bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of the Wilms' Tumor Gene WT1 in Solid Tumors and Its Involvement in Tumor Cell Growth
Authors: Oji Yusuke , Ogawa Hiroyasu , Tamaki Hiroya , Oka Yoshihiro , Tsuboi Akihiro , Kim Eui Ho , Soma Toshihiro , Tatekawa Toyoshi , Kawakami Manabu , Asada Momotaro , Kishimoto Tadamitsu , Sugiyama Haruo
Published: August 2005

To determine the role of the Wilms' tumor gene WT1 in tumorigenesis of solid tumors, expression of the WT1 gene was examined in 34 solid tumor cell lines (four gastric cancer cell lines, five colon cancer cell lines, 15 lung cancer cell lines, four breast cancer cell lines, one germ cell tumor cell line, two ovarian cancer cell lines, one uterine cancer cell line, one thyroid cancer cell line, and one hepatocellular carcinoma cell line) by means of quantitative reverse transcriptase‐polymerase chain reaction. WT1 gene expression was detected in three of the four gastric cancer cell lines, all of the five colon cancer cell lines, 12 of the 15 lung cancer cell lines, two of the four breast cancer cell lines, the germ cell tumor cell line, the two ovarian cancer cell lines, the uterine cancer cell line, the thyroid cancer cell line, and the hepatocellular carcinoma cell line. Therefore, of the 34 solid tumor cell lines examined, 28 (82%) expressed WT1. Three cell lines expressing WT1 (gastric cancer cell line AZ‐521, lung cancer cell line OS3, and ovarian cancer cell line TYK‐nu) were further analyzed for mutations and/or deletions in the WT1 gene by means of single‐strand conformation polymorphism analysis. However, no mutations or deletions were detected in the region of the WT1 gene ranging from the 3/end of exon 1 to exon 10 (the WT1 gene consists of 10 exons) in these three cell lines. Furthermore, when AZ‐521, OS3, and TYK‐nu cells were treated with WT1 antisense oligomers, the growth of these cells was significantly inhibited in association with a reduction in WT1 protein levels. Furthermore, constitutive expression of the transfected WT1 gene in cancer cells inhibited the antisense effect of WT1 antisense oligomer on cell growth. These results indicated that the WT1 gene plays an essential role in the growth of solid tumors and performs an oncogenic rather than a tumor‐suppressor gene function.
Frequent β‐Catenin Abnormalities in Bone and Soft‐tissue Tumors
Authors: Iwao Kyoko , Miyoshi Yasuo , Nawa Gen , Yoshikawa Hideki , Ochi Takahiro , Nakamura Yusuke
Published: August 2005

We have screened mutations of the β‐catenin gene by using the polymerase chain reaction‐single strand conformation polymorphism (PCR‐SSCP) method in 62 malignant bone and soft‐tissue tumors, including malignant fibrous histiocytomas (MFHs), osteosarcomas, synovial sarcomas, liposarcomas, malignant schwannomas, and other types of tumors, as well as 11 benign tumors. β‐Catenin‐activating missense mutations were found in two malignant tumors. One found in MFH occurred at codon 45 and caused an amino acid substitution from serine (one of the GSK3β‐targeted phosphorylation sites) to phenylalanine. The other, detected in synovial sarcoma at codon 32, resulted in an amino acid change from aspartic acid (located adjacent to the phosphorylation target, serine, encoded by codon 33) to tyrosine. Furthermore, we found accumulation of β‐catenin by western‐blotting analysis in 12 of 19 malignant tumors in which we found no mutation involving exon 3. Our results suggested the possible involvement of β‐catenin activation, by β‐catenin gene mutation or alteration of other factor(s), in the formation and/or progression of various types of bone and soft‐tissue tumors.
Effective Irinotecan (CPT‐11)‐containing Liposomes: Intraliposomal Conversion to the Active Metabolite SN‐38
Authors: Sadzuka Yasuyuki , Hirotsu Sachiyo , Hirota Sadao
Published: August 2005

Irinotecan hydrochloride (CPT‐11) is a prodrug of SN‐38, which is an active metabolite with anti‐tumor activity and side toxicity. The activities of CPT‐11 and SN‐38 depend on the closed lactone ring form of SN‐38. We have examined the tissue distributions of the closed and open forms of CPT‐11 and SN‐38 in Lewis lung carcinoma‐bearing mice after the administration of liposomal CPT‐11 (S‐Lip) and polyethyleneglycol (PEG)‐modified S‐Lip (S‐PEG). The plasma concentrations of closed CPT‐11 and SN‐38 were increased by liposomalization, and their blood circulation was prolonged by the PEG modification. The concentrations of closed CPT‐11 and SN‐38 in tumors were elevated by both the liposomalization and PEG modification. The closed/total ratio of SN‐38 in the tumors of the S‐PEG group was greater than that of the CPT‐11 solution (Sol) group. Thus, SN‐38 was thought to be generated in intact liposomes containing CPT‐11. The bile concentration of closed SN‐38, which is responsible for CPT‐11‐induced intestinal disorder, was decreased by liposomalization. In an in vitro experiment, the SN‐38/CPT‐11 ratio in the tumor cells of the S‐Lip group was found to be higher than that of the Sol group, and the ratio of the closed form of SN‐38 was increased by the liposomalization. Laser scanning confocal microscopy showed the generation of SN‐38 in the liposomal membrane after the incubation of S‐Lip with carboxylesterase. It is therefore considered that a part of CPT‐11 is converted to SN‐38 in the intact liposomes.
Frequent p53 Gene Mutations in Soft Tissue Sarcomas Arising in Burn Scar
Authors: Nakanishi Hirofumi , Tomita Yasuhiko , Yoshikawa Hideki , Sato Noriko , Ochi Takahiro , Aozasa Katsuyuki
Published: August 2005

Squamous cell carcinoma (SCC) is the commonest malignancy that arises in burn scars, which frequently contain p53 mutations. Soft tissue sarcoma (STS) also develops, though less frequently, in burn scars. p53 gene mutations were analyzed in paraffin‐embedded specimens from 5 patients with STS (4 males and 1 female) that had arisen in a burn scar, by means of polymerase chain reaction‐single strand conformation polymorphism (PCR‐SSCP) followed by direct sequencing. Age at burn injury ranged from 2 to 10 (median 3) years, and STS developed with a latent period ranging from 29 to 79 (median 60) years. Histologically, all were malignant fibrous histiocytoma. The PCR‐SSCP revealed aberrant bands in 4 (80%) of 5 cases. Direct sequencing revealed a total of 11 mutations in these 4 cases: 1 case had a single mutation, 1 had 2 mutations, and 2 had 4 mutations. Every tumor had at least 1 mutation that changed an amino acid, which may have provided the selection pressure for expansion. Thus, there is a high frequency of p53 gene mutations in STS appearing in burn scars. p53 mutations were also frequent in pyothorax‐associated lymphoma (PAL), a lymphoma that develops in patients with long‐standing pyothorax, so p53 mutations might be frequent in malignancies that develop in chronic inflammatory sites.
Overexpression of Proliferating Cell Nuclear Antigen in Mammalian Cells Negates Growth Arrest by Serum Starvation and Cell Contact
Authors: Fukami‐Kobayashi Junya , Mitsui Youji
Published: August 2005

Proliferating cell nuclear antigen (PCNA) functions as a processivity factor for DNA polymerase δ, and is expressed at high levels in growing normal and tumor cells. To clarify the relationship between cell proliferation and PCNA expression, we generated NIH‐3T3 cells that overexpress PCNA and analyzed the phenotype of these cells. The resulting 3T3‐PCNA cells, which overexpressed PCNA, were found to proliferate beyond the saturation density of the parental NIH‐3T3 cells. Although NIH‐3T3 cell proliferation is arrested under serum starvation conditions, 3T3‐PCNA cell proliferation is not arrested by serum starvation. The expression levels of cdk2, cdk4 and cdk6 were the same in 3T3‐PCNA and NIH‐3T3 cells. The activity of cdk4 was identical for both cell types. However, the activity of cdk2 was higher in serum‐starved 3T3‐PCNA cells than in NIH‐3T3 cells, although the expression of cyclin E decreased in both types of cells, suggesting that increases in cdk2 activity are related to negation of growth arrest in 3T3‐PCNA cells. These results indicate that increases in PCNA expression lead to the disruption of growth control and may lead to malignant transformation.
Specific Role of T and Tn Tumor‐associated Antigens in Adhesion between a Human Breast Carcinoma Cell Line and a Normal Human Breast Epithelial Cell Line
Authors: Kishikawa Teruko , Ghazizadeh Mohammad , Sasaki Yoshihiro , Springer Georg F.
Published: August 2005

The possibility that tumor‐associated antigens T and Tn act as adhesion molecules between normal and malignant breast epithelial cells at the early stages of recognition in the metastatic pathway was examined in vitro. The adhesive specificity of the antigens was assessed by means of in vitro adhesion tests between a carcinomatous breast cancer cell line (ZR75‐30) and a normal epithelial breast cell line (HLB100) using both monoclonal antibodies and lectins specific as well as nonspecific for each antigen. Adhesion assay was performed using monolayers of the normal cell line prepared on plastic culture plates and the tumor cell line labeled with a fluorescent dye as a probe. The adhesion between the two cell types occurred with significant specificity via T and Tn antigens (P < 0.001), and was temperature‐dependent. The results suggest that at the early stages of recognition by tumor cells in the metastatic process, T and Tn antigens play a role as adhesion molecules between the tumor cells and adjacent normal cells.
Inhibition of Spontaneous Rat Osteosarcoma Lung Metastasis by 3S‐[4‐(Nhydroxyamino)‐2R‐isobutylsuccinyl]amino‐1‐methoxy‐3,4‐dihydrocarbostyril, a Novel Matrix Metalloproteinase Inhibitor
Authors: Kido Akira , Tsutsumi Masahiro , Iki Katsumichi , Motoyama Masaaki , Takahama Makoto , Tsujiuchi Toshifumi , Morishita Toru , Tatsumi Kunihiko , Tamai Susumu , Konishi Yoichi
Published: August 2005

In the present experiment, we examined the effects of OPB‐3206, 3S‐[4‐(N‐hydroxyamino)‐2R isobutylsuccinyl] amino‐1‐methoxy‐3,4‐dihydrocarbostyril, a novel metalloproteinase inhibitor, on the growth and metastasis of transplantable osteosarcomas (spontaneous osteosarcoma, selected lung metastatic lesions; S‐SLM), which were previously established in rats. OPB‐3206 inhibited the activities of interstitial collagenase, gelatinases A and B, and stromelysin in vitro. After oral administration to rats, its serum concentration peaked at 40 min and the drug was no longer detectable at 8 h. When OPB‐3206 was orally administered at 0%, 0.1% and 0.4% in the diet for 4 weeks, starting 7 days after subcutaneous transplantation of osteosarcomas to male Fischer 344 rats, numbers of lung metastatic nodules were significantly reduced by the highest dose, while the growth of subcutaneous tumors was not affected. Zymographic analysis showed the presence of pro matrix metalloproteinase (proMMP)‐2, proMMP‐9 and MMP‐9 activities in S‐SLM. In animals fed 0.4% OPB‐3206, the activity of proMMP‐9 was increased, but that for MMP‐9 had become undetectable. The results thus suggest that OPB‐3206 selectively inhibits lung metastasis of rat transplantable osteosarcomas by inhibiting MMP‐9 activation.
Circumvention of 5‐Fluorouracil Resistance in Human Stomach Cancer Cells by Uracil Phosphoribosyltransferase Gene Transduction
Authors: Inaba Makoto , Sawada Hiroko , Sadata Akiko , Hamada Hirofumi
Published: August 2005

A human stomach cancer cell line with acquired resistance to 5‐fluorouracil (5‐FU), NUGC‐3/5FU/L, has been found to possess reduced ability to convert 5‐FU into active metabolites. We attempted in vitro gene therapy for this 5‐FU‐resistant cell line. NUGC‐3 and NUGC‐3/5FU/L cells were infected with recombinant adenovirus (Ad) containing Escherichia coli uracil phosphoribosyltransferase (UPRT) gene driven by CAG promoter (CA), AdCA‐UPRT, and changes in their 5‐FU metabolism and sensitivity were investigated. Activities of orotate phosphoribosyltransferase increased from 10.2 and 1.56 (nmol/mg protein/30 min) in the uninfected cells of NUGC‐3 and NUGC‐3/5FU/L to 216 and 237, respectively, after the transfection of UPRT gene. The 5‐FU nucleotide level in the acid‐insoluble fraction increased from 7.32 to 15.9 (pmol/mg protein) in NUGC‐3 cells on infection with AdCA‐UPRT, and in NUGC‐3/5FU/L cells it increased from 1.91 to 21.4. The 50% growth‐inhibition concentration (IC50) was 12.7 μmol/liter for NUGC‐3 and much higher than 100 μmol/liter for NUGC‐3/5FU/L, indicating over 8‐fold resistance. NUGC‐3/5FU/L transfected with the UPRT gene showed very high sensitivity to 5‐FU with an IC50 of 3.2 μmol/liter. The high resistance in this metabolic activation‐deficient cell line was thus completely reversed by transduction of an exogenous gene coding for a 5‐FU‐anabolizing enzyme.
Lactoferrin Inhibits Hepatitis C Virus Viremia in Patients with Chronic Hepatitis C: A Pilot Study
Authors: Tanaka Katsuaki , Ikeda Masanori , Nozaki Akito , Kato Nobuyuki , Tsuda Hiroyuki , Saito Satoru , Sekihara Hisahiko
Published: August 2005

Hepatitis C virus (HCV) is associated with the development of cirrhosis and hepatocellular carcinoma. We recently found that bovine lactoferrin, a milk protein belonging to the iron transporter family, effectively prevented HCV infection in cultured human hepatocytes (PH5CH8). We tested the hypothesis that lactoferrin inhibits HCV viremia in patients with chronic hepatitis C. Eleven patients with chronic hepatitis C received an 8‐week course of bovine lactoferrin (1.8 or 3.6 g/day). At the end of lactoferrin treatment, a decrease in serum alanine transaminase and HCV RNA concentrations was apparent in 3 (75%) of 4 patients with low pretreatment serum concentrations of HCV RNA. However, 7 patients with high pretreatment concentrations showed no significant changes in these indices. This pilot study suggests that lactoferrin is one potential candidate as an anti‐HCV reagent that may be effective for the treatment of patients with chronic hepatitis.
Phytol Is a Novel Tumor Promoter on ICR Mouse Skin
Authors: Kagoura Masayori , Matsui Chihiro , Morohashi Masaaki
Published: August 2005

Phytol is a branched, long‐chain aliphatic alcohol which has various biological effects. In this study, we examined phytol as a tumor promoter in a mouse skin initiation‐promotion model, and compared its promotion activity with that of 12‐O‐tetradecanoyl phorbol‐13‐acetate (TPA). Female ICR mice, 7 weeks of age, were initiated with 100 μg of 7,12‐dimethylbenz(a)anthracene, and were then topically promoted twice a week for 16 weeks with 100 mg of phytol or with 2.5 μg of TPA. In this model 95% of animals treated with phytol developed skin tumors within 16 weeks. The average number of lesions per mouse treated with phytol was significantly lower than that in mice treated with TPA, and this significant difference continued up to 16 weeks after the end of promotion treatment. Characterization of hyperplasia 48 h after topical application of agents showed that epidermal thickness and vertical thickness following topical application of phytol were significantly increased compared with vehicle controls, but were significantly smaller than in animals treated with TPA. Ornithine decarboxylase (ODC) activity following topical application of phytol was increased in a dose‐dependent manner and showed a weak, delayed induction (which was maximal 11–12 h after treatment) as compared with the case of TPA. The specific binding of [3H]phorbol‐12,13‐dibutyrate (PDBU) by JB6 cells was not inhibited by phytol at concentrations up to 1 mM. These results indicate that phytol has a weak tumor promoter activity compared to TPA and is a non‐TPA‐type tumor promoter in this model of mouse skin carcinogenesis.
Chemopreventive Effect of N‐(2‐Cyclohexyloxy‐4‐nitrophenyl)methane Sulfonamide (NS‐398), a Selective Cyclooxygenase‐2 Inhibitor, in Rat Colon Carcinogenesis Induced by Azoxymethane
Authors: Yoshimi Naoki , Shimizu Masahito , Matsunaga Kengo , Yamada Yasuhiro , Fujii Keiko , Hara Akira , Mori Hideki
Published: August 2005

Non‐steroidal anti‐inflammatory drugs (NSAIDs) such as sulindac and indomethacin inhibit colon carcinogenesis, and selective cyclooxygenase (COX)‐2 inhibitors are considered to be potential chemopreventive agents without the side effects of usual NSAIDs. We reported that NS‐398, N‐(2‐cyclohexyloxy‐4‐nitrophenyl)methane sulfonamide, suppressed the formation of preneoplastic lesions, aberrant crypt foci (ACF), induced by azoxymethane (AOM) in a short‐term assay of rat colon carcinogenesis. In this study, we examined the effects of long‐term NS‐398 administration on rat colon carcinogenesis. After three AOM treatments at weekly intervals, a dose of 10 mg/kg of NS‐398 in 5% Arabic gum solution was administered by gavage three times per week in group 2 until the termination of the experiment. Rats in group 1 were fed in a basal diet and given 5% Arabic gum solution alone after AOM treatment. At 40 weeks after the first AOM treatment, all rats were killed and the whole intestines including colon were examined. While the incidences of whole intestinal and colon neoplasms in group 1 were 84.6% and 80.8%, respectively, those in group 2 (given NS‐398) were 51.9% and 44.4% respectively (P=0.0177 and P=0.0103 by Fisher's exact test, respectively). The multiplicities in group 2 (0.67 ± 0.78 and 0.48 ± 0.58) were also decreased significantly compared with those (1.39 ± 1.10 and 1.08 ± 0.74) in group 1 (P < 0.01 by Welch's method and P < 0.002 by Student's t test, respectively). In immunohistochemistry for proliferative cell nuclear antigen (PCNA), the PCNA‐stained cell index (7.40 ± 0.5) in group 2 was significantly decreased from that in group 1 (14.03 ± 0.82) (P < 0.001 by Welch's method). The results suggest that NS‐398, a selective COX inhibitor, has a chemopreventive activity against colon carcinogenesis without side‐effects such as gastric ulceration.
Inhibition of Experimental Metastasis of Human Fibrosarcoma Cells by Anti‐recombinant 37‐kDa Laminin Binding Protein Antibody
Authors: Narumi Koh , Inoue Akira , Tanaka Masashi , Isemura Mamoru , Shimo‐Oka Tadashi , Abe Tatsuya , Nukiwa Toshihiro , Satoh Ken
Published: August 2005

The laminin binding protein of 37 kDa (37LBP) is regarded as a precursor protein of the high‐affinity 67‐kDa laminin receptor (67LR). Expression of 67LR/37LBP is well correlated with biological aggressiveness of cancer, particularly with invasive and metastatic potential. To investigate in detail the role of 37LBP in cancer cells, we synthesized recombinant 37LBP (r37LBP) as a fusion protein and generated an IgG‐type polyclonal antibody P4G against r37LBP. Western blot analysis with P4G showed a single band of 67LR under both nonreducing and reducing conditions using cell extract of human fibrosarcoma cells HT1080. It was shown that P4G inhibited cell attachment to immobilized laminin in a dose‐dependent manner. Further, the intravenous injection of HT1080 cells pretreated with P4G, compared with that of cells pretreated with normal rabbit serum, resulted in a reduced number of experimental metastases (3.3 ± 5.1 vs. 58.0 ± 38.0 nodules per mouse, respectively) (P < 0.005). These results suggest that P4G inhibits the colonization and growth of HT1080 cells in the lungs of mice, and that the blocking of r37LBP with the specific antibody P4G may offer a potential strategy for preventing cancer metastasis.
Differences in Substrate Specificity among Glutathione Conjugates (GS‐X) Pump Family Members: Comparison between Multidrug Resistance‐associated Protein and a Novel Transporter Expressed on a Cisplatin‐resistant Cell Line (KCP‐4)
Authors: Ueda Kaoru , Suzuki Hiroshi , Akiyama Shin‐ichi , Sugiyama Yuichi
Published: August 2005

The substrate specificity of primary active transporters expressed on two kinds of human epidermoid KB‐3‐1 derived cell lines, C‐A500 and KCP‐4, was examined; the former expresses multi‐drug resistance‐associated protein (MRP1), whereas the latter is resistant tocis‐diammine‐dichloroplatinum (II) (cisplatin). Northern blot analysis indicated that neither P‐glycoprotein, MRP1, MRP2 (canalicular multispecific organic anion transporter; cMOAT) nor MRP3 was over‐expressed on KCP‐4. Membrane vesicles isolated from C‐A500 and KCP‐4, but not from KB‐3‐1, exhibited the ATP‐dependent uptake of glutathione conjugates (GS‐X) such as leukotriene C4and 2,4‐dinitrophenyl‐S‐glutathione (DNP‐SG), indicating the presence of GS‐X pumps on these cells. The uptake of these GS‐X by membrane vesicles from C‐A500 was approximately twice that in the case of KCP‐4. Kinetic analysis indicated that theKmandVmaxvalues for DNP‐SG uptake were 2.56 and 1.43 μM, and 570 and 160 pmol/min/mg protein for C‐A500 and KCP‐4, respectively. In marked contrast, significant ATP‐dependent uptake of glutathione‐platinum complex was observed only in membrane vesicles from KCP‐4, but not those from KB‐3‐1 and C‐A500. The transport properties of estradiol‐17β‐d‐glucuronide (E217βG) were also different between the two cell lines. This was reflected in the findings that the ATP‐dependent uptake of this conjugated metabolite in membrane vesicles from C‐A500 (Km= 2.33 μM,Vmax= 34 pmol/min/mg protein) was much more extensive than that in the case of KCP‐4 (Km= 5.5 μM,Vmax= 35 pmol/min/mg protein), and that comparable uptake was observed between KCP‐4 and KB‐3‐1. Overall, a clear difference in substrate specificity among GS‐X pump family members expressed on resistant tumor cells was demonstrated.
Ex vivo Delivery of Suicide Genes into Melanoma Cells Using Epidermal Growth Factor Receptor‐specific Fab Immunogene
Authors: Ohtake Yuichiro , Chen Jiabing , Gamou Shinobu , Takayanagi Atsushi , Mashima Yukihiko , Oguchi Yoshihisa , Shimizu Nobuyoshi
Published: August 2005

The Fab fragment of monoclonal antibody B4G7 against human epidermal growth factor (EGF) receptor was conjugated with cationic poly‐l‐lysine and the resulting conjugate was further complexed with reporter genes or therapeutic genes. This Fab/DNA complex was designated as “Fab immunogene”. The Fab immunogene transfer in vitro was mediated through the EGF receptors in two melanoma cell lines. The frequency of cells expressing β‐galactosidase (β‐Gal) reporter gene was approximately 1%. The induction of suicide effects after Fab immunogene transfer of herpes simplex virus thymidine kinase (TK) or Escherichia coli cytosine deaminase (CD) gene was quite remarkable, and the growth of melanoma cells was inhibited for over 7 days in the presence of ganciclovir (GCV) or 5‐fluorocytosine (5‐FC). Similarly, when melanoma cells treated in vitro with the Fab immunogene carrying TK or CD were transplanted into the back of nude mouse, subsequent systemic administration of GCV or 5‐FC effectively suppressed the growth of tumors, indicating the occurrence of in vivo suicide effects.
Silencing of the CD44 Gene by CpG Methylation in a Human Gastric Carcinoma Cell Line
Authors: Sato Sunao , Yokozaki Hiroshi , Yasui Wataru , Nikai Hiromasa , Tahara Eiichi
Published: August 2005

We analyzed 8 human gastric carcinoma cell lines for the expression of CD44 by northern blot analysis and reverse transcription‐polymerase chain reaction (RT‐PCR), and identified 1 cell line MKN‐28 that did not express CD44. In an attempt to clarify the mechanism responsible for the inactivation of CD44 gene expression in this cell line, we investigated the methylation status around the promoter region of CD44 gene by digestion of the DNA with the methylation‐sensitive restriction enzyme Hpa II. The promoter region of CD44 in MKN‐28 revealed hypermethylation, whereas other CD44‐positive cell lines did not. Furthermore, treatment of MKN‐28 with the demethylating agent 5‐azacytidine restored the expression of the gene. These results suggest that CD44 expression is controlled by a DNA hypermethylation mechanism in MKN‐28.
Lung Cancer in Patients under 50 Years Old
Authors: Tominaga Keigo , Mori Kiyoshi , Yokoi Kohei , Noda Munehide , Goto Naomi , Machida Suguru , Nagai Masaki
Published: August 2005

A long‐term retrospective study was carried out on 790 cases of lung cancer to determine if the clinicopathologic characteristics and survival rates of lung cancer patients under the age of 50 differ from those of patients 50 years of age or older at diagnosis by analyzing data on patients registered at Tochigi Cancer Center Hospital. Of the 790 patients, 77 (9.7%) were under the age of 50 at diagnosis. The percentage of women in the younger patient group was significantly higher than that in the older patient group (39.0% vs. 27.5%; P=0.034). Tumor histology revealed a significant preponderance of adenocarcinomas (60 patients, 77.9%) and a paucity of squamous cell carcinomas (8 patients, 10.4%) in the younger age group (P<0.001). The preponderance of adenocarcinoma was significant in both males and females (male: P=0.004, female: P=0.004). Smoking rates and rate of detection by cancer screening did not differ between the two age groups. Because of the paucity of smokers among the younger female patients, causes of lung cancer other than smoking should be sought in younger patients. No difference was found in the stage of the disease at presentation, treatment methods and survival rates between the two age groups. It is suggested that the prognosis for patients with lung cancer under the age of 50 is not significantly worse than for those aged 50 years or older, as has been shown by several investigators.
Effects of d‐Galactosamine Hydrochloride and Partial Hepatectomy on Spontaneous Hepatic Injury and Hepatocarcinogenesis in Long‐Evans Cinnamon Rats
Authors: Jiao Zhongxian , Ohnishi Takamasa , Bando Yoshimi , Chone Yoshifumi , Kitaura Keisuke , Uehara Hisanori , Suzuki Yasuo , Nakamura Toshikazu , Izumi Keisuke
Published: August 2005

To examine the effect of nongenotoxic chemicals on hepatocarcinogenesis in Long‐Evans Cinnamon (LEC) rats, we gave 6‐week‐old male and female LEC rats (n=18) weekly subcutaneous injections of d‐galactosamine hydrochloride (GalN, 300 mg/kg) in 0.9% NaCl or only 0.9% NaCl for 50 weeks, and killed them in week 62. GalN‐treated male rats unexpectedly showed no lethal necrotizing hepatitis. GalN treatment increased the incidence of cholangiofibrosis in males and its severity in females, but did not cause significant increases of hepatocellular tumors in either sex. GalN treatment increased the 5‐bromo‐2′‐deoxyuridine (BrdU)‐labeling index of hepatocytes and plasma hepatocyte growth factor, and accelerated megalocytic alterations without reduction of the hepatic copper concentration. Next, male and female LEC rats were subjected to two‐thirds partial hepatectomy (PH) or sham hepatectomy in week 8 (n=12) or in week 14 (n=9), and killed in week 62. PH in week 14 inhibited lethal hepatitis, but PH in week 8 was less effective. PH reduced the hepatic copper concentration to half that of controls. The present data suggest that induction of hepatocyte regeneration by repeated injections of GalN, or by PH just before the onset of jaundice has a significant effect in prevention of hepatic injury of LEC rats, but not enhancement of spontaneous hepatocarcinogenesis.
Increased 8‐Hydroxyguanine in DNA and Its Repair Activity in Hamster and Rat Lung after Intratracheal Instillation of Crocidolite Asbestos
Authors: Yamaguchi Raizo , Hirano Takeshi , Ootsuyama Yuko , Asami Shinya , Tsurudome Yosuke , Fukada Shoko , Yamato Hiroshi , Tsuda Toru , Tanaka Isamu , Kasai Hiroshi
Published: August 2005

Asbestos and man‐made‐mineral fibers are known to increase one type of oxidative DNA damage, 8‐hydroxyguanine (8‐OH‐Gua), in vitro. In this study, we analyzed the 8‐OH‐Gua level in DNA and its repair activity after a single intratracheal instillation of fibers (crocidolite or glass) or saline to Syrian hamsters or Wistar rats. The 8‐OH‐Gua level was measured with a high‐performance liquid chromatography‐electrochemical detector (HPLC‐ECD) system. The 8‐OH‐Gua repair enzyme activity was determined with an endonuclease nicking assay using a 32P‐labeled or fluorescently labeled 22mer DNA that contains 8‐OH‐Gua at a specific position. A significant increase in the 8‐OH‐Gua level in the lung DNA was observed 1 day after the exposure to crocidolite, as compared to the saline control. The repair activity was increased significantly at 7 days. On the other hand, after exposure to glass fibers, little or no increase of these carcinogenicity indicators was detected. These assays of 8‐OH‐Gua and its repair activity in short‐term animal experiments will be useful for evaluating the carcinogenicity of fibers. This is the first report of the increase of 8‐OH‐Gua and its repair activity in the animal lung after the instillation of asbestos fibers.
Mutational Analysis of β‐Catenin Gene in Japanese Ovarian Carcinomas: Frequent Mutations in Endometrioid Carcinomas
Authors: Sagae Satoru , Kobayashi Kanji , Nishioka Yoshihiro , Sugimura Masaki , Ishioka Shinichi , Nagata Masami , Terasawa Katsuhiko , Tokino Takashi , Kudo Ryuichi
Published: August 2005

To investigate the contribution of the β‐catenin gene to the development of ovarian carcinomas, mutational analysis of exon 3 of the β‐catenin gene was conducted. We analyzed 61 primary ovarian carcinomas, consisting of 49 non‐endometrioid‐type and 12 endometrioid‐type tumors, for genetic alteration of the β‐catenin gene. Five carcinomas showed β‐catenin mutations (S37C, T41I, T41A), including 4 (33%) of 12 endometrioid‐type tumors and 1 (14%) of 7 mucinous‐type tumors. All of these mutations altered at the serine/threonine residues that are potential sites of GSK3‐β phosphorylation. We detected no carcinomas with interstitial deletion involving exon 3 of β‐catenin. Furthermore, we immunohistochemically studied 27 of the 61 ovarian carcinomas. Both nuclear and cytoplasmic β‐catenin expressions were demonstrated in 4 of the 27 ovarian carcinomas for which tissue samples were available for examination. All 4 cases exhibited mutations in exon 3 of β‐catenin, including a mucinous carcinoma. Our results suggested that β‐catenin gene mutation at potential GSK3‐β phosphorylation sites results in accumulation of β‐catenin protein within the cells and its translocation to nuclei. Accumulated β‐catenin protein may be involved in the development of endometrioid‐type ovarian carcinomas, and some mucinous‐type ovarian carcinomas.
Immunohistochemical Localization of Glutathione S‐Transferase α and π in Human Esophageal Squamous Epithelium, Barrett's Epithelium and Carcinoma
Authors: van Lieshout Esther M. M. , van Haelst Urbain J. G. M. , Wobbes Theo , Peters Wilbert H. M.
Published: August 2005

High tissue levels of glutathione S‐transferases (GSTs), a family of detoxification enzymes, are inversely correlated with cancer risk in the human gastrointestinal tract. Patients with Barrett's esophagus, wherein squamous epithelium is replaced by columnar epithelium, have an increased risk for developing esophageal adenocarcinoma. Biochemical analyses revealed that Barrett's epithelium contains lower levels of GST enzyme activity as well as some GST isoforms, as compared with squamous epithelium. So far, little information on the immunohistochemical distribution of the GST α and π isoforms in normal squamous epithelium, in Barrett's metaplastic epithelium or in adeno‐ and squamous cell carcinomas of the esophagus is available. Tissues were fixed in formalin and embedded in paraffin. Three 4 μm thick sections were used for hematoxylin and eosin staining and for immunostaining with antibodies against GST α and π. GST α and π were seen in normal squamous epithelium (0% and 75%, respectively), Barrett's epithelium (75% and 100%), adenocarcinoma (25% and 100%) and squamous cell carcinoma (27% and 91%). Staining was mainly cytoplasmic, though some nuclear staining with the GST π antibody was apparent. The varying expression of GST α and π in normal and (pre)neoplastic esophagus may have consequences for the treatment of these diseases and may contribute to an understanding of the development of these esophageal disorders.
Reduction of End‐stage Malignant Glioma by Injection with Autologous Cytotoxic T Lymphocytes
Authors: Tsurushima Hideo , Liu Shu Qin , Tuboi Koji , Matsumura Akira , Yoshii Yoshihiko , Nose Tadao , Saijo Kaoru , Ohno Tadao
Published: August 2005

Autologous cytotoxic T lymphocytes (CTL) against primary‐cultured malignant gliomas were generated from peripheral blood mononuclear cells in vitro in 4 patients. Activities of the CTL were highly specific to the corresponding autologous glioma and were inhibited, in one patient, with antibodies against CD3, CD8 and MHC‐class I molecules. When the CTL were injected 3 times into the primary‐tumor‐resected cavity via an Ommaya tube, reduction of the recurrent tumors with magnetic resonance imaging (MRI)‐measured volumes exceeding 45 cm3 was observed in 3 patients. In a patient with glioblastoma multiforme (GBM), the tumor volume (estimated, 130 cm3) was rapidly reduced to 1/3, although re‐recurrence of the tumor followed 40 days later. A slight but distinct rapid reduction of the tumor volume was observed in another GBM patient and in an anaplastic astrocytoma patient; essentially no change was observed in a further GBM patient. These results suggest that adoptive immunotherapy with autologous CTL will be clinically effective against end‐stage malignant gliomas.
Enhancement of Antiproliferative Effects of Interleukin‐1β and Tumor Necrosis Factor‐α on Human Prostate Cancer LNCaP Cells by Coculture with Normal Fibroblasts through Secreted Interleukin‐6
Authors: Kawada Manabu , Ishizuka Masaaki , Takeuchi Tomio
Published: August 2005

The cell‐cell interactions between tumor cells and stromal cells are considered to be important in the regulation of tumor development at primary and metastatic secondary sites. We studied the effects of various cytokines on the cell‐cell interactions between androgen‐dependent LNCaP or androgen‐independent PC‐3 human prostate cancer cell lines and normal fibroblasts using a coculture system. Among the tested combinations of cytokines and fibroblasts, strong modulations of cytokine actions were seen in coculture with human normal fibroblasts WI‐38. While interleukin (IL)‐1β or tumor necrosis factor‐α (TNF‐α)) partially suppressed LNCaP cell growth in monoculture, each cytokine completely inhibited it in the case of coculture with WI‐38 cells. On the other hand, they did not inhibit PC‐3 cell growth significantly, regardless of monoculture or coculture. Conditioned medium prepared from WI‐38 cells pretreated with IL‐1β or TNF‐α also strongly inhibited LNCaP cell growth. In the conditioned medium, marked IL‐6 secretion was induced from WI‐38 cells by IL‐1β or TNF‐α. Furthermore, neutralizing antibodies to IL‐6 or IL‐6 receptor abrogated the antiproliferative effects of IL‐1β‐ and TNF‐α‐pretreated WI‐38 conditioned medium. These results demonstrate that the antiproliferative effects of IL‐1β and TNF‐α on prostate cancer cells are enhanced by coculture with normal fibroblasts through some diffusible factor(s), such as IL‐6, from the stimulated fibroblasts.
N‐Acetylcysteine Modifies cis‐Dichlorodiammineplatinum‐induced Effects in Bladder Cancer Cells
Authors: Miyajima Akira , Nakashima Jun , Tachibana Masaaki , Nakamura Kaoru , Hayakawa Masamichi , Murai Masaru
Published: August 2005

We previously demonstrated a role of reactive oxygen species (ROS) in cytotoxicity induced by cis‐dichlorodiammineplatinum (CDDP) in combination with glutathione (GSH) depletors in bladder cancer cells. However, the relationship between CDDP and ROS is still unclear, although many mechanisms of drug resistance have been well characterized. The present study was undertaken to investigate the effects of N‐acetylcysteine (NAC), a GSH precursor, on the CDDP‐induced effects in bladder cancer cells (KU1). The cytotoxic effects of CDDP were significantly blunted by NAC (1 mM) in KU1 cells. The IC50 of CDDP only (10.2±1.2 μM) is significantly lower than that of CDDP with NAC (IC50: 20.3±1.6 μM) in KU1 cells. NAC also significantly increased the intracellular concentration of GSH in KU1 cells (37.2±1.6 nmol/106 cells), compared to controls (15.9±7.6 nmol/106 cells). While CDDP produced a significant increase in ROS as measured in terms of dichlorofluorescein (DCF) production in KU1 cells in a time‐dependent manner, pretreatment with NAC significantly reduced CDDP‐induced intracellular DCF in KU1 cells. Moreover, TdT‐mediated dUTP‐biotin nick‐end labeling (TUNEL) assay showed that CDDP‐induced apoptosis (31.1±3.8%) was significantly inhibited by pretreatment with NAC in KU1 cells (11.2±2.6%). These results demonstrated that NAC scavenges CDDP‐induced ROS and inhibits CDDP‐induced cytotoxicity, suggesting that ROS mediate the CDDP‐induced cytotoxicity in bladder cancer cells.
A Case Control Study on the Effectiveness of Breast Cancer Screening by Clinical Breast Examination in Japan
Authors: Kanemura Seiki , Tsuji Ichiro , Ohuchi Noriaki , Takei Hiroyuki , Yokoe Takao , Koibuchi Yukio , Ohnuki Koji , Fukao Akira , Satomi Susumu , Hisamichi Shigeru
Published: August 2005

A case‐control study was conducted in Miyagi and Gunma prefectures, Japan, to evaluate the effectiveness of breast cancer screening by clinical breast examination (CBE) alone in reducing breast cancer mortality. Case subjects, who were female and had died of breast cancer, were collected from residential registry files and medical records. Control subjects matched in sex, age and residence were randomly selected from residential registry files. The screening histories during 5 years prior to the cases having been diagnosed as breast cancer were surveyed using the examinee files of the screening facilities. Finally, the data of 93 cases and 375 controls were analyzed. The odds ratio (OR) of breast cancer death for participating in screening at least once during 5 years was 0.93 (95% confidence interval (95%CI) 0.48‐1.79). The cases were more symptomatic than the controls when screened. If the participants who had had symptoms in their breasts were classified as not screened, the OR decreased to 0.56 (95%CI 0.27‐1.18). The case control study suggests that the current screening modality (CBE) lacks effectiveness (OR=0.93), although it might be effective for an a symptomatic population (OR=0.56). The number of cases was small, and a larger case‐control study is desirable to define whether CBE is effective or not. However, it is necessary to consider the introduction of mammographic screening to reduce breast cancer mortality in Japan.
Protective Effect of Allium Vegetables against Both Esophageal and Stomach Cancer: A Simultaneous Case‐referent Study of a High‐epidemic Area in Jiangsu Province, China
Authors: Gao Chang‐Ming , Takezaki Toshiro , Ding Jian‐Hua , Li Mao‐Sheng , Tajima Kazuo
Published: August 2005

To study the relation between allium vegetable intake and cancer of the esophagus (EC) and stomach (SC) in Yangzhong city, which is one of the highest‐risk areas for these cancers in Jiangsu province, China, a simultaneous case‐referent study was conducted using histopathologically confirmed cases (EC: n=81, SC: n=153) and population‐based referents (n=234). A questionnaire was used to collect information on the general status of subjects, their dietary habits, frequency intake of allium vegetables and other foods, tea consumption, smoking and alcohol drinking. The odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by a multiple logistic regression model. The results showed that frequent intake of allium vegetables (including garlic, onion, Welsh onion and Chinese chives), raw vegetables, tomatoes and snap beans, and tea consumption were inversely associated with the risk for EC and SC. In the highest consumption category (≥1 time/ week) of garlic, onion, Welsh onion and Chinese chives, the adjusted ORs compared with the lowest category (<1 time/month) were 0.30 (CI=0.19−0.47), 0.25 (CI=0.11−0.54), 0.15 (CI=0.08−0.26), and 0.57 (CI=0.23−1.42) for EC, and 0.31 (CI=0.22−0.44), 0.17 (CI=0.08−0.36), 0.22 (CI=0.15−0.31) and 0.40 (CI = 0.17−0.94) for SC, respectively. The main results in the present study suggested that allium vegetables, like raw vegetables, may have an important protecting effect against not only stomach cancer, but also esophageal cancer.
Mutational Analysis of STK11 h Gene in Ovarian Carcinomas
Authors: Nishioka Yoshihiro , Kobayashi Kanji , Sagae Satoru , Sugimura Masaki , Ishioka Shinichi , Nagata Masami , Terasawa Katuhiko , Tokino Takashi , Kudo Ryuichi
Published: August 2005

Recently STK11,the causative gene of Peutz‐Jeghers syndrome (PJS) was identified on chromosome 19p13.3. PJS is often accompanied by several malignancies, including breast tumor, adenoma malignum of the uterine cervix, and ovarian tumor. To investigate the involvement of STK11 gene in the development of ovarian carcinomas, we analyzed 30 ovarian carcinomas for loss of hetero‐zygosity (LOH) and STK11 gene mutations. We found one missense mutation (codon 281, Pro to Leu) with heterozygous and somatic status. This mutation occurred at codon 281, which lies within the mutational hot spot (codon 279‐281) of STK11 gene previously reported in PJS. We also detected LOH in 2 (11%) of 19 informative ovarian carcinomas. Our results suggest that mutations of the STK11 gene may play a limited role in the development of ovarian carcinomas.
Relationship between Expression of p21WAF1/CIP1 and Radio resistance in Human Gliomas
Authors: Kokunai Takashi , Tamaki Norihiko
Published: August 2005

The role of p21WAF1/CIP1 (p21) in DNA repair and apoptosis following γ‐irradiation remains controversial. In this study the influence of p21 on the radiosensitivity of human brain tumors was investigated. Resected tumors were stained immunohistochemically for p21. Expression of p21 in astrocytic tumors was high, but it was low in medulloblastomas, germinomas, and primary malignant lymphomas. Glioma and medulloblastoma cell lines were transfected with pcDNA/p21 to cause p21 overexpression, then tumor‐cell colony formation and apoptosis were assessed following γ‐irradiation of the transfected and nontransfected cells. Overexpression of p21 enhanced clono‐genic survival and suppressed apoptosis after γ‐irradiation in human brain tumor cell lines with or without p53 protein deficiency. Radioresistance was acquired when p21 was overproduced in the glioma cell lines irrespective of p53 status.
Roles of Extracellular Signal‐regulated Kinase 1/2 and p38 Mitogen‐activated Protein Kinase in the Signal Transduction of Basic Fibroblast Growth Factor in Endothelial Cells during Angiogenesis
Authors: Tanaka Katsuhiro , Abe Mayumi , Sato Yasufumi
Published: August 2005

We examined the role of mitogen‐activated protein (MAP) kinases in the signal transduction of basic fibroblast growth factor (bFGF)‐mediated effects in endothelial cells (ECs). When MSS31 murine endothelial cells were stimulated with bFGF, three MAP kinase homologs, extracellular signal‐regulated kinase (ERK) 1/2, c‐Jun N‐terminal kinase (JNK) 1, and p38 MAP kinase were activated. The inhibition of the ERK1/2 pathway with PD98059, a specific inhibitor of MEK1, or of the p38 MAP kinase pathway with SB203580, a specific inhibitor of p38 MAP kinase, abrogated bFGF‐mediated tube formation by MSS31 cells in type I collagen gel. Tube formation in type I collagen gel requires proliferation and migration of these cells, and degradation of the extracellular matrix by these cells. Both PD98059 and SB203580 inhibited bFGF‐stimulated DNA synthesis as well as migration of MSS31 cells. Cell migration requires cytoskeleton reorganization and cell adhesion. bFGF induced actin reorganization and vinculin assembly in the focal adhesion plaque, both of which were inhibited by SB203580 but not by PD98059. bFGF induced the expression of the transcription factor ETS‐1 in MSS31 cells. ETS‐1 is responsible for the expression of proteases as well as integrin β3 subunit in ECs, and converts ECs to invasive phenotype. PD98059 inhibited this induction of ETS‐1, whereas SB203580 did not. These results indicate that ERK1/2 and p38 MAP kinase are requisite for the signal transduction of bFGF in ECs. The roles of these two MAP kinase homologs are not identical, but these kinases work in a coordinated fashion.
Characterization of Two Novel Retinoic Acid‐resistant Cell Lines Derived from HL‐60 Cells Following Long‐term Culture with all‐trans‐Retinoic Acid
Authors: Mori Junichirou , Suzuki Satoru , Hara Masahiro , Kaneko Atsuko , Yamashita Koh , Kumagai Mieko , Sakuma Takahiro , Kakizawa Tomoko , Yamazaki Masanori , Takeda Teiji , Miyamoto Takahide , Ichikawa Kazuo , Hashizume Kiyoshi
Published: August 2005

Either all‐trans‐retinoic acid (RA) or vitamin D3 (VD) induces differentiation of the myeloid leukemia cell line HL‐60. RA is available for the treatment of acute promyeloleukemia, although the development of resistance to the agent is a serious problem for differentiation‐inducing therapy. To approach the mechanisms of resistance to RA, we developed two novel cell lines, HL‐60–R2 and R9, which were subcloned after exposure to increasing concentrations of RA. The growth rate of HL‐60–R2 cells was significantly increased by RA treatment, whereas the growth rate of HL‐60–R9 was not affected. RA induces apoptosis in the parental HL‐60 cells. The number of apoptotic cells, however, was not increased and nitroblue tetrazolium (NBT) reduction was not altered by 1 μM RA in either of the cloned cell lines. Treatment with VD induced monocytic differentiation and increased the expression of CD11b in HL‐60 and HL‐60–R9 cells, but not in HL‐60–R2 cells. Flow cytometric and G‐banding analysis demonstrated that R2 cells were near‐triploid. The sequencing analysis revealed a deletion of three nucleotides in the sequence of the RARα gene in HL‐60–R9 cells, resulting in deletion of codon 286. No mutation was found in HL‐60–R2 cells. Taken together, these data indicate that the resistance to RA is caused by the mutation in RARα of HL‐60–R9, but by other factor(s), which also affect the VD‐response pathways, in HL‐60–R2. The abnormal response to VD may be associated with the abnormal ploidy of the R2 cells.
A Dual Topoisomerase Inhibitor, TAS‐103, Induces Apoptosis in Human Cancer Cells
Authors: Ohyama Tomoko , Li Yin , Utsugi Teruhiro , Irie Shinji , Yamada Yuji , Sato Taka‐aki
Published: August 2005

TAS‐103 (6‐[[2‐(dimethylamino)ethyl]amino]‐3‐hydroxy‐7H‐indeno[2,1‐c]quinolin‐7‐one dihydrochloride), a dual topoisomerase (topo) inhibitor, was developed as an anticancer agent by targeting topo I and topo II and has previously been shown to be effective against lung tumors. In this study, we investigated the cytotoxic activity of TAS‐103 in various human cancer cell lines (including gastric, colon, squamous, lung, and breast cancer cells) and the induction of apoptosis by TAS‐103. We next established stable transfectants of Bcl‐2 in the gastric cancer cell line AZ521 and found that Bcl‐2 blocked TAS‐103‐induced apoptosis. In addition, we demonstrated that the activities of ICE‐like and CPP32‐like proteases are involved in the signal transduction pathway of TAS‐103‐induced apoptosis. In summary, TAS‐103 is a novel type of anticancer agent with a unique mechanism and could be useful as a lead compound for development of new drugs.