authors: Shindo‐Okada Nobuko , Takeuchi Kaori , Han Beom‐Seok , Nagamachi Yasuhiro
Published: August 2005

This article reports the establishment of variant cell lines with high and low metastatic potential by the dilution plating technique. Two clones with high metastatic potential, 2S Lu‐4 and 11S Lu‐1 and two clones with low metastatic potential, 8S and 16S were established from A549 human lung adenocarcinoma. The high‐metastatic cell lines produced enhanced lung metastases, but the low‐ metastatic cell lines did not produce lung metastasis after injection into the tail vein of 5‐week‐old BALB/c nude mice. The primary tumors produced by the two high‐metastatic cells grew fast and showed enhanced angiogenesis. The high‐metastatic cells were small and flat‐shaped, while the low‐ metastatic cells were large and flat‐shaped. When the four variant cell lines and original A549 cells were embedded in collagen gels, the colonies of 2S Lu‐4, 11S Lu‐1 and A549 grew actively, whereas almost of all the colonies of 8S and 16S did not survive after 35 days in culture. These four cloned cell lines originated from heterogeneous populations of the parental A549 cells should be an excellent tool for studying the process of metastasis of lung cancer.
Copper‐transporting P‐Type Adenosine Triphosphatase (ATP7B) Is Expressed in Human Breast Carcinoma
Authors: Kanzaki Atsuko , Toi Masakazu , Neamati Nouri , Miyashita Hitoshi , Oubu Masahiro , Nakayama Kentaro , Bando Hiroko , Ogawa Kenji , Mutoh Masato , Mori Shiro , Terada Kunihiko , Sugiyama Toshihiro , Fukumoto Manabu , Takebayashi Yuji
Published: August 2005

This is the first report to show that a copper‐transporting P‐type adenosine triphosphatase, ATP7B, is expressed in certain breast carcinomas, and a priori knowledge of its expression is important for the choice of therapy. We investigated the hypothesis that ATP7B, which was shown to be associated with cisplatin resistance in vitro, is expressed in certain breast carcinomas. To test this hypothesis, ATP7B expression and protein level were examined in 41 breast carcinomas using RT‐PCR and immunohistochemistry. ATP7B gene/protein could be detected in 22.0% (9/41) of breast carcinomas and ATP7B gene expression was correlated well with the protein expression. In nine ATP7B‐positive tumors, adjacent normal breast tissue was similarly analyzed, revealing that ATP7B is upregulated in breast carcinoma. ATP7B gene expression in poorly differentiated carcinoma was significantly higher than that in well‐/moderately differentiated carcinoma (P=0.012). Furthermore, we found no association between the ATP7B gene/protein expression and that of MDR1, MRP1, LRP and BCRP. These findings suggested that ATP7B gene expression might be a chemoresistance marker for cisplatin in patients with poorly differentiated breast carcinoma.
Functional Mimicry of an Anti‐idiotypic Antibody to Nominal Antigen on Cellular Response
Authors: Ma Jie , Zhou Liqiang , Wang Daqing
Published: August 2005

One concept for immune therapy of cancer involves induction of antigen mimic antibodies to trigger the immune system into a response against the tumor cells. Anti‐idiotypic antibodies (Ab2) directed against the antigen‐combining site of other antibodies (Ab1) may functionally and even structurally mimic antigen and induce anti‐anti‐idiotypic immune response. We report here the generation of murine monoclonal antibody (mAb) WJ02 (Ab2) raised against the murine monoclonal immunoglobulin MJ01 (Ab1), which defines ovarian cancer antigen CA125. In enzyme immunoassays the binding of Ab2 to the variable region of Ab1 could be inhibited by CA125. In addition, the mimicry of mAb WJ02 to CA125 on cellular immune response was detected by human peripheral blood cells. The T cells primed by mAb WJ02 or CA125 proliferated in the presence of CA125 or mAb WJ02, respectively. Furthermore, T cells specific to mAb WJ02 could lyse ovarian cancer cells OVCAR‐3 that express CA125. Finally, we proved that a patient immunized with mAb MJ01 could induce T cells that recognize mAb WJ02. In summary, we conclude that mAb WJ02 mimics CA125 on cellular response and such functional mimicry is one of the most important criteria to select Ab2 for cancer therapy.
Anti‐tumor Effect of Chemically Synthesized Sulfolipids Based on Sea Urchin's Natural Sulfonoquinovosylmonoacylglycerols
Authors: Sahara Hiroeki , Hanashima Shinya , Yamazaki Takayuki , Takahashi Shunya , Sugawara Fumio , Ohtani Seiji , Ishikawa Masako , Mizushina Yoshiyuki , Ohta Keisuke , Shimozawa Kumiko , Gasa Shinsei , Jimbow Kouichi , Sakaguchi Kengo , Sato Noriyuki , Takahashi Nobuaki
Published: August 2005

We recently reported that 3′‐sulfonoquinovosyl‐1′‐monoacylglycerol (designatedA‐5) extracted from sea urchin intestine was effective in suppressing the growth of solid tumors. Although the major fatty acid component of A‐5 was a saturated C16 acid, there were five other fatty acids, 14:0, 18:0, 14:1, 16:1, and 18:1, which constitute minor components of A‐5. Therefore, it remains unclear as to which of these six fatty acid components of A‐5 has the anti‐tumor effect. In this study, we synthesized sulfolipids each containing only one of these six fatty acids and tested their cytotoxicity against tumor cells and in vivo anti‐tumor effects on nude‐mice bearing solid tumors of human lung adenocarcinoma cell line A‐549. The IC50 values of all products against tumor cells were more than 10‐5M, suggesting weak cytotoxic activity compared with other chemotherapeutic compounds for cancer. On the other hand, in vivo anti‐tumor assay showed that sulfoquinovosyl‐monoacylglycerols (SQMG) composed of 14:1 and 18:1 (designated SQMG(14:1) and SQMG(18:1), respectively) were significantly effective in suppressing the growth of solid tumors. Our data suggested that these two SQMGs had a substantial anti‐tumor effect in vivo, and they are of interest as candidate drugs for anti‐cancer treatment.
HER2 Is Frequently Over‐expressed in Ovarian Clear Cell Adenocarcinoma: Possible Novel Treatment Modality Using Recombinant Monoclonal Antibody against HER2, Trastuzumab
Authors: Fujimura Masaki , Katsumata Noriyuki , Tsuda Hiroshi , Uchi Naoko , Miyazaki Satomi , Hidaka Takao , Sakai Masatoshi , Saito Shigeru
Published: August 2005

Ovarian clear cell adenocarcinoma (CCA) is generally chemo‐resistant. Recently the poor prognosis and resistance to chemotherapeutic agents of HER2/neu over‐expressing tumors have become clear. Thus, we investigated the expression level of HER2 in surgically resected CCA and ovarian serous adenocarcinoma, endometrioid adenocarcinoma, and mucinous adenocarcinoma specimens, as well as CCA cell lines, by an immunohistochemical method. HER2 was over‐expressed in 42.9% of CCA (P=0.026, vs. ovarian serous adenocarcinoma), 20.8% of ovarian serous adenocarcinoma, 23.1% of ovarian endometrioid adenocarcinoma, and 30.0% of mucinous adenocarcinoma specimens. Three CCA cell lines, RMG‐1, HAC‐II and KK were also positively stained for HER2. A flow‐cytometric study of HER2 revealed 7.2‐, 6.4‐ and 4.5‐fold greater expression of HER2 than that of normal mammary gland, respectively. Trastuzumab, a humanized recombinant monoclonal antibody against HER2 significantly and dose‐dependently reduced the growth of CCA cell lines in vitro. The extent of the inhibitory effect of trastuzumab was dependent on the expression level of HER2. Trastuzumab also dose‐dependently inhibited the growth of xenografted RMG‐1 tumor. The survival period of trastuzumab‐treated mice was longer than that of the control group. From these findings, trastuzumab appears to be a candidate as a treatment modality for HER2 over‐expressing ovarian CCA.
Bromocriptine Reverses P‐Glycoprotein‐mediated Multidrug Resistance in Tumor Cells
Authors: Shiraki Nobuaki , Okamura Keiko , Tokunaga Jin , Ohmura Takafumi , Yasuda Kazuto , Kawaguchi Takeo , Hamada Akinobu , Nakano Masahiro
Published: August 2005

One of the most important causes of anticancer treatment failure is the development of multidrug resistance (MDR). The main characteristics of tumor cells displaying the MDR phenomena are cross‐resistance to structurally unrelated cytotoxic drugs having different mechanisms of action and the overexpression of the MDR1 gene, which encodes a transmembrane glycoprotein named P‐ glycoprotein (P‐gp). This study evaluated whether bromocriptine, a D2 dopaminergic receptor agonist, influenced anticancer drug cytotoxicity and P‐gp activity in a P‐gp‐expressing cell line compared to a non‐expressing subline. The Ki values for P‐gp of cyclosporine and verapamil were 1.09 and 540 μM, respectively, and that of bromocriptine was 6.52 μM in a calcein‐AM efflux assay using porcine kidney epithelial LLC‐PK1 and L‐MDR1 cells, overexpressing human P‐gp. Bromocriptine at 10 μM reduced the IC50 of doxorubicin (DXR) in K562‐DXR from 9000 to 270 ng/ml and that of vincristine (VCR) in K562‐VCR from 700 to 0.30 ng/ml, whereas the IC50 values of DXR and VCR in the K562 subline were only marginally affected by these drugs. Bromocriptine restored the anticancer effect of DXR, VCR, vinblastine, vinorelbine and etoposide on MDR‐tumor cells overexpressing P‐gp. These observations suggest that bromocriptine has the potential to reverse tumor MDR involving the efflux protein P‐gp in the clinical situation.