bluuebunny/crossref_metadata_2025 huggingface dataset:
Schedule‐dependent Interactions between Raltitrexed and Cisplatin in Human Carcinoma Cell Lines in vitro
Authors: Kano Yasuhiko , Akutsu Miyuki , Suzuki Kenichi , Yazawa Yasuo , Tsunoda Saburo
Published: August 2005

Raltitrexed (‘Tomudex”) is a new anticancer agent which inhibits thymidylate synthase. To provide a rational basis for clinical trial design of the combination of raltitrexed and cisplatin, we studied the cytotoxic effects of this combination using various schedules in vitro and four human colon cancer cell lines, Colo201, Colo320, LoVo, and WiDr. Cell growth inhibition after 5 days was determined by using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) reduction assay. The effects of drug combinations at the concentration producing 80% cell growth inhibition (IC80) level were analyzed by the isobologram method. Simultaneous exposure to raltitrexed and cisplatin for 24 h, and sequential exposure to raltitrexed followed by cisplatin produced additive effects in the Colo201, Colo320, and LoVo cells, and additive and synergistic effects in WiDr cells. Sequential exposure to cisplatin followed by raltitrexed produced additive effects in the Colo201 cells and antagonistic effects in other three cell lines. Simultaneous and continuous exposure to both agents for 5 days produced additive effects in all four cell lines. These findings suggest that the simultaneous administration of raltitrexed and cisplatin, or the sequential administration of raltitrexed followed by cisplatin, generally produce the expected cytotoxicity at the cellular level and are optimal schedules, while the sequential administration of cisplatin followed by raltitrexed produces antagonistic effects and is inappropriate for this combination. Further in vivo and clinical studies will be necessary to determine the toxicity and antitumor effects of this schedule.
Photodynamic Inactivation with Acridine Orange on a Multidrug‐resistant Mouse Osteosarcoma Cell Line
Authors: Kusuzaki Katsuyuki , Minami Ginjirou , Takeshita Hideyuki , Murata Hiroaki , Hashiguchi Shin , Nozaki Takako , Ashihara Tsukasa , Hirasawa Yasusuke
Published: August 2005

Overcoming multidrug resistance (MDR) is an urgent issue to improve the prognosis of osteosarcoma patients. In this study, we undertook to clarify the effect of photodynamic therapy (PDT) with acridine orange (AO) on the MDR mouse osteosarcoma (MOS/ADR1) cell line, by comparing the outcome with the effect on a chemosensitive osteosarcoma (MOS) cell line. Cultured cells of MOS and MOS/ADR1 cell lines were exposed to AO at various concentrations for various times, followed by long‐ or short‐term (10 or 1 min) illumination with blue light (466.5 nm) for excitation. Living cells were counted by means of the trypan blue exclusion test. The results showed that AO rapidly bound to DNA, RNA and lysosomes of living MOS and MOS/ADR1 cells and also that most tumor cells in both cell lines died rapidly (viability ratio to untreated cells: 1/1000) within 48 h under conditions of continuous or 15‐min flash exposure to AO at concentrations above 1.0 μg/ ml plus 10‐min illumination with blue light. Even after flash exposure to AO at concentrations above 1.0 μg/ml plus 1‐min illumination, the viability of MOS/ADR1 cells decreased to a viability ratio of less than 1/1000 within 72 h. Based on these results, we concluded that AO with photo excitation has a strong cytocidal effect, not only on chemosensitive mouse osteosarcoma cells, but also on MDR mouse osteosarcoma cells. These results suggested that photodynamic therapy with AO may be a new approach to treating MDR human osteosarcomas.
Biodistribution of Boron Concentration on Melanoma‐bearing Hamsters after Administration of p‐, m‐, o‐Boronophenylalanine
Authors: Hiratsuka Junichi , Yoshino Kazuo , Kondoh Hirohumi , Imajo Yoshinari , Mishima Yutaka
Published: August 2005

Although p‐boronophenylalanine (p‐BPA), a boronate analogue of tyrosine, has proven to be one of the most successful compounds for boron neutron capture therapy (BNCT) of malignant melanoma, the selective uptake mechanism of this compound into melanoma cells is not well understood. Therefore, the relationship between the structure of BPA and its specific affinity to melanoma cells appears worthy of investigation. In the present study, m‐ and o‐boronophenylalanine (m‐ and o‐BPA) were administered to melanoma‐bearing hamsters and their uptake was measured. The time courses (0.5, 2.0, 4.0 and 48.0 h) of boron concentrations in melanoma, normal skin, and blood were determined in male Syrian (golden) hamsters bearing Greene's melanomas following a single intraperitoneal injection of either p‐, m‐ or o‐BPA (100 mg/kg of BPA·fructose in 1.0 ml of saline). The boron concentrations in these tissues were measured by inductively coupled plasma‐atomic emission spectrometry (ICP‐AES). In melanoma, the order of boron uptake was p‐ >m‐>o‐BPA at all time points, and the boron concentrations obtained with p‐BPA and m‐BPA resembled each other in that they had a peak at 2 h after administration and decreased with time. The melanoma/skin boron concentration ratio of p‐BPA had a peak at 4 h after administration and the ratio ranged between 7/1 and 8/1. On the other hand, m‐BPA and o‐BPA had a peak at 2 h and their ratios ranged between 4/1 and 5/1. The difference in the accumulations of p‐BPA and m‐BPA could be due to a difference in the property of p‐BPA as a tyrosine analogue for melanin synthesis. The accumulation of m‐BPA into melanoma might indicate the baseline level of metabolism‐related amino acid transport. Our experimental findings indicate that this melanin synthesis, or the structural analogy between the boron compound and tyrosine as a precursor of melanin, is an important factor in the increased accumulation of p‐BPA in melanoma cells.
No Association of the 5′Promoter Region Polymorphism of CYP17 with Breast Cancer Risk in Japan
Authors: Hamajima Nobuyuki , Iwata Hiroji , Obata Yuichi , Matsuo Keitaro , Mizutani Mitsuhiro , Iwase Takuji , Miura Shigeto , Okuma Katashi , Ohashi Kazuhiko , Tajima Kazuo
Published: August 2005

To examine the association between breast cancer risk and a T‐to‐C substitution polymorphism at the 5′promoter region of CYP17, a case‐control study was conducted at Aichi Cancer Center Hospital in Japan. Subjects were 144 histologically confirmed breast cancer patients diagnosed in the past 4 years and 166 hospital controls without cancer. Allele frequency among controls was 44.9% (95% confidence interval; 39.5–50.2) for C allele. Odds ratio (OR) of the polymorphism relative to TT‐genotype was 0.97 (0.58‐1.64) for TC‐genotype and 0.81 (0.39–1.68) for CC‐genotype. Subgroup analyses revealed that the OR was not statistically significant for the subgroups stratified by interval after diagnosis, age at menarche, age at first birth, menopausal status, body mass index, and mother/sisters’ history of breast cancer. Consistent with previous studies conducted in other countries, the 5′promoter region polymorphism of CYP17 affected breast cancer risk of Japanese women to a limited extent. Although this is not a large‐scale case‐control study with population controls, these findings provide enough information to discourage further studies on the association between this polymorphism and breast cancer risk in Japan at large, and suggest that this polymorphism is useless for breast cancer risk estimation.
Chemoprevention by Nimesulide, a Selective Cyclooxygenase‐2 Inhibitor, of 2‐Amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP)‐induced Mammary Gland Carcinogenesis in Rats
Authors: Nakatsugi Seiichi , Ohta Toshihisa , Kawamori Toshihiko , Mutoh Michihiro , Tanigawa Tetsuya , Watanabe Kouji , Sugie Shigeyuki , Sugimura Takashi , Wakabayashi Keiji
Published: August 2005

Breast cancer is common in women all over the world, and exploration of chemopreventive approaches to this cancer is very important. Nimesulide, a selective inhibitor of cyclooxygenase‐2 (COX‐2), is a good candidate as a chemopreventive agent with low toxicity. We examined its effects on mammary tumor development in female Sprague‐Dawley rats induced with the environmental carcinogen 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP). Rats at 7 weeks of age received intragastric intubations of PhIP (85 mg/kg body weight) 4 times weekly for 2 weeks and were maintained on control diet (high fat diet) or experimental diet (high fat diet supplemented with 400 ppm nimesulide) throughout the experiment. COX‐2 protein was over‐expressed in epithelial cancer cells and stromal cells of the PhIP‐induced mammary carcinomas, but was weak or not apparent in normal mammary gland cells. The development of mammary carcinomas was clearly suppressed by administration of nimesulide. The carcinoma incidence was 51% as compared to 71% for the control diet group. The average multiplicity of carcinomas in the experimental diet group was 1.2±0.2 (P < 0.05), significantly smaller than the control diet group value (2.6±0.5). The size of carcinomas was also clearly decreased; 1.1±0.4 cm3/rat in experimental diet group (P < 0.05), 4.1±1.3 cm3/rat in the control diet group. The results therefore provide evidence that the selective COX‐2 inhibitor, nimesulide, possesses chemopreventive activity against PhIP‐induced mammary carcinogenesis in rats.
Lack of Modifying Effects of Environmental Estrogenic Compounds on the Development of Thyroid Proliferative Lesions in Male Rats Pretreated with N‐Bis(2‐hydroxypropyl)nitrosamine (DHPN)
Authors: Son Hwa‐Young , Nishikawa Akiyoshi , Ikeda Takako , Nakamura Hideaki , Miyauchi Makoto , Imazawa Takayoshi , Furukawa Fumio , Hirose Masao
Published: August 2005

The modifying effects of various environmental estrogenic compounds on thyroid carcinogenesis were investigated in a rodent two‐stage carcinogenesis model. The compounds examined were a soy isoflavone mixture (SI) and genistein (GEN) as phytoestrogens, nonylphenol (NP) as a xenoestrogen, 3‐chloro‐4‐(dichloromethyl)‐5‐hydroxy‐2(5H)‐furanone (MX) as a thyroid carcinogen and sulfadimethoxine (SDM) as a known thyroid tumor promoter. Five‐week‐old male F344 rats were given a single subcutaneous injection of N‐bis(2‐hydroxypropyl)nitrosamine (DHPN; 2800 mg/kg, body weight) or the vehicle alone. Starting one week thereafter, GEN (250 or 25 ppm in diet), SI (400 ppm in diet), NP (250 or 25 ppm in diet), MX (30 ppm, in drinking water) or SDM (1000 ppm in drinking water) was administered for 12 weeks. Major organs including the thyroid, pituitary, liver, kidney, testis, brain and pancreas were weighed and histopathological observation was performed. Thyroid weights were significantly increased (P < 0.001) only in the SDM treatment groups, especially with DHPN pretreatment. Kidney weights were slightly increased in the NP or MX treatment groups, albeit without statistical significance. Histopathologically, thyroid proliferative lesions were only observed in the SDM alone or DHPN+SDM group with significant focal hyperplasias, adenomas and adenocarcinomas limited to the combined treatment case. There were no organ weight changes or histopathological lesions in the major organs including the thyroid in the GEN, SI, NP, and MX treatment groups regardless of DHPN pretreatment. Our results thus indicate that the weakly estrogenic compounds GEN, SI and NP and the environmental rat thyroid carcinogen MX do not exert any modifying effects on thyroid carcinogenesis in rats under the present experimental conditions.
Modulation of Myogenic Differentiation in a Human Rhabdomyosarcoma Cell Line by a New Derivative of 5‐Fluorouracil (QF‐3602)
Authors: Marchal Juan Antonio , Melguizo Consolación , Prados Jose , Aránega Amelia Eva , Gómez Jose Antonio , Campos Joaquin , Gallo Miguel Angel , Espinosa Antonio , Arena Nicolo , Aránega Antonia
Published: August 2005

The in vitro study of mechanisms involved in drug‐induced maturation has made it possible to use differentiation‐based therapy in clinical practice. The goal of this new therapy is the development of specific agents to induce cancer cells to stop proliferating and express characteristics of normal cells. Recently, by structural modifications of 5‐fluorouracil (5‐FU), we synthesized a new pyrimidine acyclonucleoside‐like compound, 1‐{[3‐(3‐chloro‐2‐hydroxypropoxy)‐1‐methoxy]propyl}‐5‐fluorouracil (QF‐3602), which showed in rhabdomyosarcoma cells a low toxicity and time‐dependent growth inhibition. In this work, we compared the degree of myogenic differentiation of RD rhabdomyosarcoma (RMS) cells after treatment with QF‐3602 and 5‐FU. Scanning and transmission electron microscopy (SEM and TEM) and immunocytochemical analyses showed that QF‐3602 induced the appearance of myofilaments along the myotube‐like giant RD cells, an increase in fibronectin and a decrease in vimentin expression. In contrast, only minor changes were observed with 5‐FU. Moreover, polymerase chain reaction (PCR) analyses showed that QF‐3602 did not induce overexpression of the mdr 1 gene, a resistance mechanism that frequently appears in classical cytotoxic therapy in these tumors. Compounds obtained by structural modifications of 5‐FU may be useful in differentiation therapy as a new approach to the treatment of RMS.
Second Primary Cancers Following Breast Cancer in the Japanese Female Population
Authors: Tanaka Hideo , Tsukuma Hideaki , Koyama Hiroki , Kinoshita Yoko , Kinoshita Noriko , Oshima Akira
Published: August 2005

To assess the risk of developing second primary cancers following breast cancer in Japanese females, we performed a retrospective cohort study of 2786 patients who were newly diagnosed with breast cancer at our hospital between 1970‐1994, until the end of 1995 (average follow‐up period, 8.6 years). The expected number of each second primary cancer was calculated by multiplying the number of appropriate person‐years at risk by the corresponding age‐ and calendar period‐specific cancer incidence rates for women obtained from the Osaka Cancer Registry. One hundred and seventeen patients developed a second primary cancer other than subsequent breast cancer, yielding an observed‐to‐expected ratio (O/E) of 1.3 [95% confidence interval (CI)=1.1‐1.6]. The risk for developing a second primary cancer was significantly elevated during the first year following the diagnosis of breast cancer, and decreased with the passage of tune to unity. A significantly increased risk was noted for the development of ovarian cancer (O/E=2.4, 95% CI=1.0‐4.6), thyroid cancer (O/E=3.7, 95% CI=1.5‐7.6) and non‐Hodgkin's lymphoma (NHL) (O/E=3.5, 95% CI=1.4‐7.1) among the breast cancer patients compared with the general population. Patients who received hormonal therapy as the breast cancer treatment showed a significantly increased risk for ovarian cancer (O/E=5.5, 95% CI=1.8‐12.9). Patients who received chemotherapy as the breast cancer treatment had an increased risk for NHL (O/E=5.0, 95% CI=1.6‐11.6). These findings indicate that Japanese female patients with breast cancer had a 30% higher risk of developing a second primary cancer than the general population, the higher risk being manifested in the early period following the diagnosis of breast cancer. Medical surveillance of breast cancer patients for NHL, as well as for ovarian cancer and thyroid cancer, is required.
Apoptosis of Muscle Cells Causes Weight Loss Prior to Impairment of DNA Synthesis in Tumor‐bearing Rabbits
Authors: Ishiko Osamu , Sumi Toshiyuki , Hirai Kouzo , Honda Ken‐ichi , Nakata Shinichi , Yoshida Hiroyuki , Ogita Sachio
Published: August 2005

The mechanism of weight loss induced by the growth of malignant tumors is still unknown. We investigated it by focusing on apoptosis of skeletal muscle. VX2‐tumor was implanted into rabbits and the apoptotic index (AI) of skeletal muscle was measured by in situ end‐labeling assay. Plasma of the tumor‐bearing rabbits was perfused repeatedly through non‐coated charcoal resin. The AI reached 54.6% early after tumor implantation, when weight loss amounted to an 18% decrease in lean body mass (LBM) without change in muscle DNA synthesis or urinary 3‐methylhistidine/ creatinine ratio (3‐MH/Cr). When the decrease of LBM reached 30%, DNA synthesis was decreased by 48% and 3‐MH/Cr was increased by 104%, whereas AI was only 4.7%. The plasma perfusion did not prevent apoptosis in muscle, but unproved LBM, DNA synthesis, and 3‐MH/Cr. There may be two mechanisms of muscle depletion during the tumor growth: apoptosis in the early stage and metabolic abnormalities in muscle in the late stage.
Genetic Mapping and Allelic Loss Analysis in Mouse Thymic Lymphomas of Helios and Aiolos Belonging to the Ikaros Gene Family
Authors: Xu Hongbin , Wakabayashi Yuichi , Okano Hitomi , Saito Yuko , Miyazawa Tomonori , Kominami Ryo
Published: August 2005

The Ikaros gene undergoes bi‐allelic changes at a high frequency in γ‐ray‐induced mouse thymic lymphomas, suggesting the relevance of Ikaros to the lymphoma development. Here we test whether Helios and Aiolos, two other members of the Ikaros gene family, are also involved in lymphomagenesis. Genetic mapping showed that Helios is located between D1Mit531 and D1Mit19 on chromosome 1 and Aiolos is between D11Mit222 and D11Mit332 on chromosome 11. Analysis using polymorphic markers around the two regions revealed that neither locus exhibited allelic loss in the 78 lymphomas that were induced in p53 wild‐type mice, whereas in 102 p53(KO/+) mouse‐derived lymphomas Helios and Aiolos loci showed allelic loss in 8% (8/102) and 33% (34/102), respectively. However, 33 of the 34 lymphomas showing allelic loss at Aiolos were/j53(KO/‐) and were accompanied by loss of the p53 wild‐type allele on the same chromosome. Homozygous deletion and mutation analyses failed to detect bi‐allelic alterations. These results do not suggest any obvious contribution of Helios or Aiolos to oncogenesis of the mouse thymic lymphomas.
Influence of Previous Interferon Therapy on Recurrence after Resection of Hepatitis C Virus‐related Hepatocellular Carcinoma
Authors: Kubo Shoji , Nishiguchi Shuhei , Hirohashi Kazuhiro , Tanaka Hiromu , Tsukamoto Tadashi , Shuto Taichi , Takemura Shigekazu , Yamamoto Takatsugu , Ikebe Takashi , Wakasa Kenichi , Shiomi Susumu , Kinoshita Hiroaki
Published: August 2005

Interferon (IFN) therapy decreases the incidence of hepatocellular carcinoma (HCC) in patients with hepatitis C virus (HCV). One hundred and fifty‐nine consecutive patients who underwent liver resection for HCV‐related HCC were studied. In 17 (group 1) of the 159 patients, HCC was detected during or after IFN therapy. The incidences of recurrence after surgery in the group 1 patients and the other 142 patients (group 2) were compared. Eight patients had a complete response to IFN, 4 had a partial response, and 5 had no response. The proportion of patients without HCV viremia was significantly higher in the group 1 patients (P<0.0001). The tumor‐free survival rate was significantly higher in the group 1 patients (P=0.0010). By multivariate analysis of various risk factors for recurrence, no previous IFN was a significant independent risk factor for recurrence (risk ratio=6.336; 95% CI, 1.512‐26.50). The patients with HCC who underwent IFN therapy previously are good candidates for liver resection because recurrence after the operation was rarely observed.
Schedule‐dependent Synergism and Antagonism between Raltitrexed (“Tomudex”) and Methotrexate in Human Colon Cancer Cell Lines in vitro
Authors: Kano Yasuhiko , Akutsu Miyuki , Tsunoda Saburo , Suzuki Kenichi , Yazawa Yasuo , Furukawa Yusuke
Published: August 2005

The folate‐dependent enzymes are attractive targets for cancer chemotherapy. Methotrexate (MTX), which inhibits dihydrofolate reductase, has been widely used for the treatment of solid tumors and hematological cancers. Raltitrexed (“Tomudex”), which inhibits thymidylate synthase, is a novel anticancer agent active against colorectal cancer and some other solid tumors. We studied the optimal schedule of raltitrexed and MTX in combination against four human colon cancer cell lines Colo201, Colo320, LoVo, and WiDr. These cells were simultaneously exposed to raltitrexed and MTX for 24 h, or sequentially exposed to raltitrexed for 24 h followed by MTX for 24 h, or vice versa. Cell growth inhibition after 5 days was determined by using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. The effects of drug combinations at the concentrations of drug that produced 80% and 50% cell growth inhibition (Icg80 and IC50) were analyzed by the isobologram method (Steel and Peckham, 1979). Cytotoxic interactions between raltitrexed and MTX were schedule‐dependent. The simultaneous exposure to raltitrexed and MTX showed additive effects in Colo201, LoVo and WiDr cells and antagonistic effects in Colo320 cells. The sequential exposure to raltitrexed followed by MTX produced additive effects in all four cell lines. The sequential exposure to MTX followed by raltitrexed produced synergistic effects in Colo201, LoVo and WiDr cells and additive effects in Colo320 cells. These findings suggest that the sequential administration of MTX followed by raltitrexed produces more than the expected cytotoxicity and may be the optimal schedule at the cellular level. Further in vivo and clinical studies will be necessary to determine the toxicity and to test the antitumor effects of sequential administration of MTX followed by raltitrexed proposed on the basis of the in vitro synergism.
Vaccination Effect of Interleukin‐6‐producing Pancreatic Cancer Cells in Nude Mice: A Model of Tumor Prevention and Treatment in Immune‐compromised Patients
Authors: Yano Tomoyuki , Ishikura Hiroshi , Kato Hiroaki , Ogawa Yayoi , Kondo Satoshi , Kato Hiroyuki , Yoshiki Takashi
Published: August 2005

In an effort to explore properties important in hematogenous metastasis of pancreatic adenocarcinoma, we previously demonstrated that tumor‐derived interleukin (IL)‐6 is a crucial factor that conveys resistance to liver metastasis. Here we extend the study to examine a possible vaccination effect of tumor‐derived IL‐6 in T‐cell‐deficient nude mice, as a model for predicting the effect in immune‐compromised patients. We used a pair of IL‐6‐nonproducing and highly producing pancreatic adenocarcinoma cell lines, PCI‐43 and PCI‐43h, respectively. The reaction intensity of anti‐PCI IgG antibodies in host nude mice was maximal 28 days after inoculation of PCI‐43h cells, and remained high thereafter. A fraction of the pancreatic carcinoma cell lines, namely, PCI‐6, ‐10, and ‐43, expressed surface antigenic determinant(s) reactive with the IgG; but the others, PCI‐19, ‐24, ‐55, ‐64, ‐66, ‐68, ‐72, and ‐79, did not. Inoculation of PCI‐43h but not PCI‐43 suppressed growth of simultaneously inoculated PCI‐43, but not PCI‐24 xenografts. In addition, administration of PCI‐43h, but not PCI‐43 suppressed the growth of PCI‐43 that was xenografted 4 weeks later, thus revealing a vaccination effect of IL‐6‐producing PCI‐43h, but not IL‐6‐nonproducing PCI‐43. These data, obtained from T‐cell‐deficient nude mice, suggest an in vivo role for IL‐6 in inducing IgG‐mediated, pancreatic carcinoma‐specific vaccination against a thymus‐independent antigen.
Biological Cell Survival Mapping for Radiofrequency Intracavitary Hyperthermia Combined with Simultaneous High Dose‐rate Intracavitary Irradiation
Authors: Kurosaki Hiromasa , Sakurai Hideyuki , Mitsuhashi Norio , Tamaki Yoshio , Akimoto Tetsuo , Takahashi Takeo , Furuta Masaya , Saitoh Jun‐ichi , Hayakawa Kazushige , Niibe Hideo
Published: August 2005

We examined the best way to combine recently developed radiofrequency intracavitary hyperthermia with simultaneous high dose‐rate intracavitary brachytherapy in an original experimental model. Temperature distribution was measured with an experimental phantom which was immersed in a water bath with the temperature controlled at 37°C. Radiation dose distribution was calculated with a treatment‐planning computer. Cell survival was measured by colony assay with HeLa‐TG cells in vitro. Radiation dose response at 1‐7 Gy and time response with hyperthermia in the range of 40‐46°C were estimated. Radiation dose‐response curves in simultaneous treatment with hyperthermia for 30 min at 37 to 46°C were estimated and the surviving fractions in combined treatment were plotted against temperature. For intracavitary radiation alone, cell survival rates increased with increasing distance from the source. For intracavitary hyperthermia alone, the maximum temperature was observed at a depth of 13 mm from the surface of the applicator under suitable treatment conditions. Homogeneous cell killing from the surface of the applicator to a tumor depth of 13 mm was observed under a specific treatment condition. Our experimental model is useful for evaluating the best simultaneous combined treatment.
Alcohol, Smoking, and Dietary Status and Susceptibility to Malignant Lymphoma in Japan: Results of a Hospital‐based Case‐control Study at Aichi Cancer Center
Authors: Matsuo Keitaro , Hamajima Nobuyuki , Hirose Kaoru , Inoue Manami , Takezaki Toshiro , Kuroishi Tetsuo , Tajima Kazuo
Published: August 2005

Recent increase in the incidence of malignant lymphoma (ML) suggests possible involvement of lifestyle or environmental factors in its genesis. However, evidence for an effect of lifestyle factors, especially diet, on ML risk among Japanese is lacking. To explore the possibility that lifestyle factors exert an influence, we have conducted a hospital‐based case‐control study with 333 histo‐logically confirmed ML cases and 55 904 non‐cancer controls who first visited Aichi Cancer Center Hospital between 1988 and 1997. Multiple logistic regression analysis showed regular alcohol consumption to be associated with reduced risk of ML, whereas no risk change was observed for smoking. Some other factors including intake of vegetables (carrots and pumpkin), pork and fish showed partial associations, but their significance needs further clarification. From the previous study on genetic background for ML [Matsuo et al., Blood, 97, 3205–3209 (2001)], genetic variation combined with limited environmental factors should be targeted in future studies.
Beef Tallow, but Not Perilla or Corn Oil, Promotion of Rat Prostate and Intestinal Carcinogenesis by 3,2‐Dimethyl‐4‐aminobiphenyl
Authors: Mori Toshio , Imaida Katsumi , Tamano Seiko , Sano Masashi , Takahashi Satoru , Asamoto Makoto , Takeshita Masazumi , Ueda Hiroshi , Shirai Tomoyuki
Published: August 2005

The modifying effects of three kinds of fat (corn oil, beef tallow or perilla oil, each at 20% in the diet) on F344 rat prostate carcinogenesis induced by 3,2‐dimethyl‐4‐aminobiphenyl (DMAB) were investigated. Non‐invasive carcinomas of the ventral prostate were induced by DMAB alone and invasive carcinomas of the other prostate lobes and seminal vesicles by DMAB and testosterone propionate (TP). Eight groups of F344 rats were initiated with 50 mg/kg body weight of DMAB at 2‐week intervals for the first 20 weeks, four also receiving TP, extended until week 60. The animals received basal chow powder diet or one of three high fat diets throughout the experiment (60 weeks). One further group served as a non‐carcinogen‐treated control maintained on basal chow powder diet. Beef tallow significantly increased the development of ventral prostate carcinomas with DMAB alone (from 15 to 45%, P<0.05), while perilla oil reduced the incidence of prostatic intraepithelial neoplasia (PIN) in the ventral lobe of rats given DMAB + TP (from 70 to 10%, P<0.01), but not in those given DMAB alone. No other effects of high fats were observed regarding PIN or invasive cancers of the dorsolateral and anterior prostate or seminal vesicles. A satellite experiment demonstrated that all high fat diets for 4 weeks increased the 5‐bromo‐2‐deoxyuridine (BrdU) labeling index of prostate epithelial cells, suggesting that a high fat intake, irrespective of the fatty acid composition, may accelerate cell kinetics in the prostate. Of the three high fat diets, beef tallow was also found to increase intestinal carcinogenesis. Thus, the present data revealed carcinogenesis in the prostate and intestine to be promoted by beef tallow.
DNA Sequence of Immunoglobulin Heavy Chain Variable Region Gene in Thyroid Lymphoma
Authors: Miwa Hideaki , Takakuwa Tetsuya , Nakatsuka Shin–ichi , Tomita Yasuhiko , Matsuzuka Fumio , Aozasa Katsuyuki
Published: August 2005

Patho–epidemiological studies have shown that thyroid lymphoma (TL) develops in thyroid affected by chronic lymphocytic thyroiditis (CLTH). CLTH is categorized as an organ–specific autoimmune disease, in which activated B–lymphocytes secrete a number of autoantibodies. Because antigenic stimulation might be involved in the pathogenesis of TL, the variable region in heavy chain (VH) genes was characterized in 13 cases with TL and 3 with CLTH. Clonal rearrangement of the VH gene was found in 11 cases of TL, and cloning study with sequencing of complimen–tarity determining region (CDR) 3 revealed the presence of a major clone in 4. Three of the 4 cases used VH3 gene, with the homologous germline gene of V3–30 in two cases and VH26 in one case. A biased usage of VH3 and VH4 genes with the homologous germline gene of VH26 in VH3 gene was reported previously in cases with CLTH. A high level of somatic mutation (1–21%, average 12%) with non–random distribution of replacement and silent mutations was accumulated in all cases. The frequency of the occurrence of minor clones ranged from 29–44% per case, indicating the presence of on–going mutation. DNA sequencing of immunoglobulin VH gene suggests that TL develops among activated lymphoid cells in CLTH at the germinal center stage under antigen selection
Molecular Analysis of Tumor Suppressor Genes, Rb, p53,pl6INK4A, pl5INK4B and p14ARF in Natural Killer Cell Neoplasms
Authors: Sakajiri Sakura , Kawamata Norihiko , Egashira Motoki , Mori Kiyoshi , Oshimi Kazuo
Published: August 2005

Natural killer (NK) cell neoplasms, which are derived from mature or precursor NK cells, are rare diseases and are observed predominantly in Asian countries. We analyzed the status of the Rb, p53, p15INK4B, p16INK4A and p14ARF genes in these diseases by Southern blot, polymerase chain reaction–single strand conformational polymorphism (PCR–SSCP) and western blot analysis. We used 31 NK cell neoplasms, including four cell lines derived from NK cell neoplasms, 3 myeloid/ NK cell precursor acute leukemias, 4 blastic NK cell lymphoma/leukemias, 4 aggressive NK cell leukemia/lymphomas, 4 nasal NK cell lymphomas, and 12 chronic NK lymphocytosis. We found gene amplification of the p53 gene in one nasal NK cell lymphoma, and point mutations of the p53 gene in one blastic NK cell lymphoma/leukemia and one chronic NK lymphocytosis. In addition, homozygous deletions ofpl5,p16 andp14 genes in 5 out of 31 samples were detected; 3 were from nasal NK cell lymphoma and 2 from blastic NK cell lymphoma/leukemia. Also hemizygous deletion of the Kb gene in one blastic NK cell lymphoma was detected. Rb proteins were highly expressed in one cell line as well as two myeloid/NK cell precursor acute leukemias. In other cell lines, complete loss and an aberrant migration pattern of Rb protein expression were observed. Comparative genomic hybridization suggested that the homozygous deletions of the p15, p16 and p14 were subtle chromosomal deletions and could not be identified by standard karyotyping in some cases. Although the number of cases we analyzed was not large, alterations identified in the Rb, p53, p16, p15 and p14 genes are of significance and might be associated with tumorigenesis in NK cell neoplasms
Loss of Syndecan‐1 and Increased Expression of Heparanase in Invasive Esophageal Carcinomas
Authors: Mikami Shuji , Ohashi Kenichi , Usui Yutaka , Nemoto Tetsuo , Katsube Ken‐ichi , Yanagishita Masaki , Nakajima Motowo , Nakamura Kyouichi , Koike Morio
Published: August 2005

Heparan sulfate proteoglycans play important biological roles in cell‐cell and cell‐matrix adhesion, and are closely associated with growth factor actions. Loss of syndecan‐1, a cell surface‐bound heparan sulfate proteoglycan, has been reported for advanced head and neck carcinomas, and expression of endoglycosidic heparanase, which cleaves heparan sulfate glycosaminoglycans (HS‐GAGs), is associated with invasion and metastatic potential of malignant tumors. Paraffin sections of 103 primary esophageal squamous cell carcinomas were immunohistochemically examined for the expression of syndecan‐1 core protein, HS‐GAGs and heparanase protein, and the results were compared with various clinicopathological parameters, such as invasion depth. For 16 cases, fresh tumor samples were quantitatively analyzed for heparanase and syndecan‐1 mRNA expression by real‐time RT‐PCR in addition to the immunohistochemical studies. Syndecan‐1 core protein and HS‐GAGs expression was significantly decreased in pT2 and pT3 cases compared with their pTis and pTl counterparts. Decreased expression of core protein and HS‐GAGs was correlated with the incidence of lymphatic invasion, and venous involvement. Furthermore, decreased expression of HS‐GAGs was correlated positively with the incidence of nodal metastasis and distant organ metastasis, and negatively with the grade of tumor cell differentiation. The percentage of cytoplas‐mic heparanase protein‐positive cases increased significantly in pT2 and pT3 cases compared to that in pTis and pTl cases, and this was associated with lymphatic invasion, and venous and lymph nodal involvement. The level of heparanase mRNA was inversely correlated with the degree of HS‐GAGs expression rather than core protein. In conclusion, loss of syndecan‐1 and heparanase overexpression in esophageal squamous cell carcinomas are closely associated with malignant potential. Regarding the mechanism of loss of HS‐GAGs, heparanase upregulation appears to play an important role.
Targeting Hyperthermia for Renal Cell Carcinoma Using Human MN Antigenspecific Magnetoliposomes
Authors: Shinkai Masashige , Le Biao , Honda Hiroyuki , Yoshikawa Kazuhiro , Shimizu Kazuhiro , Saga Shinsuke , Wakabayashi Toshihiko , Yoshida Jun , Kobayashi Takeshi
Published: August 2005

Magnetoliposomes (MLs) conjugated with an antibody fragment to give specificity to a tumor were applied to hyperthermia for cancer. The Fab' fragment of the G250 antibody, which binds to MN antigen on many types of human renal cell carcinoma, was cross–linked to Af–(6–maleimidocaproyl–oxy)–dipalmitoyl phosphatidylethanolamine (EMC–DPPE) in liposomal membrane. The targetabil–ity of the G250–Fab' fragment–conjugating MLs (G250–FMLs) was investigated using the mouse renal cell carcinoma (mRCC) and MN antigen–presenting cell, MN–mRCC. The amount of G250–FMLs uptake reached 67 pg/cell against MN–mRCC cells in an in vitro experiment using plastic dishes and this value was about 6 tunes higher than that in the case of MLs. In an in vivo experiment using MN–mRCC–harboring mice, 1.5 mg of the FMLs per carcinoma tissue accumulated (tumor weight was 0.19 g), which corresponded to approximately 50% of the total injection. This value was 27 tunes higher than that of the MLs. After injection of the FMLs, mice were exposed to intracellular hyperthermia using alternating magnetic field irradiation. The temperature of tumor tissue increased to 43°C and the growth of the carcinoma was strongly arrested for at least 2 weeks. These results indicate the G250–FMLs could target renal cell carcinoma cells in vitro and in vivo, and are efficiently applicable to the hyper thermic treatment of carcinoma.
Signal Transduction Pathways through TRK‐A and TRK‐B Receptors in Human Neuroblastoma Cells
Authors: Sugimoto Tohru , Kuroda Hiroshi , Horii Yoshihiro , Moritake Hiroshi , Tanaka Takeo , Hattori Seisuke
Published: August 2005

Little is known about the signal transduction pathways of TRK family receptors in neuroblastoma (NB) cells. In this study, an NB cell line, designated MP‐N‐TS, was established from an adrenal tumor taken from a 2‐year‐old boy. This cell line expressed both TRK‐A and TRK‐B receptors, which is rare in a single NB cell line. Therefore, the MP‐N‐TS cell line was used to determine whether the signal transduction through these constitutive receptors is functional. Three neurotrophins, nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF) and neurotrophin‐4/ 5 (NT‐4/5), induced tyrosine phosphorylation of panTRK, and BDNF and NT‐4/5 induced tyrosine phosphorylation of TRK‐B. Tyrosine phosphorylation of panTRK and/or TRK‐B by the neurotro‐phins was inhibited in the presence of a tyrosine kinase inhibitor K252a. Tyrosine phosphorylation of Src homologous and collagen (She), extracellular signal‐regulated kinase (ERK)‐l and ERK‐2, and phospholipase C‐γl (PLC‐γl) was increased by the three neurotrophins and the increase was inhibited in the presence of K252a. Activation of Ras, detected as the GTP‐bound form of Ras, was induced by the three neurotrophins. The neurotrophins did not modulate the expressions of TRK‐A or TRK‐B mRNA, but they did induce the expression of c‐fos mRNA. Exogenous NGF induced weak neurite outgrowth, whereas exogenous BDNF and NT‐4/5 induced distinct neurite outgrowth. Exogenous BDNF and NT‐4/5 increased the number of viable cells, while NGF did not. Our results demonstrate that the signal transduction pathways through TRK‐A and TRK‐B in MP‐N‐TS cells are functional and similar, and the main downstream signaling pathways from the three neurotrophins are mitogen‐activated protein kinase (MAPK) cascades through She, activated Ras, ERK‐1 and ERK‐2, and the transduction pathway through PLC‐γl. Further, BDNF and NT‐4/5 increased cell viability. The MP‐N‐TS cell line should be useful for clarifying the TRK‐A and TRK‐B signaling pathways responsible for the different prognoses in patients with NB.
Expression of MAGE‐B Genes in Esophageal Squamous Cell Carcinoma
Authors: Nagashima Hideki , Sadanaga Noriaki , Mashino Kohjiro , Yamashita Keishi , Inoue Hiroshi , Mori Masaki , Sugimachi Keizo
Published: August 2005

The MAGE‐B (MAGE‐B1, ‐B2, ‐B3, and ‐B4) genes share strong homology with the MAGE‐A gene family. MAGE‐B1 and ‐B2 encode common tumor‐specific peptide antigens. There is, however, still very little information about the expression of these genes in human gastro‐intestinal carcinomas. We investigated the expression of MAGE‐B1 and ‐B2 genes in 29 cell lines and 53 clinical tumor samples of esophageal squamous cell carcinoma by reverse transcription polymerase chain reaction (RT‐PCR). MAGE‐B1 and ‐B2 gene transcripts were detected by RT‐PCR in 1 (3%) and 6 (21%) cell lines, and in 9 (17%) and 17 (32%) clinical samples, respectively. Among them, 7/29 (24%) cell lines and 19/53 (36%) clinical samples expressed at least either MAGE‐B1 or ‐B2. A significant correlation was found between negative MAGE‐B gene expression and vascular invasion (P=0.008). In 45 out of 53 esophageal carcinoma RNA samples, the MAGE‐A1, ‐A2, and ‐A3 genes were detected in 27 (60%), 23 (51%), and 30 (67%) samples, respectively, while the MAGE‐B genes were detected in 18 (40%) samples. The frequency of MAGE‐B gene expression in esophageal carcinoma was relatively higher than that observed for gastric or colorectal carcinomas (12% and 2%, respectively). Therefore, the MAGE‐B genes could be used as targets in specific immunotherapy of esophageal squamous cell carcinomas.
Examination of in vitro Chemosensitivity Test Using Collagen Gel Droplet Culture Method with Colorimetric Endpoint Quantification
Authors: Kobayashi Hisayuki , Higashiyama Masahiko , Minamigawa Kazuhiko , Tanisaka Keizo , Takano Toshikazu , Yokouchi Hideoki , Kodama Ken , Hata Taeko
Published: August 2005

To develop a simpler method of performing the collagen gel droplet‐embedded culture drug sensitivity test (CD‐DST), we examined the introduction of colorimetric quantitative determination of images for evaluation of anticancer effect against cancer cells alone in the presence of fibroblasts, based on differences in proliferative morphology and stainability with neutral red of cells within collagen gel drops determined using a video‐microscope and NIH Image software. In examinations using a human cancer cell line and a fibroblast cell line, a high degree of linearity between number of cancer cells and image‐optical density was found within the range of 102–106 cells/droplet (r2=0.933). Using NIH Image, fibroblast cells could be eliminated at a cut‐off value of 128, and an immunocytochemical method demonstrated that the cells eliminated from the image were indeed fibroblasts, and those remaining were cancer cells. CD‐DST was carried out with mixtures of cancer cells with fibroblasts at various ratios, and the feasibility of evaluating anticancer activity in cancer cells alone with no effect of fibroblasts at any mixing ratio was confirmed. In addition, for CD‐DST of primary cell cultures of human lung cancers collected at the time of surgery, a high correlation between results obtained with the volume supplementation method, a current cell quantification method, and those with the imaging colorimetric quantification method was obtained (r=0.933). These results indicate that introduction of imaging colorimetric quantification utilizing NIH Image makes CD‐DST a quick and simple method that should be highly useful for clinical chemosensitivity testing using primary cell cultures of human cancers.
Sex Hormone Dependency of Diethylnitrosamine‐induced Liver Tumors in Mice and Chemoprevention by Leuprorelin
Authors: Nakatani Tamaki , Roy Goutam , Fujimoto Nariaki , Asahara Toshimasa , Ito Akihiro
Published: August 2005

The prevalence of liver tumors throughout the world makes it imperative to seek chemopreventive agents. This tumor appears to be hormone‐responsive and hormonal manipulations may therefore be beneficial. On this basis, both sexes of 12‐day‐old B6C3FJ mice were injected i.p. with diethylnitrosamine (DEN) at the dose of 2.5 μg/g body weight and observed for 32 weeks (males) or 36 weeks (females). In 100% of male mice, liver tumors were observed with an average diameter of 2.72 mm and multiplicity of 60.8. Orchidectomy at 6 weeks of age in these mice inhibited the incidence, multiplicity and size to 63%, 5.6 and 1.54 mm, respectively. By further implantation with an E2 pellet at monthly intervals, these parameters were reduced to 26%, 0.6 and 0.61 mm, respectively. Administration of a gonadotropin‐blocking chemical, leuprorelin, to DEN‐treated male mice significantly reduced the multiplicity and size of tumors to 18.3 and 2.54 mm (P<0.01 compared to those of DEN only). In female mice, the incidence of liver tumor was significantly smaller than that of males. However, ovariectomy and/or testosterone supplement significantly increased the occurrence of liver tumor. An anti‐estrogen, toremifene, caused a marked further decrease of liver tumors. Mitotic indices with bromodeoxyuridine in tumor tissues paralleled the occurrence of liver tumors. Serum testosterone levels were significantly reduced by orchidectomy or by leuprorelin administration. These results further confirm that liver tumor is testosterone‐responsive and hormonal manipulation by surgical orchidectomy or by chemical orchidectomy i.e. by leuprorelin, could substantially prevent the appearance of liver tumors.
Targets of Transcriptional Regulation by Transforming Growth Factor‐β: Expression Profile Analysis Using Oligonucleotide Arrays
Authors: Akiyoshi Shingo , Ishii Masami , Nemoto Nobuo , Kawabata Masahiro , Aburatani Hiroyuki , Miyazono Kohei
Published: August 2005

Transforming growth factor‐βs (TGF‐βs) are potent inhibitors of cell proliferation, and disruption of components of the TGF‐β signaling pathway leads to tumorigenesis. Mutations of transmem‐brane receptors and Smads mediating intracellular signaling have been reported in various cancers. To identify transcriptional targets of TGF‐β, we conducted an expression profile analysis. HaCaT cells derived from human keratinocytes and highly sensitive to TGF‐β were treated with TGF‐β in the absence or presence of cycloheximide (CHX). mRNAs extracted from the HaCaT cells were used for hybridization of oligonucleotide arrays representing approximately 5600 human genes. TGF‐β increased the expression of PAI‐1, junB, p21 cdk inhibitor, Smad7, βIG‐H3, and involucrin that have been reported to be up‐regulated by TGF‐β, validating the usefulness of this approach. The induction of βIG‐H3 by TGF‐β was completely abolished by CHX, suggesting that the transcription of βIG‐H3 is not directly regulated by TGF‐β. Unexpectedly, we identified more genes down‐regulated by TGF‐β than up‐regulated ones. TGF‐β repressed the expression of epithelial specific Ets that may be involved in breast and lung tumorigenesis, which could contribute to tumor suppression by TGF‐β. Among a panel of cell cycle regulators, TGF‐β induced the expression of p21 cdk inhibitor; however, the induction of other cdk inhibitors was not significant in the present study. Taken together, the results suggest that TGF‐β may suppress tumorigenesis through positive and negative regulation of transcription.
Glutathione S‐Transferases in Small Intestinal Mucosa of Patients with Coeliac Disease
Authors: Wahab Peter J. , Peters Wilbert H. M. , Roelofs Hennie M. J. , Jansen Jan B. M. J.
Published: August 2005

Patients with villous atrophy due to coeliac disease have an increased risk of developing small intestinal malignancies. Intestinal glutathione (GSH) and glutathione S‐transferases (GST) are involved in the protection against carcinogenesis. The aim of this study was to evaluate GSH content and GST enzyme activity in small intestinal mucosa of untreated coeliacs compared to controls. We evaluated GSH content and GST enzyme activity, including the levels of GST classes α, μ, π, θ in small intestinal biopsies of untreated coeliacs (flat mucosa, Marsh IHC, n=12) compared to normal subjects (n=23). Next, we evaluated GSH and GST's in coeliacs in remission (Marsh 0‐1, n=11), coeliacs with persisting villous atrophy while on a gluten‐free diet (partial villous atrophy, Marsh IIIA (n=5); subtotal villous atrophy, Marsh IIIB (n=6) and patients with infiltrative/crypt‐hyperplastic Marsh II lesions (n=4). Total GST enzyme activity and content of GSTa are markedly suppressed in Marsh IIIC lesions compared to controls (resp. 220±79 vs. 4641189 nmol/mg protein‐min (P<0.001) and 2.79±2.46 vs. 6.47±2.29 μg/mg protein (P<0.001). In coeliacs in remission these levels normalized. Total GST enzyme activity and GSTα levels are proportionately lowered according to the degree of mucosal pathology in Marsh II, IIIA and IIIB. (Spearman's σ correlation coefficient for total GST, ‐0.596, P<0.001; GSTα, ‐0.620, P<0.001). GSTμ, π and θ and GSH levels are not significantly different in the selected study groups of mucosal pathology compared to controls. Total GST enzyme activity and content of GSTα in small intestinal mucosa are significantly lower in untreated coeliac disease compared to controls. In Marsh II, IIIA and IIIB, GST enzyme activity and GSTα content are proportionally lower according to the degree of mucosal pathology. Normal values are seen in coeliacs in remission. This correlation between coeliac disease and a suppressed GSH/GST detoxification system may explain in part the carcinogenic risk in untreated coeliac disease.