bluuebunny/crossref_metadata_2025 huggingface dataset:
Uptake and Intracellular Distribution of Amrubicin, a Novel 9‐Amino‐anthracycline, and Its Active Metabolite Amrubicinol in P388 Murine Leukemia Cells
Authors: Yamaoka Takashi , Hanada Mitsuharu , Ichii Shinji , Morisada Shinya , Noguchi Toshihiro , Yanagi Yoshikazu
Published: August 2005

Amrubicin, a 9‐aminoanthracycline anti‐cancer drug, and its C‐13 hydroxyl metabolite amrubicinol, were examined for growth‐inhibitory activity as well as cellular uptake and distribution in P388 murine leukemia cells and doxorubicin‐resistant P388 cells. Also discussed are the differences in the mechanisms of action among amrubicin, amrubicinol and doxorubicin in terms of their cellular pharmacokinetic character. In P388 cells, amrubicinol was about 80 times as potent as amrubicin, and about 2 times more potent than doxorubicin in a 1‐h drug exposure growth‐inhibition test. A clear cross‐resistance was observed to both amrubicin and amrubicinol in doxorubicin‐resistant P388 cells, though the resistance index was lower for amrubicin. The intracellular concentration of amrubicinol was about 6 times and 2 times higher than those of amrubicin and doxorubicin, respectively. Compared to doxorubicin, amrubicin and amrubicinol were released rapidly after removal of the drugs from the medium. A clear correlation was found between the growth‐inhibiting activity and the cellular level of amrubicin and amrubicinol in P388 cells. About 10 to 20% of amrubicin or amrubicinol taken up by the cells was detected in the cell nuclear fraction, whereas 70 to 80% of doxorubicin was localized in this fraction. These results suggest that amrubicin and amrubicinol exert cytotoxic activity via a different mechanism from that of doxorubicin.
Effect of Functional Magnetic Particles on Radiofrequency Capacitive Heating
Authors: Shinkai Masashige , Ueda Kousuke , Ohtsu Shinji , Honda Hiroyuki , Kohri Kenjirou , Kobayashi1 Takeshi
Published: August 2005

Magnetic particles (magnetite) were used to make radio frequency (RF) capacitive hyperthermia effective to a specific site. In an agar phantom experiment, a magnetite‐containing agar piece was buried in a large agar phantom and heated by an 8 MHz‐RF capacitive heating device. The magnetite‐containing agar piece was heated more than the magnetite‐free agar phantom, and the specific adsorption rate in the phantom was increased 1.5 times by the magnetite particles. The temperature distribution in the large agar phantom showed that the highest temperature was obtained at the center of the magnetite‐containing piece. The rate of temperature increase was approximately proportional to the magnetite concentration to the power 0.8. This method was applied to an in vivo experiment using a pig. Magnetite was prepared as a colloidal material dispersed in a carboxymethylcellulose solution (CMC‐Mag) and intramuscularly injected in the pig femur. As a result of 8 MHz‐RF heating, the temperature at the CMC‐Mag‐injected point increased to over 43°C after 7 min, while the temperature at a point without magnetite was under 40°C at the same time. The specific adsorption rate in the magnetite‐containing tissue was twice that of the magnetite‐ free tissue. In addition, the time required to reach a temperature of over 43°C was only 7 min, while it was over 15 min in the case without the CMC‐Mag.
Genotype Difference of Aldehyde Dehydrogenase 2 Gene in Alcohol Drinkers Influences the Incidence of Japanese Colorectal Cancer Patients
Authors: Murata Motoi , Tagawa Masatoshi , Watanabe Satoshi , Kimura Hideki , Takeshita Tatsuya , Morimoto Kanehisa
Published: August 2005

A case‐control study was conducted to explore the possible etiologic role of alcohol and aldehyde dehydrogenase 2 (ALDH2) gene among Japanese colorectal cancer patients. Information on their drinking, smoking and dietary habits was collected from 265 colon and 164 rectum cancer patients, and 794 non‐cancer patients as a control group. Genotypes of the ALDH2 gene at codon 487, glutamic acid (ALDH2*1)as a wild‐type or lysine (ALDH2*2)as a mutated type with reduced enzyme activity, were analyzed by polymerase chain reaction in 160 colon and 110 rectum cancer patients and 121 control persons. Univariate analysis with the χ2 statistical test showed that heavy alcohol drinking (P < 0.01), frequent meat intake (P < 0.001), and irregular (P < 0.01), hasty (P < 0.01) and excessive (P <0.001) eating habits were associated with the incidence of both colon and rectum cancers, whereas heavier smoking (P < 0.05) and infrequent fish (P < 0.03)and fruit (P < 0.01) intake were solely associated with incidence of rectum cancer. Infrequent green vegetable intake was not correlated with the incidence of colorectal cancer. Multivariate unconditional logistic regression analysis confirmed the association of alcohol consumption (P < 0.01) and meat intake (P < 0.05). Homozygous and heterozygous carriers of ALDH2*2 allele tended to be found in colon (trend P= 0.04)but not in rectum cancer patients compared to controls. Risk elevation for colon cancer due to alcohol consumption was pronounced among the heterozygotes and it was statistically significant especially for distal colon cancer (trend P=0.02). We conclude that alcohol consumption is a risk factor for colorectal cancer and the risk can be enhanced in ALDH2 heterozygotes.
Preventive Effects of Glycyrrhizae radix Extract on Estrogen‐related Endometrial Carcinogenesis in Mice
Authors: Niwa Kenji , Hashimoto Midori , Morishita Shigeo , Yokoyama Yasuhiro , Mori Hideki , Tamaya Teruhiko
Published: August 2005

Short‐ and long‐term experiments were conducted to examine the effects of Glycyrrhizae radix (Gl radix) extract on mouse endometrial carcinogenesis. Gl radix treatment (2 weeks) decreased the levels of c‐fos/jun mRNA and the corresponding oncoproteins induced by estradiol‐17β(E2) in castrated mice uteri, as determined by reverse transcription‐polymerase chain reaction and Southern blot analysis, and immunohistochemical methods, respectively. For the long‐term assays, 98 female ICR mice were given N‐methyl‐N‐nitrosourea (MNU) solution (1 mg/100 g body wt.) and normal saline (as controls) into their left and right uterine corpora, respectively. They were divided into four groups as follows: group 1 was given 0.625%Gl radix‐ and 5 ppm E2–containing diet; group 2, 5 ppm E2–containing diet; group 3, 0.625%Gl radix‐containing diet; and group 4, the basal diet alone. Gl radix treatment significantly decreased uterine weights and the incidences of uterine endometrial atypical hyperplastic and malignant lesions. It is suggested that Gl radix has inhibitory effects on E2‐related endometrial carcinogenesis in mice, through suppression of estrogen‐induced c‐fos/jun‐expressions.
Detection of Disseminated Urothelial Cancer Cells in Peripheral Venous Blood by a Cytokeratin 20‐specific Nested Reverse Transcriptase‐Polymerase Chain Reaction
Authors: Fujii Yasuhisa , Kageyama Yukio , Kawakami Satoru , Kihara Kazunori , Oshima Hiroyuki
Published: August 2005

More than half of all patients with invasive urothelial cancer subsequently develop metastatic disease even after radical resection of the primary cancer. In these patients, neoplastic cells may be disseminated prior to or during the operation. A nested reverse transcriptase‐polymerase chain reaction (nested RT‐PCR) assay which amplifies cytokeratin (CK) 20 transcripts was used to detect cancer cells in the peripheral blood of urothelial cancer patients. This assay was able to detect 10 bladder cancer cell line cells in a sample of ten million peripheral‐blood mononuclear (PBMN) cells. CK 20‐specific signals were detected in 9 (22.5%) of 40 PBMN cell samples prepared from 40 urothelial cancer patients in relation to the tumor stage, including 0/13 patients with a superficial tumor, 4/21 (19%) with a regionally invasive tumor and 5/6 (83%) with a metastatic tumor (P=0.0002 in χ2 test). No signals were detected in any of 25 healthy donor PBMN cell samples. The present results indicate that the CK 20 RT‐PCR assay is applicable for detection of urothelial cancer cells in the peripheral blood. The assay also confirms that hematogenic dissemination occurs in invasive urothelial cancers but rarely in superficial ones.
Prevention by Chitosan of Myelotoxicity, Gastrointestinal Toxicity and Immunocompetent Organic Toxicity Induced by 5‐Fluorouracil without Loss of Antitumor Activity in Mice
Authors: Kimura Yoshiyuki , Okuda Hiromichi
Published: August 2005

We examined the antitumor activity and side effects (myelotoxicity, immunocompetent organic toxicity and gastrointestinal toxicity) of combined treatment with the cancer chemotherapy drug 5‐fluorouracil (5‐FU) and dietary fiber chitosan in sarcoma 180‐bearing mice. 5‐FU (12.5 mg/kg×2/ day) plus chitosan (150, 375 and 750 mg/kg×2/day) inhibited the tumor growth as well as 5‐FU alone. Chitosan (150 and 750 mg/kg×2/day) blocked the reduction of blood leukocyte number caused by 5‐FU administration, and it prevented the injury of the small intestinal mucosa membrane and delayed the onset of diarrhea induced by 5‐FU. Furthermore, chitosan (750 mg/kg×2/ day) prevented the reduction of spleen weight induced by 5‐FU in sarcoma 180‐bearing mice, and the reduction of lymphocyte and CD8+ T cell numbers induced by 5‐FU was also prevented by the oral administration of chitosan (750 mg/kg×2/day) in C57BL/6 mice. Chitosan (150 and/or 750 mg/kg×2/day) reduced the 5‐FU incorporation into RNA fractions of small intestine and spleen without affecting the 5‐FU incorporation into the tumor in sarcoma 180‐bearing mice. These findings suggest that prevention of the 5‐FU side effects by chitosan might be partly due to the selective inhibition of 5‐FU uptake into the small intestine and spleen, resulting in the reduction of immune function toxicity, myelotoxicity and gastrointestinal toxicity of 5‐FU. Therefore, it is concluded that the combination of chitosan and 5‐FU might be useful for the prevention of side effects such as gastrointestinal toxicity, immunotoxicity and myelotoxicity caused by 5‐FU.
Antitumor Effect on Human Gastric Cancer and Induction of Apoptosis by Vascular Endothelial Growth Factor Neutralizing Antibody
Authors: Kamiya Kinji , Konno Hiroyuki , Tanaka Tatsuo , Baba Megumi , Matsumoto Keigo , Sakaguchi Takanori , Yukita Ayako , Asano Makoto , Suzuki Hideo , Arai Tomio , Nakamura Satoshi
Published: August 2005

Induction of apoptosis by antiangiogenic therapy has been suggested as a new anticancer strategy. To clarify the mechanism of the antitumor effect achieved by inhibition of vascular endothelial growth factor (VEGF), which is a major mediator of angiogenesis, we used an orthotopic transplantation model of human gastric carcinoma line (MT2) treated with a monoclonal VEGF neutralizing antibody (VEGF Ab). We histologically examined the microvessel density (MVD) and the apoptotic index (AI) in this model. Transplanted tumor growth was significantly inhibited by the VEGF Ab (P= 0.03), and there was a significant decrease in the number of mice with liver metastasis (P= 0.004). The MVD detected by immunohistochemical staining with ER‐MP12 antibody was 33.6 ± 8.0 in the control group and 21.1 ± 5.4 in the treated group, and the difference was significant (P < 0.0001). The AI values of the control and treated groups were 4.73 ± 1.11 and 7.26 ± 1.62, respectively, and this difference is also significant (P < 0.0001). However, the expression of VEGF mRNA in transplanted tumors did not show a significant difference between the control and treated groups. These results suggest that the antitumor effect of the VEGF Ab on human gastric carcinoma is exerted by inducing mild hypoxia followed by apoptosis, which does not influence VEGF mRNA expression in the carcinoma.
Suicide Risk among Cancer Patients: Experience at One Medical Center in Japan, 1978–1994
Authors: Tanaka Hideo , Tsukuma Hideaki , Masaoka Tohru , Ajiki Wakiko , Koyama Yoko , Kinoshita Noriko , Hasuo Seiko , Oshima Akira
Published: August 2005

In order to identify the risks, methods and locations of suicide among Japanese cancer patients, we followed up 23,979 cancer patients aged 15 years and older, who were newly diagnosed at one medical center between 1978–94. Suicide risk was evaluated by comparing the observed number (O) with the expected number (E), which was calculated from the suicide mortality rate among Osaka residents. By the end of 1994, there were 48 suicide death among 109,374 person‐years of follow‐up. Suicide risk was significantly increased within the first five years following cancer diagnosis (male: O/E=1.62, 95% confidence interval (CI)=1.03–2.43, female: O/E=2.13, 95%CI=1.22–3.47), and decreased to unity after five years had elapsed following the cancer diagnosis. Patients in their fifties (O/E=1.93, 95%CI=1.48–4.37), diagnosed between 1978–85 (O/E=1.93, 95%CI=1.19–2.95), having remote metastasis at their initial diagnosis (O/E=4.66, 95%CI=2.12–8.84), the site being in the female genital organs (O/E=3.58, 95%CI=1.54–7.06) or in the male genital organs (O/E= 5.86, 95%CI= 1.58–15.05), had a significantly higher suicide risk within five years following cancer diagnosis. The highest suicide mortality was observed between the third and fifth months after cancer diagnosis (O/E=4.35, 95%CI=1.87–8.58); most of these suicides (7/8) occurred soon after discharge from hospital. Among the 39 patients who committed suicide within five years after their diagnosis, the most common method among those who took their lives while in hospital was by jumping to their death (10/16), and that among those who committed suicide in other locations was by hanging (14/23). These findings indicate that Japanese patients with cancer run the highest risk of suicide soon after having been discharged from hospital. Clinicians should assess cancer patients' psychological distress and potential risk of suicide before approving discharge from hospital.
Loss of Heterozygosity in (Lewis×F344)F1 Rat Urinary Bladder Tumors Induced with N‐Butyl‐N‐(4‐hydroxybutyl)nitrosamine Followed by Dimethylarsinic Acid or Sodium L‐Ascorbate
Authors: Chen Tianxin , Na Yifei , Wanibuchi Hideki , Yamamoto Shinji , Lee Chyi Chia R. , Fukushima Shoji
Published: August 2005

Dimethylarsinic acid (DMA), a main metabolite of arsenicals which are carcinogenic in man, exerts tumor‐promoting activity on rat urinary bladder carcinogenesis initiated with N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine (BBN). Sodium L‐ascorbate (Na‐AsA) is also a strong tumor promoter in this animal model. In this study, we used (Lewis×F344)F1 rats to compare molecular alterations in urinary bladder tumors caused by BBN followed by DMA or Na‐AsA. Male, 6‐week‐old rats were given 0.05% BBN in their drinking water for 4 weeks, and then the rats in group 1 were maintained with no further treatment for 40 weeks. The animals of groups 2 and 3 were administered 0.01% DMA in their drinking water (group 2) or 5% Na‐AsA in the powder diet (group 3) after the BBN treatment. Group 4 rats were given 0.05% BBN continuously for 36 weeks. At weeks 12, 20, 36 and 44, subgroups of rats were killed. Histopathological examination revealed promoting activity for DMA and, to a greater extent, Na‐AsA on urinary bladder carcinogenesis. Loss of heterozygosity (LOH), detected with the polymerase chain reaction using 36 microsatellite markers, was found to be present in 2 of 9 (22%) urinary bladder tumors after treatment with DMA and 3 of 22 (14%) induced by continuous administration with BBN. No LOH was, however, detected in urinary bladder tumors after treatment with Na‐AsA. The results thus suggest that the mechanisms of action of these two promoters, DMA and Na‐AsA, may differ in rat urinary bladder carcinogenesis.
β‐Catenin (Ctnnb1) Gene Mutations in Diethylnitrosamine (DEN)‐induced Liver Tumors in Male F344 Rats
Authors: Yamada Yasuhiro , Yoshimi Naoki , Sugie Shigeyuki , Suzui Masumi , Matsunaga Kengo , Kawabata Kunihiro , Hara Akira , Mori Hideki
Published: August 2005

Alterations in multiple phosphorylation sites on exon 3 of the β‐catenin gene have recently been implicated in hepatocarcinogenesis in humans as well as mice. To identify genetic alterations which could be involved in the chemical‐induced hepatocarcinogenesis of rats, we analyzed the status of the sites in the β‐catenin gene (Ctnnb1) of liver neoplasms induced by diethylnitrosamine (DEN) in male F344 rats, using the polymerase chain reaction‐single strand conformation polymorphism method. In the present investigation, we examined 35 hepatocellular neoplasms (28 adenomas and 7 carcinomas) for the expression of mutations in the region of the β‐catenin gene. Point mutation at codon 32, 35, 37 or 41, which has been reported in human and mouse liver cell carcinomas and/or other cancers, was recognized in eleven (31%) out of 35 lesions (8 adenomas and 3 carcinomas). Our results indicate that Ctnnb1 mutations may contribute to hepatocarcinogenesis in rats. Our finding that Ctnnb1 mutation was present in adenomas as well as carcinomas also suggests that the mutation is a relatively early event in DEN‐induced hepatocarcinogenesis in rats.
Mutational Inactivation of Mitotic Checkpoint Genes, hsMAD2 and hBUB1, Is Rare in Sporadic Digestive Tract Cancers
Authors: Imai Yasuo , Shiratori Yasushi , Kato Naoya , Inoue Tohru , Omata Masao
Published: August 2005

Genetic instability is a key mechanism of tumorigenesis, and the instability exists at two distinct levels, the nucleotide and the chromosome levels. Disruption of the mitotic spindle checkpoint is one of the underlying mechanisms leading to aneuploidy and alterations of hsMAD2 and hBUB1, assumed to take part in the spindle checkpoint in human cells, have been found to be associated with chromosomal instability in some tumor cell lines. Therefore, we investigated the mutational status of the hsMAD2 and hBUB1 genes in 32 sporadic digestive tract cancers by reverse transcription‐ polymerase chain reaction‐single strand conformation polymorphism analysis. The entire coding sequence of the hsMAD2 gene, and conserved regions (codons 21–152 and codons 732–1043) presumed to be functionally important in the hBUB1 gene were analyzed. Mutation of the hsMAD2 gene was not observed at all and missense mutation of the hBUB1 gene was noted in one rectal cancer case. Sequencing analysis revealed an AGT‐to‐GGT missense mutation, substituting glycine for serine, at codon 950, which is conserved between budding yeast and human. These results indicate that mutations of the hsMAD2 and hBUB1 genes are very rare and presumably play a very restricted role in tumor development of sporadic cancers.
CDR3 Sequences of MALT Lymphoma Show Homology with Those of Autoreactive B‐Cell Lines
Authors: Yumoto Norio , Kurosu Katsushi , Furukawa Masakazu , Mikata Atsuo
Published: August 2005

We have examined the CDR3 sequence and adjacent regions of immunoglobulin genes from B‐cell lymphoma of mucosa‐associated lymphoid tissue (MALT). Twenty‐nine sequences (15 sequences from 13 low‐grade MALT lymphomas, marginal zone B‐cell lymphomas; 7 sequences from 6 highgrade MALT lymphomas; 7 sequences from 7 diffuse large cell lymphomas) were obtained after cloning of the polymerase chain reaction‐amplified segments. In the low‐grade MALT, high‐grade MALT and diffuse large cell lymphomas, the mean length of the CDR3 region was 47.6 ± 10.31 (range 21 to 60), 38.71 ± 10.37 (range 27 to 57) and 40.86 ± 3.34 (range 39 to 48) nucleotides, respectively. The length of the CDR3 region was significantly greater in the low‐grade MALT lymphoma group than in the other two groups. CDR3 sequences in lymphoma cell clones of 14 cases showed 60 to 81% homology with autoantibody‐associated lymphocyte clones including rheumatoid factor. The incidences of these autoantibody‐associated lymphocyte clones were higher in the high‐grade MALT (4/6) and diffuse large lymphomas (5/7) than in the low‐grade MALT lymphoma (5/13). Cases with more than 70% homology at the nucleotide level were found to have 71 to 82% homology with autoantibodies at the protein level in the low‐grade MALT lymphomas (2/13), and 67% homology in the high‐grade MALT lymphomas (2/7). These results indicate that MALT lymphomas may be derived from the malignant transformation of autoreactive B‐cells.
Increased Serum Levels of Vascular Endothelial Growth Factor in Patients with Renal Cell Carcinoma
Authors: Sato Kazunari , Tsuchiya Norihiko , Sasaki Ryusei , Shimoda Naotake , Satoh Shigeru , Ogawa Osamu , Kato Tetsuro
Published: August 2005

Neovascularization, an essential event for the growth of solid tumors, is regulated by a number of angiogenic factors. One such factor, vascular endothelial growth factor (VEGF), is considered to exert a potent angiogenic activity, as indicated by immunohistochemical and molecular evidence. In this study we investigated the serum VEGF level (s‐VEGF) in patients with renal cell carcinoma (RCC). s‐VEGF in peripheral blood samples was analyzed in 40 RCC patients and 40 patients without cancer (controls) using a sandwich enzyme‐linked immunoassay. In 20 RCC patients, serum samples were obtained separately from the bilateral renal veins. s‐VEGF was also measured before, 4 and 8 weeks after nephrectomy in 11 patients. There were significant differences in s‐VEGF between the RCC patients and the controls (207.3 ± 32.9 vs. 71.5 ± 9.1 pg/ml, mean ± SE) (P < 0.005), between the tumor‐bearing renal veins and the contralateral ones (P < 0.01), between the pre‐ and post‐nephrectomy situations (P < 0.01) and among the various parameters of tumor status such as tumor extent (P < 0.001) and existence of metastasis (P < 0.001). s‐VEGF significantly correlated with the tumor volume obtained by a three‐dimensional measurement (r= 0.802, P < 0.0001). The sensitivity and specificity of s‐VEGF at the cut‐off level of 100 pg/ml, as determined by the receiver‐operating‐characteristics curve, were 80.0% and 72.5%, respectively. The results indicate that tumor tissue of RCC liberates VEGF into the systemic blood flow and that s‐VEGF is a possible marker for RCC.
Detection of Hypoxic Cells in Murine Tumors Using the Comet Assay: Comparison with a Conventional Radiobiological Assay
Authors: Shibuya Keiko , Sasai Keisuke , Xie Xuejun , Utsumi Hiroshi , Shibata Toru , Hiraoka Masahiro
Published: August 2005

The comet (single‐cell electrophoresis) assay has been developed as a method for measuring DNA damage in single cells after irradiation. We have developed our own methods and image analysis system for the comet assay to identify hypoxic fractions. In vitro, we tested our system using a cultured tumor cell line (SCCVII). In vivo, we compared the hypoxic fractions detected by this assay with those determined by the in vivo‐in vitro clonogenic assay using two rodent tumors (SCCVII/C3H, EMT6/KU/balb/c), which exhibit different types of hypoxia: acute and chronic. In vitro, our method could differentiate hypoxic cells from oxic cells, using the parameter of tail moment. In vivo, there were good correlations between the hypoxic fractions determined by the comet assay and by the clonogenic assay, in SCCVII/C3H (r= 0.85) and in EMT6/KU/balb/c (r= 0.75) tumors. By comparison of the two methods in chronically hypoxic and acutely hypoxic tumors, we further confirmed that the comet assay is clinically useful for estimating hypoxic fractions of solid tumors.
Combination Chemotherapy with Nedaplatin and Cyclophosphamide in Human Ovarian Cancer Model
Authors: Uchida Naomi , Yoshida Hiroshi , Yamada Hajime , Wada Tohru , Daikatsu Kouji , Ikeuchi Isao , Maekawa Ryuji , Sugita Kenji , Yoshioka Takayuki
Published: August 2005

The antitumor efficacy of the combination of nedaplatin (NDP) with cyclophosphamide (CPM) was evaluated using human ovarian cancer models. Since NDP has been found to have greater anti‐tumor activity and lower nephrotoxicity than cisplatin (CDDP), we also compared the antitumor activity of NDP plus CPM with that of CDDP plus CPM. Increasing doses of NDP (16.5, 33 and 66 mg/kg as a total dose) and a fixed amount of CPM (174 or 348 mg/kg as a total dose) were injected three times at intervals of 7 days via the tail vein into mice implanted with RMUG‐S, OC9‐JCK or KF‐28 human ovarian cancer. Simultaneous administration of NDP with CPM resulted in markedly enhanced inhibition of tumor growth for all cancers tested. The growth inhibition and survival effect of the combination therapy of NDP with CPM against KF‐28 and OC9‐JCK were as potent as those of CDDP plus CPM. Neither increased hematotoxicity nor a significant difference in maximum concentration, half time or area under the curve of platinum or CPM in plasma between the single and combined treatment was found. These results suggest that the combination of NDP with CPM may be clinically effective.
Hypomethylation of CpG Sites and c‐myc Gene Overexpression in Hepatocellular Carcinomas, but Not Hyperplastic Nodules, Induced by a Choline‐deficient L‐Amino Acid‐defined Diet in Rats
Authors: Tsujiuchi Toshifumi , Tsutsumi Masahiro , Sasaki Yasutaka , Takahama Makoto , Konishi Yoichi
Published: August 2005

We have investigated aberrant methylation of CpG nucleotides (CpG sites) and gene expression of c‐myc during hepatocarcinogenesis induced by a choline‐deficient, L‐amino acid‐defined (CDAA) diet in rats. Male Fischer 344 rats, 6 weeks old, were continuously given a CDAA diet for 50 and 75 weeks and then killed. Macroscopically detectable nodules, which were histologically confirmed to be hyperplastic nodules (HNs) or well‐differentiated hepatocellular carcinomas (HCCs), were dissected free from the surrounding tissue. Normal control liver was obtained from 6‐week‐old rats. Methylation of CpG sites of the c‐myc gene was investigated in bisulfite‐treated DNA isolated from normal liver, HNs and HCCs. All 33 cytosines in the 5′‐upstream region of the c‐myc gene were fully methylated in control liver and the 4 HNs. In contrast, these cytosines were completely unmethylated in 5 HCCs. Examination of the c‐myc expression by reverse transcription‐polymerase chain reaction (RT‐PCR) analysis also showed a marked increase as compared to the low levels in normal livers and HNs. These results suggest that hypomethylation of the c‐myc gene might play a critical role in malignant transformation from HN to HCC during CDAA diet‐induced hepatocarcinogenesis in rats.
Serum n‐3 Fatty Acids, Fish Consumption and Cancer Mortality in Six Japanese Populations in Japan and Brazil
Authors: Kobayashi Minatsu , Sasaki Satoshi , Hamada Gerson S. , Tsugane Shoichiro
Published: August 2005

Japanese people consume significant amounts of long chain n‐3 polyunsaturated fatty acids (PUFAs) derived from fish, but the association of PUFAs with cancer mortality has not been fully investigated. To study geographic differences in n‐3 PUFAs intake, we compared serum fatty acid and dietary fish intake among various Japanese populations having different rates of cancer mortality. The subjects were 50 men from each of five regions in Japan and 47 Japanese men from Sao Paulo, Brazil. All were randomly selected and aged 40 to 49 years. Serum fatty acids were measured by gas chromatography and the frequency of fish intake was obtained by a food frequency questionnaire. Significant geographic differences in serum fatty acid levels (% of total fatty acids) and fish intake (days/4 weeks) were observed. The percentages of serum total PUFA were similar in the six regions, though there was an almost three‐fold difference in n‐3 PUFAs content between Brazil (3.9%) and Akita (10.9%). The frequency of total fish intake corresponded to serum n‐3 PUFAs composition. The relationship between cancer mortality and serum n‐3 PUFAs levels was not clear, though an inverse association between prostate cancer and serum n‐3 PUFAs levels appeared to exist. The results suggest that although serum n‐3 PUFAs varied significantly, the observed geographic difference did not account for the different cancer risks at the population level.
Prevention of Lung Metastasis by Intra‐tumoral Injection of Cepharanthin and Staphylococcal Enterotoxin B in Transplantable Rat Osteosarcoma
Authors: Okada Kyoji , Sakusabe Noboru , Kobayashi Akira , Hoshi Naoto , Sato Kozo
Published: August 2005

The antitumor effect of intra‐tumoral injection of Cepharanthin, a biscoclaurin alkaloid extracted from Stephania cephalanta Hayata, and staphylococcal enterotoxin B was evaluated using F344 male rats bearing transplantable rat osteosarcoma, S‐SLM. A macroscopic lung metastatic nodule of tumor was transplanted into the subcutaneous back space, and 0.5 mg of Cepharanthin and 2 pg of staphylococcal enterotoxin B were injected into the tumor on days 12, 13 and 14. On day 28, all animals were killed with an overdose of pentobarbital sodium, and the transplanted tumors and lungs were examined. The wet weight of the lungs of the rats treated with Cepharanthin and staphylococcal enterotoxin B was significantly lower, and apoptosis in the lung metastatic nodules was significantly higher than that of the control or that of rats treated with only Cepharanthin or staphylococcal enterotoxin B. In the transplanted tumors, infiltration of TRAP (tartrate‐resistant acid phosphatase)‐positive multinucleated giant cells was prominent in the rats treated with Cepharanthin and staphylococcal enterotoxin B. These findings indicate that intra‐tumoral injection of Cepharanthin and staphylococcal enterotoxin B induced infiltration of TRAP‐positive multinucleated giant cells within the transplanted rat osteosarcoma, and reduced lung metastasis.
Rare‐type Mutations of MMAC1 Tumor Suppressor Gene in Human Glioma Cell Lines and Their Tumors of Origin
Authors: Zhang Shu‐Jing , Endo Sumio , Ichikawa Tomio , Yoshimura Junichi , Onda Kiyoshi , Tanaka Ryuichi , Washiyama Kazuo , Kumanishi Toshiro
Published: August 2005

A total of 10 glioma cell lines were examined to evaluate the status of the MMAC1 gene, a candidate tumor suppressor gene. Six cell lines showed mutations with presumed loss of heterozygosity and 1 cell line showed no mRNA expression. The 6 mutations consisted of 3 3‐bp deletions (codons 17, 101 or 199), 1 missense mutation (codon 252) and 2 truncation mutations (1 nonsense mutation at codon 233 and 1 2‐bp insertion at codon 241). Among them, the 3‐bp deletions, which are a rare type of mutation in MMAC1 gene, were located in the N‐terminal half (codons 1–212) of the coding region, which is considered important in MMAC1 function. The missense mutation was located unusually in the C‐terminal half (codons 212–403), but it was in a small region in which some other reported missense mutations are clustered. Thus, these 4 mutations were suggested to have functional effects on the MMAC1 activity, like the other 2 mutations with predicted protein truncations. By sequence analysis of cDNA clones, we confirmed that all the mutations including these 4 rare ones were in the MMAC1 gene, not in the PTH2 pseudogene. In 2 cases, we also examined the primary glioma tissues from which the cell lines had been derived and found the same mutations as in the cell lines in both cases. This suggested that the mutations in these cell lines were derived from the primary glioma tissues, but not from artifacts arising during long‐term in vitro cultivation.
Localization of a Target Region of Allelic Loss to a 1‐cM Interval on Chromosome 16p.13.13 in Hepatocellular Carcinoma
Authors: Koyama Masaaki , Nagai Hisaki , Bando Koichi , Ito Masahide , Moriyama Yuukichi , Emi Mitsuru
Published: August 2005

To identify the location of the putative tumor suppressor gene on chromosome 16p that may be involved in hepatocellular carcinoma (HCC), we examined 96 primary HCCs and evaluated their patterns of allelic loss at 10 microsatellite marker loci distributed along this chromosome arm. Allelic loss at one or more loci was observed in 46 (48%) of these tumors. Through detailed deletion mapping of tumors having partial or interstitial deletions, we identified a commonly deleted region at a 1‐cM interval, flanked by D16S519 and D16S3078 at 16p13.13, defining the location of a putative tumor suppressor gene for HCC. This region contains the gene for JAB (JAK‐binding protein), which is responsible for negative‐feedback regulation of the JAK‐STAT pathway induced by cytokine stimulation, raising the possibility that inactivation of this gene may participate in hepatocarcinogenesis via genetic and/or epigenetic changes.
Down‐regulation of KAI1 Messenger RNA Expression Is Not Associated with Loss of Heterozygosity of the KAI1 Gene Region in Lung Adenocarcinoma
Authors: Tagawa Kohei , Arihiro Koji , Takeshima Yukio , Hiyama Eiso , Yamasaki Masahiro , Inai Kouki
Published: August 2005

KAI1, a metastasis suppressor gene of prostate cancer, is located on human chromosome 11p11.2. Down‐regulation of KAI1 mRNA during tumor progression and metastasis has been reported for several kinds of cancer, but the mechanism of this down‐regulation is not known. In the present study, our aim was to ascertain the relationship between down‐regulation of KAI1 mRNA expression and KAI1 gene alterations in lung cancer. Forty‐nine cases of adenocarcinoma of the lung were studied by reverse‐transcriptase polymerase chain reaction (RT‐PCR) assay of KAI1 mRNA and by immunohistochemical detection of KAI1 protein. In addition, markers of the microsatellite loci D11S1344 and D11S1326 were used to investigate loss of heterozygosity (LOH) and replication errors (RERs) of the KAI1 gene region. The RT‐PCR assay showed that there was no correlation between KAI1 mRNA expression and either the age of the patients or tumor size. By contrast, KAI1 mRNA expression was significantly correlated with gender (P=0.047), metastasis to the lymph nodes or other organs (P=0.004), the histological grade of the tumor (P=0.036) and the pathological stage (P=0.049). Immunohistochemical staining showed that in one case without metastasis, loss of KAI1 mRNA was associated with invasion of the stroma by KAI1 protein‐negative cancer cells. The numbers of informative cases by microsatellite analysis were 14 (28.6%) of 49 at D11S1344 and 27 (55.1%) of 49 at D11S1326; none of 49 adenocarcinomas showed LOH or RERs at these loci. These results suggest that down‐regulation of KAI1 mRNA expression rarely if ever involves LOH or RERs of the KAI1 gene region in primary lung adenocarcinoma.
A Novel Combretastatin A‐4 Derivative, AC‐7700, Shows Marked Antitumor Activity against Advanced Solid Tumors and Orthotopically Transplanted Tumors
Authors: Nihei Yukio , Suga Yasuyo , Morinaga Yoshihiro , Ohishi Kazuo , Okano Akira , Ohsumi Koji , Hatanaka Toshihiro , Nakagawa Ryusuke , Tsuji Takashi , Akiyama Yukio , Saito Sachiko , Hori Katsuyoshi , Sato Yasufumi , Tsuruo Takashi
Published: August 2005

AC‐7700, a novel combretastatin A‐4 derivative, suppresses the growth of solid tumors by inhibiting tumor perfusion. We evaluated the antitumor activity of AC‐7700 on solid tumors in two experimental models, an advanced tumor model (murine colon 26 (c26) adenocarcinoma, colon 38 (c38) adenocarcinoma, MethA fibrosarcoma, Sarcoma 180 (S180), Lewis lung carcinoma (3LL), human LS180 adenocarcinoma) and an orthotopically transplanted tumor model (c26), compared with that of cisplatin (CDDP). The maximum tolerable dose (MTD) of CDDP suppressed early‐stage c26 and c38 tumor growth when treatment was started after the tumor volume (TV) reached 0.2–0.5 cm3, but it showed reduced activity against the same tumors at an advanced growth stage when TV exceeded 2 cm3. At its MTD, AC‐7700 was active against all tumors tested except 3LL in both early and advanced growth stages, reducing the tumor mass and having a curative effect in advanced c38 tumors. AC‐7700 was also effective on orthotopically transplanted c26 tumors, showing a comparable activity to that on subcutaneous tumors. Unlike flavon acetic acid, which damages tumor vasculature by inducing endogenous tumor necrosis factor‐α production, AC‐7700 potently suppressed the growth of advanced c26 tumors in athymic as well as euthymic mice. These results suggest that AC‐7700 is a novel antivascular agent that may have potent activity against advanced‐stage cancer in the clinical setting.
Overexpression of Midkine in Pancreatic Duct Adenocarcinomas Induced by N‐Nitrosobis(2‐oxopropyl)amine in Hamsters and Their Cell Lines
Authors: Tsutsumi Masahiro , Kadomatsu Kenji , Tsujiuchi Toshifumi , Sakitani Hiroyuki , Ikematsu Shinya , Kubozoe Tadahiko , Yoshimoto Masatoshi , Muramatsu Takashi , Sakuma Sadatoshi , Konishi Yoichi
Published: August 2005

The expression of midkine (MK) was investigated in pancreatic ductal hyperplasias, atypical hyperplasias and adenocarcinomas induced by N‐nitrosobis(2‐oxopropyl)amine (BOP) in hamsters, and in hamster ductal adenocarcinoma cell lines (HPD‐1NR, ‐2NR and ‐3NR). MK mRNA was clearly overexpressed in invasive pancreatic duct adenocarcinomas (PCs) and the three cell lines as assessed by northern blot analysis, and MK protein expression increased from ductal hyperplasia through atypical hyperplasias, intraductal carcinomas and invasive PCs by immunohistochemistry. The extent of overexpression of MK mRNA in PCs was almost the same as in hamster whole embryonic tissue. MK is reported to be a retinoid‐responsive gene, but MK mRNA expression was not affected by treatment with all‐trans retinoic acid (tRA) or N‐(4‐hydroxyphenyl)retinamide (4‐ HPR) in HPD‐1NR cells. The results thus suggest that MK expression is involved in the development and progression of pancreatic ductal adenocarcinomas induced by BOP in hamsters, with loss of upregulation by retinoic acid.
Increased Frequencies of Gene and Chromosome Mutations after X‐Irradiation in Mouse Embryonal Carcinoma Cells Transfected with the bcl‐2 Gene
Authors: Taga Masataka , Shiraishi Kazunori , Shimura Tsutomu , Uematsu Norio , Oshimura Mitsuo , Niwa Ohtsura
Published: August 2005

Preimplantation stage mouse embryos are known to be highly sensitive to the killing effect of DNA damaging agents such as radiation. Interestingly, however, this stage of development is well protected from radiation induction of malformation and carcinogenesis in postnatal life. In recent years, it has become clear that the stem cells of preimplantation stage embryos undergo extensive apoptosis after DNA damage. It has been postulated that this apoptosis is likely to be responsible for the resistance to malformation, by excluding cells carrying deleterious DNA damage. We have tested the possible role of apoptosis in elimination of gene and chromosome mutations in undifferentiated mouse embryonal carcinoma cell line, F9, transfected with human bcl‐2 cDNA. The colony radiosensitivity of F9 cells was not affected by overexpression of the bcl‐2 gene, but the apoptotic cell death was suppressed, as examined by DNA ladder assay and Hoechst staining. This suppression was accompanied by an increase in the frequencies of hprt mutation and micronucleus formation after X‐irradiation. These results support the idea that maintenance of genomic integrity during early development is likely to be executed by apoptotic elimination of cells at risk.
Adenovirus‐mediated Transfer of Fas Ligand Gene Augments Radiation‐induced Apoptosis in U‐373MG Glioma Cells
Authors: Shinoura Nobusada , Yamamoto Naoki , Asai Akio , Kirino Takaaki , Hamada Hirofumi
Published: August 2005

Most malignant astrocytomas (gliomas) express a high level of Fas, whereas the surrounding normal tissues such as neurons and astrocytes express a very low level of Fas. Thus, transduction of Fas ligand would selectively kill malignant astrocytoma cells. On the other hand, glioma cells harboring p53 mutation have been reported to be resistant to conventional therapies including radiation. To override the resistance mechanism of glioma cells with p53 mutation to radiation, we transduced U‐373MG malignant astrocytoma (glioma) cells harboring mutant p53 with Fas ligand via an adenovirus (Adv) vector in combination with X‐ray irradiation, and evaluated the degree of apoptosis. The degree of apoptosis in U‐373MG cells infected with the Adv for Fas ligand (Adv‐FL) and treated with irradiation (81%) was much higher than that in U‐373MG cells infected with Adv‐FL and not treated with irradiation (0.8%) or that in U‐373MG cells infected with the control Adv for lacZ and treated with irradiation (5.0%). In U‐373MG cells infected with Adv‐FL, irradiation increased the expression of Fas ligand. Coincident with the increase in Fas ligand, there was a marked reduction in the caspase‐3 level and a marked increase in the cleaved form of poly(ADP‐ribose) polymerase (PARP), which are downstream components of Fas ligand‐mediated apoptosis. This suggests that the enhanced activation of caspase‐3 by the transduction of Fas ligand combined with irradiation, induced extensive apoptosis in U‐373MG cells. In summary, transduction of Fas ligand may override the resistance mechanism to radiotherapy in glioma cells harboring p53 mutation.
Protection against Malignant Progression of Spontaneously Developing Liver Tumors in Transgenic Mice Expressing O6‐Methylguanine‐DNA Methyltransferase
Authors: Qin Xiusheng , Zhang Shaomin , Matsukuma Shoichi , Zarkovic Mirjana , Shimizu Seiichiro , Ishikawa Takatoshi , Nakatsuru Yoko
Published: August 2005

To study the effect of O6‐methylguanine‐DNA methyltransferase (MGMT) on carcinogenesis, we have previously generated MGMT transgenic mice overexpressing the bacterial MGMT gene, ada, and demonstrated that high MGMT levels in the liver suppress induction of liver tumors after treatment with an alkylating hepatocarcinogen. To examine the effects of life‐long elevation of MGMT activity on mouse spontaneous liver tumor development, ada‐transgenic and control nontransgenic mice were compared. We also examined mutations at codon 61 of the H‐ras oncogene, reported as a hot spot in mouse liver tumors, using a direct DNA sequencing method. The results revealed no significant difference in tumor incidence or mutation spectrum, but interestingly, ada‐transgenic mice were found to have fewer malignant tumors and survived longer, indicating a possible protective role of MGMT against malignant conversion.
Mutations and Reduced Expression of the Transforming Growth Factor‐β Receptor II Gene in Rat Lung Adenocarcinomas Induced by N‐Nitrosobis‐(2‐hydroxypropyl)amine
Authors: Tsujiuchi Toshifumi , Sasaki Yasutaka , Tsutsumi Masahiro , Konishi Yoichi
Published: August 2005

Mutations and expression of the transforming growth factor‐β receptor type II (TGF‐βRII) gene were investigated in lung adenocarcinomas induced by N‐nitrosobis(2‐hydroxypropyl)amine (BHP) in rats. Males of the Wistar strain, 6 weeks old, were given 2000 ppm of BHP in their drinking water for 12 weeks and then maintained without further treatment until killed at week 25. Total RNA was extracted from 12 adenocarcinomas and mutations in TGF‐βRII were investigated by RT‐PCR‐restriction‐SSCP analysis followed by sequencing analysis. Two out of 12 adenocarcinomas showed band shifts, indicative of mutations (16.7%). One was a CTG‐to‐TTG (Leu to Leu) transition at codon 308 without amino acid alteration and the other a frameshift deletion of one of two guanines at nucleotides 1434 to 1435 (codon 477 to 478). Semi‐quantitative RT‐PCR analysis demonstrated significantly reduced TGF‐βRII expression in adenocarcinomas, as compared with normal lung tissue. These results suggest that TGF‐βRII alterations may play a role in the acquisition of growth advantage by lung adenocarcinomas induced by BHP in rats.
A Novel “Nihon'’Rat Model of a Mendelian Dominantly Inherited Renal Cell Carcinoma
Authors: Okimoto Kazuo , Kouchi Mami , Kikawa Emi , Toyosawa Kaoru , Koujitani Takatoshi , Tanaka Kohji , Matsuoka Nobuo , Sakurai Junko , Hino Okio
Published: August 2005

A novel rat model of hereditary renal cell carcinoma (RC) was found in a rat colony of the Sprague‐Dawley strain in Japan, and named the “Nihon'’rat. In this strain, RCs develop from early preneoplastic lesions, which begin to appear at 4 weeks of age, forming adenomas by the age of 16 weeks. The RCs are predominantly of clear cell type. Southern blot, northern blot and SSCP analyses revealed no change in the Tsc1, Tsc2, VHL, and c‐Met genes. Thus, the Nihon rat should be a valuable experimental model for understanding renal carcinogenesis, especially clear cell type, which is common among human RCs.
Evaluation of the Reliability of Chromosomal Imbalances Detected by Combined Use of Universal DNA Amplification and Comparative Genomic Hybridization
Authors: Harada Tomohiko , Shiraishi Kei , Kusano Noriyoshi , Umayahara Kenji , Kondoh Satoshi , Okita Kiwamu , Sasaki Kohsuke
Published: August 2005

Comparative genomic hybridization (CGH) analysis of microscopic tumor samples is allowed by universal DNA amplification using degenerate oligonucleotide primed‐PCR (DOP‐PCR). To evaluate the reliablity of DOP‐PCR CGH, we performed DOP‐PCR CGH and standard CGH in parallel using DNAs extracted from 10 malignant tumors of the hepatobiliary tract and pancreas. Similar results were obtained by both methods with a few exceptions, indicating that DOP‐PCR CGH provides cytogenetic information equivalent to that obtained from standard CGH. We also investigated the sensitivity of DOP‐PCR CGH using sequential dilutions of DNA from microdissected tumor cells. DOP‐PCR using 100 to 800 pg of template DNA yielded successful CGH results. However, less than 50 pg of template DNA was not suitable because of the small amount of generated DNA. These findings suggest that DOP‐PCR CGH is applicable for CGH analysis of tiny specimens which are too small for standard CGH. Accordingly, DOP‐PCR CGH analysis may become a useful method in clinical laboratory examination.
Overexpression of Human Telomerase RNA in Helicobacter pylori‐infected Human Gastric Mucosa
Authors: Hur Kwon , Gazdar Adi F. , Rathi Asha , Jang Ja‐June , Choi Jae‐Hoon , Kim Dae‐Yong
Published: August 2005

Telomerase, an enzyme associated with cellular immortality and malignancy, plays an important role in cellular immortalization and tumorigenesis. Furthermore, overexpression of the RNA component of the telomerase, called human telomerase RNA (hTR), has been demonstrated in various human cancers as an early event. The pattern of hTR expression following Helicobacter pylori (H. pylori) infection in human gastric mucosa was investigated by a radioactive in situ hybridization (ISH) assay. Paraffin‐embedded sections of 50 biopsy specimens taken from the gastric antrum of individual patients infected to different extents with H. pylori, as well as normal gastric mucosa, were studied. In normal gastric mucosa, only weak hTR expression was noted and the expression was limited to basal cells of the gastric glands. However, the degree of hTR expression gradually increased in parallel with the degree of H. pylori infection. The mean scores of gastric mucosa with mild, moderate and severe degrees of H. pylori infection were 2.3, 2.8, and 3.7 times higher than that of normal gastric mucosa, respectively. The results of this study suggested that up‐regulation of hTR expression is a frequent and early event associated with H. pylori infection in the gastric mucosa and may play some role in gastric carcinogenesis. Sufficient synthesis of hTR during this early stage may be a prerequisite for telomerase reactivation to occur in gastric cancer.