bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanistic Aspects of Green Tea as a Cancer Preventive: Effect of Components on Human Stomach Cancer Cell Lines
Authors: Okabe Sachiko , Ochiai Yumiko , Aida Miwa , Park Keunchil , Kim Seong‐Jin , Nomura Taro , Suganuma Masami , Fujiki Hirota
Published: August 2005

It is now well accepted that (–)‐epigallocatechin gallate (EGCG) inhibits carcinogenesis in the digestive tract in rodents. To understand the mechanisms of anticarcinogenesis, we first studied growth inhibition by EGCG in human stomach cancer cell lines established at Seoul National University(SNU cell lines). Inhibition by EGCG of [3H]thymidine incorporation into eight SNU celllines was examined, in relation to transforming growth factor‐β (TGF‐β) responsiveness. Various tea polyphenols derived from green tea and black tea induced growth inhibition and apoptosis of human stomach cancer cell line KATO III, and inhibition of tumor necrosis factor‐α (TNF‐α)release from the cells, in the order of (–)‐epicatechin gallate (ECG), EGCG, (–)‐epigallocatechin(EGC), teaflavins (TF) and (–)‐epicatechin (EC). In addition, we demonstrated that EGCG inhibited TNF‐α gene expression in KATO III cells, as well as okadaic acid‐induced AP‐1 and NF‐κB activation. The inhibitory potencies of EGCG for AP‐1 and NF‐κB binding to DNA were different between KATO III cells and mouse fibroblast cell line BALB/3T3. Thus, EGCG and other teapolyphenols may interact with various transcription factors, in addition to AP‐1 and NF‐κB, in nuclei of various cells, resulting in inhibition of TNF‐α gene expression and TNF‐α release.
Inhibitory Role of Plasminogen Activator Inhibitor‐1 in Invasion and Proliferation of HLE Hepatocellular Carcinoma Cells
Authors: Morita Yasushi , Hayashi Yoshitake , Kanamaru Taichi , Itoh Takashi , Suzuki Satoshi , Yamamoto Masahiro , Kuroda Yoshikazu , Itoh Hiroshi
Published: August 2005

Plasminogen activator inhibitor (PAI)‐1, a serine protease inhibitor, inactivates urokinase‐type plasminogen activator (uPA) and regulates degradation of the extracellular matrix; whether it functions for or against tumor progression, however, has been the subject of controversy. To assess the role of PAI‐1 in invasion and proliferation of hepatocellular carcinoma (HCC) cells, HLE cells were transfected with a vector capable of expressing an antisense PAI‐1 transcript. Analysis of seven stably transfected clones (PAI‐1−) showed reductions of 81% in PAI‐1 mRNA by northern blot analysis and 63% in the cellular PAI‐1 antigen level by enzyme‐linked immunosorbent assay(ELISA). There was no change in the levels of secreted PAI‐1 or PAI‐2. The activity of cellular uPA increased by 54%, without change in the protein level or the secreted uPA activity evaluated by ELISA. Morphologically, PAI‐1 antisense induced a spindle shape with narrower cytoplasmic processes in HLE cells. The forced inhibition of PAI‐1 increased the invasion and the growth of PAI‐1− cells by 75% and 82%, respectively. These results suggest that PAI‐1 plays a role in inhibiting invasion and proliferation, and the balance between uPA and PAI‐1 expression is important to assess the invasiveness of HCC cells.
Indomethacin Suppresses the Growth of Colon 26, Meth‐A and FM3A Tumors in Mice by Reducing the Prostaglandin E2 Content and Telomerase Activity in Tumor Tissues
Authors: Ogino Mitsuharu , Hisatomi Hisashi , Murata Minoru , Hanazono Makoto
Published: August 2005

The antitumor effect of indomethacin on Colon 26, Meth‐A and FM3A tumors was investigated in mice. The prostaglandin E2 content in tumor tissues was assayed to find out if indomethacin acts on tumors, and the telomerase activity in tumors and somatic tissues (testis, liver, spleen and colon) was also monitored during indomethacin treatment. Growth of Colon 26, Meth‐A and FM3A tumors was significantly (P < 0.001‐0.05) suppressed by indomethacin compared to the untreated controls. The prostaglandin E2 content in the three tumors was markedly (P < 0.001) reduced by indomethacin. Telomerase activity in Colon 26 and FM3A tumors was significantly (P < 0.001) lower than that of untreated tumors (80% and 45% decrease versus the controls, respectively), and the activity in Meth‐A tumor was slightly decreased (10% decrease versus the control) by indomethacin. Telomerase activity in the somatic tissues was not significantly affected by indomethacin. In summary, this study shows the effectiveness of indomethacin as an antitumor agent against three types of tumors, and suggests that indomethacin affects telomerase activity in tumors in vivo.
Contribution of the Nucleoside Transport System to Doxorubicin Transport in HL60 Cells but Not in Mononuclear Cells
Authors: Nagasawa Kazuki , Fumihara Tomohiko , Ohnishi Noriaki , Yokoyama Teruyoshi
Published: August 2005

Previously, we reported that pirarubicin (THP), an anthracycline, was transported, at least in part, via a nucleoside transport system in human leukemic HL60 cells, but not in mononuclear cells (MNCs). In this study, the contribution of the nucleoside transport system to the transport of other anthracyclines, doxorubicin (DOX), daunorubicin (DNR) and idarubicin (IDA), in HL60 cells and MNCs was investigated. The experiments were performed after both types of cells had been pretreated with a metabolic inhibitor, 2,4‐dinitrophenol, to deplete cellular ATP. The DOX uptake by HL60 cells was partially inhibited by inhibitors of equilibrative nucleoside transporters. In HL60 cells, moreover, the uptake of DOX depended on an inwardly directed Na+‐gradient, and was inhibited by concentrative nucleoside transporters, but there was no change in the DNR or IDA uptake under any of these conditions. On the other hand, the uptake of the three drugs by MNCs was not affected by any inhibitors of the nucleoside transporters, and there was no dependence of the uptake on an Na+‐gradient. These results suggested that DOX, but not DNR or IDA, was partially transported in HL60 cells via the nucleoside transport system, whereas in MNCs the system did not contribute to the uptake of any of these three drugs. Thus, nucleoside transport systems contributing to the transport of anthracyclines may be different among different derivatives and cell types.
Natural Killer T Cell Ligand α‐Galactosylceramide Inhibited Lymph Node Metastasis of Highly Metastatic Melanoma Cells
Authors: Nakui Minoru , Iwakabe Kenji , Ohta Akio , Sekimoto Masashi , Sato Marimo , Makuuchi Hiroyasu , Kawano Tetsu , Taniguchi Masaru , Nishimura Takashi
Published: August 2005

The role of natural killer T (NKT) cells in the prevention of multiple tumor metastasis was examined. The i.v. inoculation of a highly metastatic subline of B16‐BL6 (B16‐BL6‐HM) melanoma cells resulted in the formation of metastatic nodules in lymph nodes in addition to lung, intrapleural cavity, and ovary. However, treatment of the mice with the NKT cell ligand α‐galactosylceramide (α‐GalCer) three times from 1 day after B16‐BL6‐HM melanoma inoculation caused a significant inhibition of multiple metastasis. Lymph node metastasis of B16‐BL6‐HM was almost completely blocked by α‐GalCer treatment. This antimetastatic effect of α‐GalCer was abolished in NKT celldeficient mice. These results suggest that α‐GalCer‐activated NKT cells played a critical role in the prevention of lymph node metastasis of melanoma cells.
Obesity, Weight Gain and Risk of Colon Adenomas in Japanese Men
Authors: Kono Suminori , Handa Koichi , Hayabuchi Hitomi , Kiyohara Chikako , Inoue Hisako , Marugame Tomomi , Shinomiya Sachiko , Hamada Hiroaki , Onuma Kazuya , Koga Hiroko
Published: August 2005

Obesity has been related to increased risk of colon cancer or adenomas, but the epidemiologic findings are not entirely consistent. We examined the relation of not only body mass index (BMI) but also waist‐to‐hip ratio (WHR) and weight gain to colon adenoma risk in men who received a preretirement health examination at the Japan Self Defense Forces (SDF) Fukuoka and Kumamoto Hospitals during the period from 995 to 1996. In the series of 803 men at age 47–55 years, 189 cases of colon adenomas and 226 controls with normal total colonoscopy were identified. Weight at 10 years before was ascertained by referring to the recorded data. After allowance for hospital, rank in the SDF, smoking and alcohol use, weight gain over the past 10 years was significantly associated with increased risk of colon adenomas (odds ratio for ≥ 6 kg versus ≤−2 kg = 2.2; 95% confidence interval 1.0–4.8). High BMI and high WHR were each associated with increased risk, but only WHR was related to the risk independently of weight gain. In particular, weight gain accompanied with a high WHR was associated with a significant increase in the risk. Men with high physical activity tended to have lower risk. Associations with obesity‐related variables and physical activity were not materially differential as regards the location and size of adenoma. The findings indicate that weight gain in middle age leading to abdominal obesity increases the risk of colon adenomas, and consequently of colon cancer.
Multiple K‐ras Mutations in Hyperplasia and Carcinoma in Cases of Human Pancreatic Carcinoma
Authors: Matsubayashi Hiroyuki , Watanabe Hidenobu , Yamaguchi Toshikazu , Ajioka Yoichi , Nishikura Ken , Iwafuchi Mitsuya , Yamano Miki , Kijima Hiroshi , Saito Toshihiko
Published: August 2005

Mucous cell hyperplasia (MCH) has been considered an important precursor of pancreatic ductal carcinoma based on histological and molecular research, although various K‐ras mutations rates are seen among cases with pancreatic carcinoma, chronic pancreatitis and normal pancreas, with a wide range of histological characters. To investigate the premalignant potential of MCH and the multicentricity of pancreatic carcinoma, we analyzed K‐ras mutation at codon 12 in carcinoma foci of 82 cases of surgically‐resected pancreatic carcinoma [67 solid‐type carcinomas (SCs) and 15 ductectatic‐type carcinomas (DCs)], as well as in both MCH and carcinoma foci in 42 cases (30 SCs and 12 DCs), using an enriched polymerase chain reaction (PCR)‐enzyme linked mini‐sequence assay (ELMA). K‐ras mutation was recognized in 85% (57/67) of SCs and 73% (11/15) of DCs, and multiple K‐ras mutations in 12% (8/67) of SCs and in 20% (3/15) of DCs. Multiple K‐ras mutations were also recognized in MCHs in 47% (14/30) of SCs and in 42% (5/12) of DCs. Moreover, the same sequence at K‐ras codon 12 in MCH and carcinoma was identified in 76% (32/42) of carcinoma cases and it was more frequently recognized in hyperplasias with histological atypia (51%, 37 of 72 foci) than those without atypia (24%, 16 of 68 foci) (P < 0.0007). These results further support the idea of multicentric carcinogenesis and premalignant potential of atypical hyperplasia in the human pancreas, although about half of the hyperplasias around carcinomas were not thought to be direct precursors.
Expression of Tumor Necrosis Factor‐α and Interleukin‐6 in Oral Squamous Cell Carcinoma
Authors: Nakano Yasuko , Kobayashi Wataru , Sugai Satoshi , Kimura Hiroto , Yagihashil Soroku
Published: August 2005

To explore the role of cytokines in tumor development and clinical manifestations, we examined the expressions of tumor necrosis factor‐α (TNF‐α) and interleukin‐6 (IL‐6) in tumor tissues obtained from 57 patients with oral squamous cell carcinoma (OSCC) and their relationships to pathological grade and staging. Enzyme‐linked immunosorbent assay on the tumor tissues demonstrated elevated concentrations of TNF‐α and IL‐6 proteins and upregulated mRNA levels were detected by the reverse transcription‐polymerase chain reaction method when compared to those in normal control tissues. These cytokines and their transcripts were localized in stromal macrophages and in the tumor cells in particular of the front area of tumor issues, possibly indicating active synthesis of these cytokines by tumor cells. Larger‐sized tumors (T3, 4) contained significantly greater levels of IL‐6 proteins than small‐sized tumors (T1, 2) (P < 0.05). The levels of these cytokines were significantly reduced in cases with effective pre‐treatment with radiation or anti‐cancer agents compared to those in the less effective group (P < 0.05, grade IIa vs. grade IV for both TNF‐α and IL‐6). The present study thus demonstrated enhanced expression of cytokines in OSCC tissues.
Detection of Three Genetic Polymorphisms in the 5′‐Flanking Region and Intron 1 of Human CYP1A2 in the Japanese Population
Authors: Chida Michihiro , Yokoi Tsuyoshi , Fukui Takafumi , Kinoshita Moritoshi , Yokota Jun , Kamataki Tetsuya
Published: August 2005

Interindividual variability of the activity of CYP1A2 may be expected to affect cancer susceptibility, since the enzyme is capable of activating several carcinogens. In the present study, we found three new polymorphisms in the 5′‐flanking region (CYP1A2/B) and intron 1 (CYP1A2/C and CYP1A2/D) of CYP1A2 in Japanese by using polymerase chain reaction‐single strand conformation polymorphism. We developed methods to detect these polymorphisms by polymerase chain reaction‐restriction fragment length polymorphism and performed a population study (159 subjects) to estimate the frequencies of the alleles. The frequencies of the CYP1A2/A (adenine), CYP1A2/B (thymine‐deleted), CYP1A2/C (guanine) and CYP1A2/D (adenine) variants were 21.1, 42.0, 8.2 and 61.3%, respectively. The results of family study supported the idea that these CYP1A2 genotypes are inherited with an autosomal codominant transmission.
Expression of the DMBT1 Gene Is Frequently Suppressed in Human Lung Cancer
Authors: Takeshita Hiroaki , Sato Masami , Shiwaku Hiromi O. , Semba Shuho , Sakurada Akira , Hoshi Masato , Hayashi Yutaka , Tagawa Yutaka , Ayabe Hiroyoshi , Horii Akira
Published: August 2005

DMBT1 (deleted in malignant brain tumors) is a candidate tumor suppressor gene that has been mapped to chromosome 10q25.3‐q26.1, a region in which frequent loss of heterozygosity (LOH) has been observed in several human tumors. Since DMBT1 is highly expressed in the lung, we analyzed LOH at the DMBT1 locus and expression of this gene in lung cancer. Thirty‐five (53%) of 66 primary lung cancers showed LOH, and diminished expression of DMBT1 was observed in 20 (91%) of 22 lung cancer cell lines: three (14%) of them showed loss of expression. We further determined the primary structure of DMBT1 and analyzed genetic alterations in this gene using 23 lung cancer cell lines. Two (9%) of them had homozygous deletion within the gene, and two cell lines had genetic aberrations: one was a rearrangement involving exons 5 and 6, and the other was a missense mutation at codon 52. These results suggest that inactivation of the DMBT1 gene plays an important role in human lung carcinogenesis.
Two Target Regions of Allelic Loss on Chromosome 9 in Urinary‐bladder Cancer
Authors: Ohgaki Kenji , Minobe Kaori , Kurose Keisuke , Iida Aritoshi , Habuchi Tomonori , Ogawa Osamu , Kubota Yoshinobu , Akimoto Masao , Emi Mitsuru
Published: August 2005

Allelic losses on chromosome 9 are common in a wide variety of human tumors; moreover, two predisposing loci for some inherited cancer syndromes, i.e., familial malignant melanoma and Gorlin syndrome, have been identified on this chromosome. To define the location of putative tumor suppressor genes involved in cancer of the urinary bladder, 85 bladder cancers were examined for allelic loss at 18 microsatellite loci on chromosome 9. Correlations were also sought between loss of heterozygosity on chromosome 9 and several clinicopathological parameters. Allelic loss was observed in 54 of the tumors (64%) and deletion mapping identified two target regions; one at an interval on 9p21 flanked by D9S736 and D9S165, and the other at an interval on 9q31‐34 flanked by D9S58 and D9S61. No subtle mutation was detected in the PTCH gene which lies in the latter interval. Allelic loss on chromosome 9 was observed frequently in low grade and non‐invasive tumors as well as in tumors of more advanced phenotype. Inactivation of tumor suppressor genes lying in either of two regions of common deletion identified on chromosome 9 might affect carcinogenic mechanisms at an early stage of tumor development in the urinary bladder.
Clonal Emergence in Uremic Parathyroid Hyperplasia Is Not Related to MEN1 Gene Abnormality
Authors: Shan Liang , Nakamura Yasushi , Murakami Maki , Nakamura Misa , Naito Akihiro , Kawahara Katsuhiko , Utsunomiya Hirotoshi , Mori Ichiro , Kakudo Kennichi
Published: August 2005

It is difficult to differentiate between parathyroid neoplasia and hyperplasia. In an attempt to elucidate the clonality of uremic parathyroid hyperplasia and the molecular genetic abnormalities accounting for clonal emergence, we analyzed 20 cases of uremic parathyroid hyperplasia. Clonalities were determined using the X‐chromosome‐linked human androgen receptor (HUMARA) gene and the phosphoglycerate kinase (PGK) gene, and multiple endocrine neoplasia type 1 (MEN1) gene abnormality was analyzed by studying loss of heterozygosity (LOH) in 11q13 and somatic mutations in the MEN1 gene. As a positive control, a case of MEN1 with Zollinger‐Ellison syndrome was analyzed simultaneously. Our analysis revealed that a majority (75%) of the uremic parathyroid hyperplasia tissues, including an autograft with recurrent hyperparathyroidism, was of monoclonal origin. Clonality did not correlate with serum carboxyl‐terminal parathyroid hormone (C‐PTH) level, calcium level, hemodialytic duration, gland weight or pathological features. Neither LOH in 11q13 nor somatic mutation in the MEN1 gene was detected. For the MEN1 case, a germline mutation (W198X) was detected in exon 3. We concluded that a majority of the uremic parathyroid hyperplasia cases was in fact monoclonal neoplasia. MEN1 gene abnormality played a minor role, if any, in the clonal emergence in uremic parathyroid hyperplasia.
Expression of Vascular Endothelial Growth Factor (VEGF) Family Members in Breast Cancer
Authors: Kurebayashi Junichi , Otsuki Takemi , Kunisue Hironori , Mikami Yoshinori , Tanaka Katsuhiko , Yamamoto Shigeru , Sonoo Hiroshi
Published: August 2005

Vascular endothelial growth factor (VEGF)‐A is known to play an important role in tumor angiogenesis. Three additional members of the VEGF family, VEGF‐B, ‐C and ‐D, have recently been discovered. VEGF‐C and VEGF‐D are ligands for VEGF receptor‐3, which is expressed in the endothelium of lymphatic vessels. The expression of VEGF‐C is known to be associated with the development of lymphatic vessels. Therefore, it is conceivable that VEGF‐C and VEGF‐D might play a role in the development of lymphatic vessels in solid tumors. To obtain some clue as to this role, we developed a semi‐quantitative reverse transcription‐polymerase chain reaction method to investigate the mRNA expression levels of each VEGF family member in breast cancer. All the VEGF family members were expressed at different levels in seven human breast cancer cell lines explored. Although VEGF‐A and VEGF‐B expressions were detected in both node‐positive and node‐negative breast tumors, VEGF‐C expression was detected only in node‐positive tumors. VEGF‐D expression was detected only in an inflammatory breast cancer and a tumor which developed an inflammatory skin metastasis. These findings suggest a possible relationship between the expression level of VEGF‐C and/or VEGF‐D and the development of lymphatic tumor spread.
Antitumor Effects due to Irreversible Stoppage of Tumor Tissue Blood Flow: Evaluation of a Novel Combretastatin A‐4 Derivative, AC7700
Authors: Hori Katsuyoshi , Saito Sachiko , Nihei Yukio , Suzuki Manabu , Sato Yasufumi
Published: August 2005

The relation between tumor tissue blood flow (tBF) reduction and antitumor effects was investigated. Changes in tBF of normal tissues (liver, kidney cortex, bone marrow and brain cortex) and tumors (Yoshida sarcoma subline, LY80 and Sato lung carcinoma, SLC) due to i.v. administration of AC7700 (1, 3, 10 mg/kg), one of the combretastatin A‐4 derivatives, were measured with the hydrogen clearance method. The change in blood flow in tumor microfoci was also observed directly using a rat transparent chamber. Chemotherapy against the solid tumors (LY80, SLC) was performed by administering AC7700 7 times at intervals of 3 days and the effect on the tumor growth, the histological effect, the effect on lymph node metastasis and the survival rate were investigated. Tumor tBF showed a dose‐dependent response to AC7700. Although tumor tBF decreased markedly at a dose of 1 mg/kg, it tended to recover partly within several hours. At 10 mg/kg, however, tumor tBF completely stopped within approximately 30 min and never recovered in many regions. The irreversible stoppage of tumor tBF was observed in large s.c. tumors and in microfoci as well. On the other hand, in normal tissues, tBF changes due to AC7700 were not uniform. In the liver, although tBF decreased by approximately 50% at 10 mg/kg AC7700, it recovered within 8 h. In the brain, although the mean maximum reduction was 35%, the blood flow recovered to the original level within 24 h. The blood flow in the kidney cortex did not change at all. In the bone marrow, tBF decreased by approximately 80%. Generally, the blood flow reduction in normal tissues tended to be reversible. The effect on tumor growth and the histological effect were also dependent on the dose of AC7700. The tumor growth was markedly inhibited by 10 mg/kg AC7700 and extensive necrosis was induced. Lymph node metastases were significantly inhibited and survival was prolonged significantly. In the control group, all 8 SLC tumor‐bearing rats died of cancer, the presence of which was verified by gross and microscopic evaluation, within 45 days after tumor implantation. On the other hand, in the treated group, 2 of 8 rats recovered completely and survived. No obvious side effects such as body weight loss, anemia or diarrhea were observed at the dose used in this experiment. From these results, we conclude that strong antitumor effects are obtained by stopping tumor tBF irreversibly and by shutting off the nutritional supply into tumor tissue. AC7700 has been demonstrated to be a promising anticancer compound which has such an action.
In situ Gene Transfer and Suicide Gene Therapy of Gastric Cancer Induced by N‐Ethyl‐N′‐nitro‐N‐nitrosoguanidine in Dogs
Authors: Matsukura Norio , Hoshino Arichika , Igarashi Takehito , Hasegawa Hirokazu , Okino Tetsuya , Onda Masahiko , Iijima Osamu , Akiyama Katsuhiko , Goto Takashi , Takubo Kaiyo , Suzuki Satoru , Shimada Takashi
Published: August 2005

Gene therapy could potentially revolutionize the treatment of gastrointestinal (GI) tract cancer. The aim of this study was to establish a practical method of gene transfer which would be applicable to human gastric cancer. Retrovirus or/and adenovirus vectors carrying the lacZ marker gene were transferred in situ by needle through an endoscopic biopsy channel into primary gastric cancer in six male beagle dogs that had been treated with N‐ethyl‐N′‐nitro‐N‐nitrosoguanidine (ENNG). In addition, an adenovirus vector carrying the herpes simplex virus thymidine kinase (Ad.CAGHSV‐TK) gene was introduced in situ into cancer tissues in the stomach of three dog, and the animals were treated with intravenous ganciclovir (GCV). Retrovirus‐producing cells which expressed the lacZ gene were specifically localized to the injection site in the stomach. The lacZ gene was more widely transferred into the tumor by the adenovirus vector than by retrovirus‐producing cells. Improvement of the needle used for gene transfer and the use of multiple injections per tumor led to more diffuse transfer of the vector into the tumor. The Ad.CAG lacZ gene was also transferred into regional lymph nodes of the stomach. Moderate to diffuse degeneration of the primary cancer tissues of the stomach was found after Ad.CAGHSV‐TK/GCV gene therapy. Moreover, almost complete tissue degeneration was observed in the regional lymph nodes of the stomach. An adverse effect of HSV‐TK/GCV gene therapy was acute hepatotoxicity, which was not found after Ad.CAG lacZ gene transfer, but was found after high‐titer Ad.CAGHSV‐TK gene transfer followed by GCV. These findings suggest that in situ gene transfer of a suicide gene followed by prodrug treatment may be applicable not only to primary tumors, but also to lymph node metastases of gastric cancer, though further study of both beneficial and adverse effects is required before clinical usage.
Lack of Modification by Environmental Estrogenic Compounds of Thyroid Carcinogenesis in Ovariectomized Rats Pretreated with N‐bis(2‐hydroxypropyl)nitrosamine (DHPN)
Authors: Son Hwa‐Young , Nishikawa Akiyoshi , Ikeda Takako , Furukawa Fumio , Hirose Masao
Published: August 2005

The effects of environmental estrogenic compounds, soy isoflavone mixture (SI), genistein (GEN), and nonylphenol (NP), and the possible goitrogen 3‐chloro‐4‐(dichloromethyl)‐5‐hydroxy‐2(5H)‐furanone (MX), on thyroid carcinogenesis were investigated in ovariectomized (OVX) female rats. Five‐week‐old OVX F344 rats were given a single subcutaneous injection of N‐bis(2‐hydroxypropyl) nitrosamine (DHPN; 2400 mg/kg, body weight) or vehicle alone. Starting 1 week later, 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), sulfadimethoxine (SDM), a known thyroid tumor‐promoter (1000 ppm in drinking water), or β‐estradiol 3‐benzoate (EB), a synthetic estrogen (0.5 mg in cholesterol pellet, s.c.) were administered for 12 weeks. SDM and EB were included as positive controls. At sacrifice the major organs including the thyroid, pituitary, liver, kidney, uterus, vagina, brain and pancreas were collected and histopathological observation was performed. Thyroid weights were significantly increased (P < 0.001) only in the SDM treatment group and pituitary weights were elevated with SDM (P < 0.05) and EB (P < 0.001). Kidney and uterus weights were also significantly increased (P < 0.05) by EB. Histopathologically, proliferative lesions of the thyroid were only observed in the SDM treatment group and of the pituitary in the SDM or EB treatment groups. Renal tubule lesions, uterine squamous metaplasia, vaginal keratinization and telangiectasia of pancreatic islets were also observed with EB. There were no organ weight changes or histopathological lesions in the major organs, including the thyroid, in the GEN, SI, MX or NP treatment groups. Our results thus indicated a lack of modifying effects on thyroid carcinogenesis in female OVX rats, in agreement with our previous finding in males.
Overexpression of DA41 in v‐Ha‐ras‐3Y1 Cells Causes Growth Suppression
Authors: Ozaki Toshinori , Nakamura Yohko , Hanaoka Eiji , Nakagawara Akira , Sakiyama Shigeru
Published: August 2005

We have recently found that DA41 exhibits marked homology with mouse PLIC‐1, PLIC‐2, frog XDRP1 and yeast DSK2. XDRP1 has been shown to be associated with cyclin A, and blocks cell division of frog embryo. In the present study, we examined the biological role(s) of DA41 in mammalian cells by overexpressing it in v‐Ha‐ras‐transformed 3Y1 cells (ras‐3Y1). Transfectants which expressed a high level of DA41 mRNA exhibited a decrease in growth rate, a reduction in saturation density, and a suppression of colony formation in soft agar medium. To clarify the molecular mechanism(s) by which DA41 affects cell growth, the effect of DA41 expression on the levels of various cell cycle‐regulatory proteins was examined. The forced expression of DA41 gene resulted in a remarkable reduction in CDK2 activity, while the amount of CDK2 did not change. These observations indicate that DA41 is involved in cell cycle regulation in ras‐3Y1 cells.
The Specific Expression of Three Novel Splice Variant Forms of Human Metalloprotease‐like Disintegrin‐like Cysteine‐rich Protein 2 Gene in Brain Tissues and Glioma
Authors: Harada Taishi , Nishie Akihiro , Torigoe Kiyoyuki , Ikezaki Kiyonobu , Shono Tadahisa , Maehara Yoshihiko , Kuwano Michihiko , Wada Morimasa
Published: August 2005

We have previously identified 67 exons on a yeast artificial chromosome contig spanning 1.5 Mb around the multidrug resistance 1 gene region of human chromosome 7q21.1. In this study, we identified three novel cytoplasmic variants (MDC2‐γ, MDC2‐δ, and MDC2‐γ) of the human metalloprotease‐like disintegrin‐like cysteine‐rich protein 2 (MDC2) among these exons by screening a human brain cDNA library and also by using a reverse transcription polymerase chain reaction. Genomic sequence analysis strongly supported the idea that the variations in the cytoplasmic domain were generated by alternative splicing. The expression of MDC2 variant forms in human brain tissue and gliomas was examined by reverse transcription polymerase chain reaction and RNase protection assay. MDC2‐& ccar was expressed only in the cortical and hippocampal regions in human brain, but not in gliomas. In contrast, MDC2‐γ was a major form expressed in human gliomas. Specific expression of these cytoplasmic variants of MDC2 in human brain and its malignancies is discussed.
Centrosomal Kinases, HsAIRK1 and HsAIRK3, are Overexpressed in Primary Colorectal Cancers
Authors: Takahashi Takao , Futamura Manabu , Yoshimi Naoki , Sano Jun , Katada Masahiro , Takagi Yukihiro , Kimura Masashi , Yoshioka Takashi , Okano Yukio , Saji Shigetoyo
Published: August 2005

Members of the recently identified family of Homo sapiens Aurora/Ipl1‐related kinases (HsAIRKs), homologous to chromosome segregation kinases, fly Aurora and yeast Ipl1, are highly expressed during M phase, and have been suggested to regulate centrosome function, chromosome segregation, and cytokinesis. In the present study, immunohistochemical analyses were performed of HsAIRK1 and HsAIRK3 expression in 78 primary colorectal cancers and 36 colorectal adenomas as well as 15 normal colorectal specimens. In normal colon mucosa, some crypt cells showed weak positive staining in 10 and 12 out of 15 cases for HsAIRK1 and HsAIRK3, respectively, the remaining cases being negative. Elevated expression of HsAIRK1 was observed in 53 (67.9%) of the colorectal cancers, and of HsAIRK3 in 40 (51.3%). Furthermore, colorectal adenomas showed high expression of HsAIRK1 and HsAIRK3 in 11 (30.6%) and 7 (19.4%) cases, respectively, thus being intermediate between colorectal cancers and normal colorectal mucosa. Interestingly, HsAIRK1 overexpression was significantly associated with pT (primary tumor invasion) and p53 accumulation in colorectal cancers. There was no significant correlation between proliferating cell nuclear antigen‐labeling index (PCNA‐LI) and the levels of these proteins. The results suggest that overexpression of HsAIRK1 and HsAIRK3 might be involved in tumorigenesis and/or progression of colorectal cancers.
Activation of Intestinal Mucosal Immunity in Tumor‐bearing Mice by Lactoferrin
Authors: Wang Wen‐Ping , Iigo Masaaki , Sato Jun , Sekine Kazunori , Adachi Isamu , Tsuda Hiroyuki
Published: August 2005

We have previously demonstrated that oral administration of bovine lactoferrin (bLF) markedly increases CD4+ and CD8+ T cells and NK (asialoGM1+) cells in the blood of tumor‐bearing mice and enhances anti‐metastatic activity. In this paper, we document that oral administration of bLF and bLF‐hydrolysate (bLFH) is associated with strong increases in CD4+ and CD8+ T, as well as asialoGM1+ cells in lymphoid tissues and lamina propria of the small intestine in mice, especially in tumor‐bearing animals in which Co26Lu cells were implanted subcutaneously. Moreover, IgM+ and IgA+ B cells in lamina propria of the small intestine were also significantly increased by bLF and bLFH. Bovine apo‐transferrin (bTF) did not exhibit such activity. In the colon, only CD8+ cells were significantly increased by treatment with bLF, while asialoGM1+ cells were significantly decreased. bLF and bLFH induced cytokines to activate T, B and asialoGM1+ cells. Administration of bLF and bLFH, but not bTF, increased production of interleukin‐18 (IL‐18), interferon‐gamma (IFN‐γ) and caspase‐1 in the mucosa of the small intestine. Particularly high levels of IL‐18 were found in the epithelial cells of the small intestine. Moreover, administration of bLF and bLFH, but not bTF, induced IFN‐γ presenting cells in the small intestine. Caspase‐1, which processes proIL‐18 to mature IL‐18, was also induced in the epithelial cells of the small intestine following treatment with bLF and bLFH, but not with bTF. These results suggest that enhanced production of IL‐18 and IFN‐γ and caspase‐1 induction by treatment with bLF may be important for elevation of intestinal mucosal immunity.
Transduction of a Fiber‐mutant Adenovirus for the HSVtk Gene Highly Augments the Cytopathic Effect towards Gliomas
Authors: Shinoura Nobusada , Sakurai Shinya , Asai Akio , Kirino Takaaki , Hamada Hirofumi
Published: August 2005

Suicide gene therapy utilizing the herpes simplex thymidine kinase (HSVtk)/ganciclovir (GCV) system has been performed to kill cancer cells. However, the low transduction efficiency of HSVtk gene into cancer cells critically limits its efficacy in cancer treatment in clinical situations. To improve delivery of the HSVtk gene into cancer cells, we transduced U‐87MG and U‐373MG glioma cells with adenovirus (Adv) vectors with a fiber mutant, F/K20, which has a stretch of 20 lysine residues added at the C‐terminus of the fiber, for the HSVtk gene (Adv‐TK‐F/K20), and compared the cytopathic effect of Adv‐TK‐F/K20 with that of the Adv for HSVtk with wild‐type fiber (Adv‐TK). The cytopathic effect of Adv‐TK‐F/K20 in U‐87MG and U‐373MG cells was approximately 140 and 40 times, respectively, stronger than that of Adv‐TK. At the same multiplicity of infection (MOI) in each cell line, Adv‐TK‐F/K20 induced a higher degree of apoptosis (U‐87MG, 35%; U‐373MG, 77%) than Adv‐TK (U‐87MG, 0.11%; U‐373MG, 27%) in U‐87MG (MOI 0.03) and U‐373MG cells (MOI 0.1). Cleavage of poly(ADP‐ribose)polymerase (PARP) was more marked in the cells that were infected with Adv‐TK‐F/K20 than in cells that were infected with Adv‐TK. These results indicate that gene therapy utilizing Adv‐TK‐F/K20 may be a promising therapeutic modality for the treatment of gliomas.
Production of a Single‐chain Variable Fragment Antibody Recognizing Type III Mutant Epidermal Growth Factor Receptor
Authors: Nakayashiki Norihisa , Yoshikawa Kazuhiro , Nakamura Kazuyasu , Hanai Nobuo , Okamoto Kenta , Okamoto Sho , Mizuno Masaaki , Wakabayashi Toshihiko , Saga Sinsuke , Yoshida Jun , Takahashi Toshitada
Published: August 2005

The type III deletion mutant of the epidermal growth factor receptor (EGFR) is a potential target in diagnostic and therapeutic approaches for those glioblastomas characterized by its expression. We previously raised a mouse monoclonal antibody, 3C10 (IgG2b) specifically recognizing this mutant EGFR. In this study, a single‐chain variable fragment (scFv) antibody was produced. Partial determination of its N‐terminal amino acid sequence and preparation of adequate primers for variable heavy chain (VH) and variable light chain (VL) genes were performed to allow cloning by means of reverse transcriptase‐polymerase chain reaction. The genes cloned were assembled with a linker, (Gly4Ser)3, and ligated into a bacterial expression vector to express the scFv as cytoplasmic inclusion bodies. After appropriate refolding, the antibody activity of the VH‐VL scFv was examined in an enzyme‐linked immunosorbent assay. 3C10 scFv showed a selective reactivity with the mutant peptide, similarly to the parental 3C10 antibody. A mouse transfectant expressing the type III mutant EGFR and a glioblastoma with type III deletion‐mutant EGFR were positively stained by immunofluorescence. By Biacore analysis, the affinity (KA) of the parental 3C10 for the mutant peptide was 9.7x107M‐1, while that of 3C10 scFv was 2.45‐2.48x107M‐1, being approximately 4‐fold weaker. The results together suggested that the scFv antibody retained the appropriate structure to recognize a conformational epitope of the mutant receptor, similarly to the parental antibody.
Association of Preoperative Radiation Effect with Tumor Angiogenesis and Vascular Endothelial Growth Factor in Oral Squamous Cell Carcinoma
Authors: Shintani Satoru , Kiyota Akihisa , Mihara Mariko , Nakahara Yuuji , Terakado Nagaaki , Ueyama Yoshiya , Matsumura Tomohiro
Published: August 2005

This study examined the relationship between tumor angiogenesis and the radiation‐induced response, evaluated based on pathological changes, in oral squamous cell carcinoma patients treated with preoperative radiation therapy. Forty‐one cases of squamous cell carcinoma treated with preoperative radiation therapy were investigated. Tumor angiogenesis was assessed by scoring the intratumor microvessel density (IMVD). Expression of vascular endothelial growth factor (VEGF) was also evaluated before and after preoperative radiotherapy. There was no correlation between IMVD in the specimens before therapy and the pathological response to radiation therapy. However, radiation therapy decreased IMVD in the specimens after therapy. A significant association was observed between VEGF expression and resistance to radiation therapy: only 4 of the 21 patients whose tumors exhibited a high level (2+ or 3+) of VEGF staining experienced a major (3+ or 4+) pathological response to radiation therapy. Furthermore, an increasing level of VEGF expression after radiation therapy was observed in non‐effective (0 to 2+) response cases. These results suggest that VEGF expression and the induction of this protein are related to radiosensitivity and could be used to predict the effects of preoperative radiation therapy on oral squamous cell carcinoma.
The Effects of Boron Neutron Capture Therapy on Liver Tumors and Normal Hepatocytes in Mice
Authors: Suzuki Minoru , Masunaga Shin‐Ichiro , Kinashi Yuko , Takagaki Masao , Sakurai Yoshinori , Kobayashi Toru , Ono Koji
Published: August 2005

To explore the feasibility of employing boron neutron capture therapy (BNCT) to treat liver tumors, the effects of BNCT were investigated by using liver tumor models and normal hepatocytes in mice. Liver tumor models in C3H mice were developed by intrasplenic injection of SCCVII tumor cells. After borocaptate sodium (BSH) and boronophenylalanine (BPA) administration, 10B concentrations were measured in tumors and liver and the liver was irradiated with thermal neutrons. The effects of BNCT on the tumor and normal hepatocytes were studied by using colony formation assay and micronucleus assay, respectively. To compare the effects of BSH‐BNCT and BPA‐BNCT, the compound biological effectiveness (CBE) factor was determined. The CBE factors for BSH on the tumor were 4.22 and 2.29 using D10 and D0 as endpoints, respectively. Those for BPA were 9.94 and 5.64. In the case of hepatocytes, the CBE factors for BSH and BPA were 0.94 and 4.25, respectively. Tumor‐to‐liver ratios of boron concentration following BSH and BPA administration were 0.3 and 2.8, respectively. Considering the accumulation ratios of 10B, the therapeutic gain factors for BSH and BPA were 0.7‐1.3 and 3.8‐6.6, respectively. Therefore, it may be feasible to treat liver tumors with BPA‐BNCT.
Mechanism of Anti‐tumor Effect of Combination of Bleomycin and Shock Waves
Authors: Kato Masanori , Ioritani Naomasa , Suzuki Takashi , Kambe Mariko , Inaba Yasuo , Watanabe Ryuji , Sasano Hironobu , Orikasa Seiichi
Published: August 2005

We have previously reported marked enhancement of the cytocidal effect of bleomycin (BLM) on cancer cell suspensions in vitro by the combination with shock waves. In this study, we evaluated the synergistic effects on cancer cell proliferation and apoptosis in solid tumors. A spherical piezoceramic element was used as the shock wave source, with a pressure peak of 40 MPa. A human colon cancer cell line, SW480 was implanted onto the back of nude mice. Two thousand shock waves were administered to the tumor immediately following an intravenous injection of BLM at a dose of one‐tenth of the LD50. The tumor was extirpated at 3, 6, 12, 24, 72 h and 1 week following shock exposure. Cell proliferation and apoptosis were detected by Ki‐67 using antibody MIB‐1 and by the terminal deoxynucleotidyl transferase (TdT)‐mediated dUTP‐biotin nick‐end labeling (TUNEL) method. The lowest percentage (35.7%) of Ki‐67‐positive cells appeared 24 h following the treatment. The maximum apoptotic index was detected within 6 h following the treatment. Moreover, numerous large cells with enlarged nuclei were detected histologically. These results suggest that shock waves may enhance chemotherapeutic effects by increasing apoptosis and decreasing cell proliferation in the tumor tissue.
Structure‐Activity Relationship of a Novel Group of Mammalian DNA Polymerase Inhibitors, Synthetic Sulfoquinovosylacylglycerols
Authors: Hanashima Shinya , Mizushina Yoshiyuki , Ohta Keisuke , Yamazaki Takayuki , Sugawara Fumio , Sakaguchi Kengo
Published: August 2005

We reported previously that sulfolipids in the sulfoquinovosylacylglycerol class from a fern and an alga are potent inhibitors of DNA polymerase α and β and potent anti‐neoplastic agents. In developing a procedure for chemical synthesis of sulfolipids, we synthesized many derivatives and stereoisomers of sulfoquinovosylmonoacylglycerol (SQMG)/sulfoquinovosyldiacylglycerol (SQDG). Some of these molecules were stronger inhibitors than the SQMG/SQDG originally reported as natural compounds. In this study, we examined the structure‐inhibitory function relationship of synthetic SQMG/SQDG and its relationship to cytotoxic activity. The inhibitory effect is probably mainly dependent on the fatty acid effect, which we reported previously, although each of the SQMG/SQDG was a much stronger inhibitor than the fatty acid alone that was present in the SQMG/SQDG. The inhibitory effect could be influenced by the chain size of fatty acids in the SQMG/SQDG. The sulfate moiety in the quinovose was also important for the inhibition. Lineweaver‐Burk plots of SQMG/SQDG indicated that DNA polymerase α was non‐competitively inhibited, but the SQMG/SQDG were effective as antagonists of both template‐primer DNA‐binding and nucleotide substrate‐binding of DNA polymerase β. The SQMG had an cytotoxic effect, but the SQDG tested did not. The SQDG might not be able to penetrate into cells. Based on these results, we discuss the molecular action of SQMG/SQDG and propose drug design strategies for developing new anti‐neoplastic agents.
Nuclear Localization of Immunoreactive β‐Catenin Is Specific to Familial Adenomatous Polyposis in Papillary Thyroid Carcinoma
Authors: Kurihara Katsumi , Shimizu Shin‐Ichiro , Chong Ja‐Mun , Hishima Tunekazu , Funata Nobuaki , Kashiwagi Hiroshi , Nagai Hideaki , Miyaki Michiko , Fukayama Masashi
Published: August 2005

Thyroid carcinoma is the first symptom in some patients with familial adenomatous polyposis (FAP). We evaluated the cellular localization of β‐catenin in thyroid carcinomas associated (n=4) or not associated (n=173) with FAP, since loss of functional protein of the adenomatous polyposis coli (APC) gene leads to nuclear accumulation of β‐catenin in adenomas and carcinomas of the FAP colon. Immunoreactive β‐catenin was demonstrated at the cell membrane of glandular cells of the non‐neoplastic thyroid and non‐FAP carcinomas. On the other hand, cytoplasmic and nuclear accumulation of β‐catenin is specific to FAP‐associated papillary carcinomas. The abnormality in the APC/β‐catenin pathway is thus also important in FAP‐associated thyroid carcinoma, and β‐catenin immunohistochemistry is a feasible screening method to identify occult FAP in young patients with thyroid tumors.
Identification of Genes Associated with the Progression of Adult T Cell Leukemia (ATL)
Authors: Kohno Tomoko , Moriuchi Ryozo , Katamine Shigeru , Yamada Yasuaki , Tomonaga Masao , Matsuyama Toshifumi
Published: August 2005

Patients with adult T‐cell leukemia/lymphoma (ATL) exhibit a variety of clinical features, and this disease is therefore clinically subclassified into acute, lymphomatous, chronic, and smoldering types. Acute ATL is a typical leukemic form of ATL with rapid progression, and chronic ATL is a less aggressive clinical form allowing long‐term survival even without chemotherapy. In the present study, we used fresh peripheral blood mononuclear cells (PBMC) from both types of ATL patients to identify molecules that may contribute to the difference between acute and chronic ATL. Isolated mRNAs expressed differentially between the two types of ATL include a T‐cell differentiation antigen (MAL), a lymphoid‐specific member of the G‐protein‐coupled receptor family (EBI‐1/CCR7), a novel human homologue to a subunit (MNLL) of the bovine ubiquinone oxidoreductase complex, and a human fibrinogen‐like protein (hpT49). We found that the former three are upregulated in acute ATL and the last is down‐regulated in both chronic and acute ATL. We speculate that dysregulation of the genes may account for the malignant features of ATL cells, in terms of growth, energy metabolism, and motility.
Nonrandom Chromosomal Imbalances in Esophageal Squamous Cell Carcinoma Cell Lines: Possible Involvement of the ATF3 and CENPF Genes in the 1q32 Amplicon
Authors: Pimkhaokham Atiphan , Shimada Yutaka , Fukuda Yohji , Kurihara Naoki , Imoto Issei , Yang Zeng‐Quan , Imamura Masayuki , Nakamura Yusuke , Amagasa Teruo , Inazawa Johji
Published: August 2005

Using comparative genomic hybridization (CGH), we investigated copy number aberrations in 29 esophageal squamous cell carcinoma (ESC) cell lines. All lines displayed numerous chromosome imbalances. The most frequent losses were observed on chromosome 18q (65.5%), Xp (48.3%), 3p (44.8%), 4q (44.8%), 8p (41.4%), 11q23–25 (34.5%) and 4p (27.6%), whereas the most common copy number gains were noted at 8q (86.2%), 3q (82.8%), 5p (69%), 7p (69%), 20q (65.5%), 9q (55.2%), 11q (55.2%), 1q (48.3%), Xq (44.8%) and 18p (37.9%). High‐level gains (HLGs) were detected at 3q26 (9 cases), 8q23 (6 cases), 5p14–15 (6 cases), 18p11.2–11.3 (6 cases), 3q27–28 (5 cases), 5p13 (3 cases), 7p14–15 (3 cases), 20q12–13 (3 cases), 11q13 (3 cases), 14q21 (2 cases), 20p11.2 (2 cases), 13q32 (2 case), and 1q32 (1 case). Among them, HLGs of 1q32 have been reported in other types of cancer, including glioblastoma and breast cancers. We successfully narrowed down the smallest common amplicon involving 1q‐gain to the genomic segment between D1S414 and D1S2860 by fluorescence in situ hybridization (FISH). Southern and northern blot analysis clearly demonstrated that ATF3, human activating transcription factor‐3 and CENPF, centromere protein F, mapped within this region, were significantly amplified and over‐expressed in 1q32 amplicon.
Intratumor Heterogeneity of Centromere Numerical Abnormality in Multiple Primary Gastric Cancers: Application of Fluorescence in situ Hybridization with Intermittent Microwave Irradiation on Paraffin‐embedded Tissue
Authors: Kobayashi Keiko , Kitayama Yasuhiko , Igarashi Hisaki , Yoshino Goro , Kobayashi Toshihiko , Kazui Teruhisa , Sugimura Haruhiko
Published: August 2005

Our recent success in retrieving distinct fluorescence signals in response to centromere specific probing of paraffin‐embedded tissues after intermittent microwave (MW) treatment provided the opportunity to analyze chromosome numbers or centromere abnormality in situ in human tumors in various clinicopathological settings. In this study, centromere numerical abnormality (CNA) was investigated by fluorescence in situ hybridization (FISH) in a case of multiple gastric cancer having intratumor histological heterogeneity. The different profiles as determined using a total of 20 specific probes on 4 multifocal lesions in the stomach confirmed the multi‐clonality of these tumors. FISH with probes specific for chromosomes 10, 11, 16 and 18 revealed intratumor heterogeneity of the CNA, which corresponded to the histological heterogeneity. Our report clearly demonstrates, for the first time, intratumor heterogeneity of CNA and its association with the histological picture, and substantiates the applicability of the MW‐assisted FISH protocol to paraffin‐embedded pathological specimens.