bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulin‐like Growth Factor (IGF)‐I, IGF‐binding Protein‐3 and Colorectal Adenomas in Japanese Men
Authors: Teramukai Satoshi , Rohan Thomas , Lee Kyong‐Yeon , Eguchi Hiroyuki , Oda Takashi , Kono Suminori
Published: August 2005

Several epidemiological studies have found that high levels of plasma insulin‐like growth factor (IGF)‐I and low levels of IGF‐binding protein (IGFBP)‐3 are related to an increased risk of colorectal cancer or late‐stage adenomas. We examined the relation of body mass index, fasting and 2‐h postload plasma glucose levels and plasma concentrations of IGF‐I and IGFBP‐3 to colorectal adenomas in middle‐aged Japanese men. The study subjects comprised 157 cases of histologically diagnosed colorectal adenomas and 311 controls with normal colonoscopy or non‐polyp benign lesions in a consecutive series of 803 men receiving a preretirement health examination at two hospitals of the Self Defense Forces (SDF). After adjustment for rank in the SDF, hospital, smoking and IGFBP‐3, a statistically nonsignificant modest increase in the prevalence odds of colorectal adenomas was observed for the highest versus the lowest quartile level of IGF‐I. The increase was slightly greater with further adjustment for 2‐h glucose concentrations (adjusted odds ratio 1.8, 95% confidence interval 1.0–4.5, trend P=0.06). Men with high levels of IGFBP‐3 showed only a minimal decrease in risk after adjustment for IGF‐I. The association with IGF‐I was less evident for advanced adenomas (≥5 mm in size or tubulovillous/villous). Fasting and 2‐h glucose and body mass index were more strongly positively associated with colorectal adenomas than IGF‐I, especially with advanced adenomas, independently of IGF‐I and IGFBP‐3. The findings suggest that plasma IGF‐I and IGFBP‐3 may be involved in colorectal tumorigenesis regardless of the stage in growth of adenoma, but not as a mediator for the effects of being overweight or of hyperglycemia.
Reduced Expression and Promoter Methylation p16 Gene in Epstein‐Barr Virus‐associated Gastric Carcinoma
Authors: Osawa Toshiya , Chong Ja‐Mun , Sudo Makoto , Sakuma Kazuya , Uozaki Hiroshi , Shibahara Junji , Nagai Hideo , Funata Nobuaki , Fukayama Masashi
Published: August 2005

Epstein‐Barr virus (EBV)‐associated gastric carcinoma (EBVaGC) is a unique type of gastric carcinoma (GC), which is considered to develop in a different pathway from EBV‐negative GC. To evaluate a possible role of p16, an inhibitor of G1/S transition of the cell cycle, in the carcinogenesis of EBVaGC, pl6‐immunohistochemistry and methylation‐specific PCR analysis (MSP) were applied to surgically resected gastric carcinomas. When the percentage of p16‐positive cells in more than 1000 carcinoma cells was expressed as p16 labeling index (p16‐LI), it ranged from 2.5 to 88.1 (mean 42.9±24.4) in 70 gastric carcinomas. EBVaGC showed significantly lower values (n=15, 26.1±22.1) than EBV‐negative GC (n=55, 47.5±23.2) (P=0.0036). Fresh frozen tissues of 55 gastric carcinomas (16 EBVaGC and 39 EBV‐negative GC) were further subjected to MSP, to evaluate abnormal methylation of the promoter region of the p16 gene. The frequency of methylation was significantly higher in EBVaGC (14/16) than in EBV‐negative GC (9/39) (<0.0001). The methylation‐positive carcinomas showed significantly lower p16‐LI (35.9±21.6) than the unmethylated ones (55.2±22.7) (P=0.0014). Thus, a marked decrease of p16 expression, caused by the aberrant methylation of the p16 gene promoter, is closely associated with the development of EBVaGC.
Fuzzy Neural Network Applied to Gene Expression Profiling for Predicting the Prognosis of Diffuse Large B‐cell Lymphoma
Authors: Ando Tatsuya , Suguro Miyuki , Hanai Taizo , Kobayashi Takeshi , Honda Hiroyuki , Seto Masao
Published: August 2005

Diffuse large B‐cell lymphoma (DLBCL) is the largest category of aggressive lymphomas. Less than 50% of patients can be cured by combination chemotherapy. Microarray technologies have recently shown that the response to chemotherapy reflects the molecular heterogeneity in DLBCL. On the basis of published microarray data, we attempted to develop a long‐overdue method for the precise and simple prediction of survival of DLBCL patients. We developed a fuzzy neural network (FNN) model to analyze gene expression profiling data for DLBCL. From data on 5857 genes, this model identified four genes (CD10, AA807551, AA805611 and IRF‐4) that could be used to predict prognosis with 93% accuracy. FNNs are powerful tools for extracting significant biological markers affecting prognosis, and are applicable to various kinds of expression profiling data for any malignancy.
Functional Polymorphism of the Thymidylate Synthase Gene in Colorectal Cancer Accompanied by Frequent Loss of Heterozygosity
Authors: Kawakami Kazuyuki , Ishida Yoshinori , Danenberg Kathleen D. , Omura Kenji , Watanabe Go , Danenberg Peter V.
Published: August 2005

The thymidylate synthase (TS) gene has a polymorphic repeated sequence in its 5′‐untranslated region. The repeat length is associated with TS protein expression, which suggests that we may be able to predict the efficacy of 5‐fluorouracil (S‐FU)‐based chemotherapy from a patient's TS genotype determined through analysis of normal tissue obtained non‐invasively. However, it is not yet elucidated whether the TS genotype is identical in tumor and normal tissue. In this study, we investigated the TS genotype in 151 matched tumor and normal DNA samples isolated from colorectal cancer and adjacent normal tissues by PCR analysis. The results showed that TS genotypes are identical in normal and tumor tissues of homozygous individuals, suggesting that the repeat sequence is stable through carcinogenesis. However, in heterozygous samples, an unbalance between the 2R and 3R alleles in the tumor DNA was frequently observed, suggesting loss of heterozygosity (LOH) at the TS locus. Detailed LOH analysis revealed that 62% (31 of 50) of 2R/3R‐heterozygous samples had LOH. Frequent LOH at the TS locus was confirmed by RT‐PCR of TS mRNA and microsatellite analysis using the marker D18S59, located on 18p11.3. There was no difference in the expressions of TS mRNA and TS protein between LOH and non‐LOH samples. However, when the heterozygous genotype bearing LOH was subdivided according to the number of repeats, the cancer tissue with 2R/loss genotype expressed a significantly lower level of TS protein than did that with 3R/loss genotype. The results suggest that the difference in TS genotype between tumor and normal tissue due to LOH should be considered when the genotype is analyzed with normal tissue, such as peripheral blood cells, because it is important for TS protein expression.
Quantitative Analysis of Multidrug‐resistance mdr1 Gene Expression in Head and Neck Cancer by Real‐time RT‐PCR
Authors: Tseng Ching‐Ping , Cheng Ann‐Joy , Chang Joseph Tung‐Chieh , Tseng Chin‐Hsiao , Wang Hung‐Ming , Liao Chun‐Ta , Chen I‐How , Tseng Kelvin Chingchung
Published: August 2005

Progression of head and neck cancer is always associated with changes of gene expression profile. In this study, we characterized the expression of multidrug‐resistance mdr1 gene, which may play a role in tumorigenesis and multidrug resistance in head and neck cancer. A TaqMan one‐step RT‐PCR with a linear range for quantification across at least a 5 log scale of concentration of mdr1 mRNA was designed to determine the level of mdr1 expression in 50 pairs of normal vs. malignant head and neck tissues. Both the absolute level of mdr1 mRNA in tumor (T) and the relative mdr1 expression between tumor and its normal counterpart (T/N) were measured and their associations with several clinical variables were analyzed. Among the clinical variables analyzed, only the clinical stage of tumor was found to be associated with mdr1 expression. The distribution of clinical stages differed significantly (P<0.01) among the 27 specimens that had a T/N>1, with 59.3%, 22.2%, 14.8% and 3.7% in stage IV, III, H, and I, respectively. In addition, 76% of stage IV and 75% of stage III tumors had a T/N>1 compared to 25% of stage II and 20% of stage I tumors (P=0.004). Multivariate logistic regression analysis also indicated a significant difference of mdr1 expression between the early (I and II) and advanced (III and IV) stages tumors. The adjusted odds ratios (95% confidence intervals) were 1.477 (1.084–2.012) and 1.001 (1.000–1.002) for T/N (P<0.05) and T (P<0.05) treated as continuous variables, and 15.521 (3.414–70.550) and 5.074 (1.154–22.311) for T/N (P<0.001) and T (P<0.05) treated as binary variables, respectively. Taken together, the data presented here indicated that real‐time RT‐PCR provides a quantitative way to monitor mdr1 gene expression. The differential expression of mdr1 between early and advanced stages of head and neck cancer may shed light on the process of tumorigenicity and offer clues to the planning of new treatments.
Multiparametric in situ mRNA Hybridization Analysis of Gastric Biopsies Predicts Lymph Node Metastasis in Patients with Gastric Carcinoma
Authors: Takahashi Yutaka , Kitadai Yasuhiko , Ellis Lee M. , Bucana Corazon D. , Fidler Isaiah J. , Mai Masayoshi
Published: August 2005

We examined the expression level of several genes that regulate different steps in metastasis formation in formalin‐fixed, paraffin‐embedded biopsies of 189 primary human gastric carcinomas prior to surgical resection in patients in whom lymph node metastasis was not evident by endoscopic ultrasound or computed tomography (CT) scan. The expressions of epidermal growth factor receptor (EGF‐R), vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)‐2 and E‐cadherin were examined by a colorimetric in situ mRNA hybridization technique. The integrity of the mRNAs was verified, leaving 161 (85.2%) patients for study. After gastrectomy, 82 patients had positive lymph nodes and 79 patients had negative lymph nodes. The concurrent expression levels of MMP‐2 and E‐cadherin mRNAs were significantly higher and lower, respectively, in the metastatic tumors than the non‐metastatic tumors. Expression of EGF‐R and VEGF was not different between the metastatic and non‐metastatic tumors. However, when only the intestinal‐type of gastric cancer was evaluated, the level of VEGF mRNA was significantly higher in tumors associated with lymph node metastasis than in those without metastasis. However, a high MMP‐2:E‐cadherin ratio significantly correlated with lymph node metastasis in both types of gastric cancer. These results suggest that multiparametric in situ hybridization analysis for several metastasis‐related genes may allow the preoperative prediction of lymph node metastasis from gastric cancer.
Serum Insulin‐like Growth Factors, Insulin‐like Growth Factor‐binding Protein‐3, and Risk of Lung Cancer Death: A Case‐control Study Nested in the Japan Collaborative Cohort (JACC) Study
Authors: Wakai Kenji , Ito Yoshinori , Suzuki Koji , Tamakoshi Akiko , Seki Nao , Ando Masahiko , Ozasa Kotaro , Watanabe Yoshiyuki , Kondo Takaaki , Nishino Yoshikazu , Ohno Yoshiyuki ,   for the JACC Study Group
Published: August 2005

To elucidate the roles of insulin‐like growth factors (IGFs) in the development of lung cancer, we conducted a case‐control study nested within the Japan Collaborative Cohort Study. Serum samples were collected at baseline from 39 140 men and women between 1988 and 1990. We measured serum IGF‐I, IGF‐II, and IGF‐binding protein‐3 (IGFBP‐3) in 194 case subjects who subsequently died from lung cancer during an 8‐year follow‐up and in 9351 controls. The odds ratios (ORs), adjusted for smoking and other covariates, were smaller with higher levels of IGF‐II and IGFBP‐3. The ORs across quartiles were 0.41 (95% confidence interval [CI], 0.27–0.63), 0.47 (0.31–0.71), and 0.67 (0.46–0.98) for IGF‐II (trend P=0.018), and 0.55 (95% CI, 0.37–0.81), 0.54 (0.36–0.82), and 0.67 (0.45–1.01) for IGFBP‐3 (trend P=0.037). These peptides were not independently related to lung cancer risk when mutually adjusted. The risk was increased in the highest vs. the lowest quartile of IGF‐I only after controlling for IGFBP‐3 (OR, 1.74; 95% CI, 1.08–2.81). Limiting subjects to those followed for ≥3 years strengthened the negative associations of IGF‐II and IGFBP‐3, whereas the ORs for IGF‐I generally decreased. A higher level of circulating IGFBP‐3 and/or IGF‐II may decrease lung cancer risk. Elevated serum IGF‐I may increase the risk, but this could partly be attributable to latent tumors.
Earlier Helicobacter pylori Infection Increases the Risk for the N‐Methyl‐N‐nitrosourea‐induced Stomach Carcinogenesis in Mongolian Gerbils
Authors: Cao Xueyuan , Tsukamoto Tetsuya , Nozaki Koji , Tanaka Harunari , Shimizu Nobuyuki , Kaminishi Michio , Kumagai Toshiko , Tatematsu Masae
Published: August 2005

Helicobacter pylori (H. pylori) is now well known to be associated with stomach cancer, with infection during childhood rather than as an adult considered to be more important for carcinogenesis. To evaluate the difference in susceptibility to stomach carcinogenesis in relation to age of acquisition of H. pylori infection, we designed an experiment involving inoculation of H. pylori ATCC43504 followed by N‐methyl‐N‐nitrosourea (MNU) treatment at different ages. Four‐week‐ old male Mongolian gerbils (MGs) were divided into twelve groups. H. pylori was inoculated at 4, 18 and 32 weeks of age, as representatives of early, middle and late infection, respectively. Two weeks later, the animals were treated with MNU. Groups without H. pylori and/or MNU were included as controls. The incidences of adenocarcinomas at 52 weeks after the inoculation in the early (H. pylori+MNU), middle (H. pylori+MNU), and late (H. pylori+MNU) group were 60% (12/ 20), 18.4% (2/11), and 10% (2/20), respectively. The corresponding figures were 14.8% (4/27), 0% (0/11), and 0% (0/21) in the MNU‐alone groups. A higher titer of serum IgG for H. pylori and higher gastrin level were seen in the early‐infected compared to the middle and the late groups (P<0.01). The results clearly demonstrated that early acquisition of H. pylori significantly increases gastric chemical carcinogenesis with MNU, as compared to the case with later infection, possibly because of differences in host gastric mucosal factors and immunologic responses.
Effects of Antioxidant 1‐O‐Hexyl‐2,3,5‐trimethylhydroquinone or Ascorbic Acid on Carcinogenesis Induced by Administration of Aminopyrine and Sodium Nitrite in a Rat Multi‐organ Carcinogenesis Model
Authors: Yada Hideaki , Hirose Masao , Tamano Seiko , Kawabe Mayumi , Sano Masashi , Takahashi Satoru , Futakuchi Mitsuru , Miki Tokutaro , Shirai Tomoyuki
Published: August 2005

The effect of antioxidant, 0.25% 1‐O‐hexyl‐2,3,5‐trimethylhydroquinone (HTHQ) or 0.25% ascorbic acid (AsA), on Carcinogenesis induced by administration of 0.05% aminopyrine (AP) and 0.05% sodium nitrite (NaNO2), was examined using a rat multi‐organ carcinogenesis model. Groups of twenty F344 male rats were treated sequentially with an initiation regimen of N‐diethylnitrosamine, N‐methyl‐N‐nitrosourea, N‐butyl‐N‐(4‐hydroxybutyl) nitrosamine, N, N′‐dimethylhydrazine and 2,2′‐dihydroxy‐di‐n‐propylnitrosamine during the first 4 weeks, followed by AP+NaNO2, AP+NaNO2+HTHQ, AP+NaNO2+AsA, NaNO2+HTHQ, NaNO2+AsA, each of the individual chemicals alone or basal diet and tap water as a control. All surviving animals were killed at week 28, and major organs were examined histopathologically for development of preneoplastic and neoplastic lesions. In the AP+NaNO2 group, the incidences of hepatocelluar adenomas and hemangiosarcomas were 95% and 35%, respectively. When HTHQ or AsA was simultaneously administered, the incidences decreased to 58% and 11%, or to 80% and 15%, respectively. On the other hand, in the AP+NaNO2 group and the NaNO2‐alone group, when HTHQ, but not AsA, was simultaneously administered, the incidence of carcinomas in the forestomach significantly increased. The results suggest that HTHQ can prevent tumor production induced by AP and NaNO2 more effectively than AsA. On the other hand, an enhancing or possible carcinogenic effect of simultaneous administration of HTHQ and NaNO2 only on the forestomach is suggested, while simultaneous treatment with the same dose of AsA and NaNO2 may not be carcinogenic to the fore‐stomach or other organs.
Estrogen Receptor Expression and Estrogen Receptor‐independent Cytotoxic Effects of Tamoxifen on Malignant Rhabdoid Tumor Cells in vitro
Authors: Koshida Shigeki , Narita Tsutomu , Kato Hirofumi , Yoshida Shinobu , Taga Takashi , Ohta Shigeru , Takeuchi Yoshihiro
Published: August 2005

Recent studies have shown that the antiestrogen tamoxifen (TAM) can be used in the treatment of malignant neoplasms other than breast cancer. In the present study, we investigated the expression of estrogen receptor (ER) in six malignant rhabdoid tumor (MRT) cell lines. Alterations in MRT cell growth in response to estrogen or antiestrogens (4‐hydroxytamoxifen (4‐OHT), TAM, and ICI 182 780) were also investigated. RT‐PCR and western blotting showed that ER‐a was expressed in three of the six MRT cell lines. While 17‐β‐estradiol (E2) did not significantly alter MRT cell line proliferation, the hydroxylated tamoxifen metabolite 4‐OHT significantly inhibited the growth of all 6 MRT cell lines. However, the steroidal antiestrogen ICI 182 780 did not alter the proliferation of any of the MRT cell lines. 4‐OHT induced apoptosis in both ER‐α‐negative and ER‐α‐positive MRT cell lines, as assessed by nuclear morphology and DNA fragmentation. Neither growth inhibition nor induction of apoptosis due to 4‐OHT was blocked by the addition of excess E2. Our data suggested that 4‐OHT induced cytotoxic effects against MRT cells, and that these effects were independent of ER expression.
Peroxisome Proliferator‐activated Receptor Gamma Activation Induces Cell Cycle Arrest via the p53‐independent Pathway in Human Anaplastic Thyroid Cancer Cells
Authors: Chung Sung Hwa , Onoda Naoyoshi , Ishikawa Tetsuro , Ogisawa Kana , Takenaka Chiemi , Yano Yoshihisa , Hato Fumihiko , Hirakawa Kosei
Published: August 2005

Anaplastic thyroid carcinoma is one of the most aggressive human malignancies. Outcomes of intensive multimodal therapy have been far from satisfactory. Furthermore, p53 gene dysfunction, often found in this type of cancer, is known to impair the efficacy of the therapeutic agents. Specific ligands for peroxisome proliferator activated receptor gamma (PPAR‐γ) induce growth suppression in some tumor cells. In this study, we investigated the role of PPAR‐γ in anaplastic thyroid cancer cell lines (OCUT‐1, ACT‐1). PPAR‐γ was expressed and functional in both cell lines. Activation of PPAR‐γ with its specific ligands, troglitazone and 15‐deoxy‐Δ12, 14‐prostaglandin J2, inhibited cell growth in a dose‐dependent manner through inducing G1 cell cycle arrest. P53 protein expression differed in OCUT‐1 and in ACT‐1, though the levels stayed constant irrespective of ligand exposure in both cell lines. In contrast, p21 and p27 proteins were induced in a dose‐dependent manner in both situations. This study showed that PPAR‐γ ligands were able to induce growth suppression in anaplastic thyroid cancer cells via a p53‐independent, but p21‐ and p27‐dependent cytostatic pathway. These tumor‐suppressive effects of PPAR‐γ may provide a novel approach to the treatment of anaplastic thyroid cancer.
Microarray‐based Analysis of Anti‐angiogenic Activity of Demethoxycurcumin on Human Umbilical Vein Endothelial Cells: Crucial Involvement of the Down‐regulation of Matrix Metalloproteinase
Authors: Kim Jin Hee , Shim Joong Sup , Lee Seok‐Ki , Kim Kyu‐Won , Rha Sun Young , Chung Hyun Cheol , Kwon Ho Jeong
Published: August 2005

cDNA microarray‐based gene expression analysis has been successfully employed to explore the action mechanism and to validate the targets of several drugs. In the present study, we evaluated anti‐angiogenic activity of demethoxycurcumin (DC), a structural analog of curcumin, isolated from Curcuma aromatica, and investigated the effect of DC on genetic reprogramming in cultured human umbilical vein endothelial cells (HUVECs) using cDNA microarray analysis. Of 1024 human cancer‐focused genes arrayed, 187 genes were up‐regulated and 72 genes were down‐regulated at least 2‐fold by DC. Interestingly, 9 angiogenesis‐related genes were down‐regulated over 5‐fold in response to DC, suggesting that the genetic reprogramming was crucially involved in anti‐angiogenesis by the compound. To verify the results obtained from cDNA microarray analysis, matrix metalloproteinase‐9 (MMP‐9), the product of one of the angiogenesis‐related genes down‐regulated over 5‐fold by DC, was investigated using gelatin zymography. DC potently inhibited the expression of MMP‐9, yet showed no direct effect on its activity. These data show that gene expressional change of MMP‐9 is a major mediator for angiogenesis inhibition by DC. All genes identified and microarray data are available on the web at http://dasan.sejong.ac.kr/bioprobe/.
Helicobacter pylori Does Not Promote N‐Methyl‐N‐nitrosourea‐induced Gastric Carcinogenesis in SPF C57BL/6 Mice
Authors: Nakamura Yoshihiro , Sakagami Takashi , Yamamoto Noriyasu , Yokota Yoshiro , Koizuka Hiromasa , Hori Kazutoshi , Fukuda Yoshihiro , Tanida Noritoshi , Kobayashi Takehiko , Shimoyama Takashi
Published: August 2005

Helicobacter pylori (H. pylori) infection has been acknowledged as a promoter and an initiator for gastric carcinogenesis in experimental models using Mongolian gerbils with H. pylori strains TN2GF4 and ATCC 43504, which have +ve cagA and vacA phenotype s1/ml. To get more insight into the role of H. pylori in gastric carcinogenesis, we studied the effect of H. pylori SS1, which has +ve cagA and vacA phenotype s2/m2, on Af‐methyl‐N‐nitrosourea (MNU)‐induced chemical gastric carcinogenesis using SPF C57BL/6 mice. Thus, H. pylori SSI was inoculated 1 week after the completion of MNU treatment to examine the promoting effect of this bacterium. The incidences of polypoid lesions, differentiated adenocarcinomas, and adenomatous hyperplasias were 67% (10/ 15), 47% (7/15) and 80% (12/15), respectively, in the MNU‐alone group. The corresponding figures were 31% (8/26), 23% (6/26) and 35% (9/26) in the MNU+H. pylori group. The incidences of polypoid lesions and adenomatous hyperplasia were significantly different between the groups. Thus, the results indicate that H. pylori SSI infection reduced susceptibility to chemical gastric carcinogenesis in this model. The discrepancy between the present result and previous results is likely to have been caused by differences in host factors and bacterial factors. Further study of the relationship between gastric carcinogenesis and H. pylori infection is needed.
Uterine Adenocarcinoma in N‐Ethyl‐N‐nitro‐N‐nitrosoguanidine‐treated Rats with High‐dose Exposure to p‐tert‐Octylphenol during Adulthood
Authors: Katsuda Shin‐ichi , Yoshida Midori , Kuroda Hiroyuki , Ando Jin , Takahashi Masakazu , Kurokawa Yuji , Watanabe Gen , Taya Kazuyoshi , Maekawa Akihiko
Published: August 2005

Since many risk factors are associated with the development of uterine adenocarcinomas in humans, the etiology is unclear in most cases, although it has been pointed out that estrogen may play essential roles. To clarify the effects of exposure to p‐tert‐octylphenol (OP), an environmental xenoestrogen, on uterine carcinogenesis, adult Donryu rats were initiated with a single intrauterine treatment of N‐ethyl‐N‐nitro‐N‐nitrosoguanidine (ENNG) at 11 weeks of age and exposed thereafter to 100 mg/kg OP by s.c. injection until 15 months of age. Adult ovariectomized (OVX) rats were also treated in a similar way. OP had no effect on occurrence of persistent estrus in middle age, although uterotrophic effects were obvious in OVX rats. At the termination, development of uterine adenocarcinomas was significantly increased in animals exposed to OP during adulthood. No tumors, but a few focal hyperplasias, developed in OVX rats. These findings suggest that OP has tumor‐promoting effects on ENNG‐treated endometrium of rats, possibly due to direct action on the uterus, as indicated by the uterotrophic effect when a high dose of OP was given. The results provide clues to the mechanisms of influence of hormonal disrupters on uterine carcinogenesis.
Development of Hepatocellular Adenomas and Carcinomas Associated with Fibrosis in C57BL/6J Male Mice Given a Choline‐deficient, L‐Amino Acid‐defined Diet
Authors: Denda Ayumi , Kitayama Wakashi , Kishida Hideki , Murata Nao , Tsutsumi Masahiro , Tsujiuchi Toshifumi , Nakae Dai , Konishi Yoichi
Published: August 2005

Development of hepatocellular carcinomas in rats caused by a choline‐deficient, L‐amino acid defined (CDAA) diet, usually associated with fatty liver, fibrosis, cirrhosis and oxidative DNA damage, has been recognized as a useful model of hepatocarcinogenesis caused by endogenous factors. In the present study, in order to further explore involved factors and genes, we established an equivalent model in spontaneous liver tumor‐resistant C57BL/6J mice. Six‐week‐old males and females were continuously fed the CDAA diet and histological liver lesions and oxidative DNA damage due to 8‐hydroxydeoxyguanosine (8‐OHdG) were examined after 22, 65 and 84 weeks. In male mice, fatty change and fibrosis were evident at 22 weeks, and preneoplastic foci of altered hepatocytes were seen at an incidence of 8/8 (100%) and a multiplicity of 6.6±4.0 per mouse at 65 weeks. Hepatocellular adenomas and carcinomas developed at incidences of 16/24 (66.7%) and 5/ 24 (20.8%), and multiplicities of 1.421±1.32 and 0.29±0.62, respectively, at 84 weeks. The female mice exhibited resistance to development of these lesions. The CDAA diet also increased 8‐OHdG levels in male but not female mice. These results indicate that a CDAA diet causes hepatocellular preneoplastic foci, adenomas and carcinomas associated with fibrosis and oxidative DNA damage in mice, as in rats, providing a hepatocarcinogenesis model caused by endogenous factors in mice.
Chemoprevention of N‐Methylnitrosourea‐induced Colon Carcinogenesis by Ursodeoxycholic Acid‐5‐aminosalicylic Acid Conjugate in F344 Rats
Authors: Narisawa Tomio , Fukaura Yoko , Takeba Naomi , Nakai Keiko
Published: August 2005

Bile acids enhance colon carcinogenesis in animal models, whereas ursodeoxycholic acid (UDCA) suppresses it. Nonsteroid anti‐inflammatory drugs prevent colon cancer development in animals and humans. The aim of the present study was to explore the inhibitory effect of UDCA conjugate with 5‐aminosalicylic acid (5‐ASA), UDCA‐5‐ASA conjugate (UDCA‐5‐ASA), against colon carcinogenesis in rats. One‐hundred‐and‐twenty‐nine 7‐week‐old F344 rats received an intrarectal instillation of 2 mg of N‐methylnitrosourea 3 tunes a week for 3 weeks, and were fed a 0% (control), 0.11% or 0.02% UDCA‐5‐ASA‐, 0.08% UDCA‐ or 0.03% 5‐ASA‐supplemented diet for the next 27 weeks. The test diets contained an equimolar amount of a test agent, 2.0 mmol/kg diet, except for the 0.02% UDCA‐5‐ASA diet. The tumor incidence and the mean number of tumors/rat at week 30 were significantly lower and smaller in the UDCA‐5‐ASA diet groups, 48% and 0.7 in both, and marginally lower in the UDCA and 5‐ASA diet groups, 56% and 0.9, and 64% and 0.8, compared to the control group, 83% and 1.3. All the tumors were polypoid in shape, and most of them were differentiated adenocarcinomas restricted to the mucosa or submucosa. An analysis by HPLC for bile acids and 5‐ASA in the feces and serum collected at week 30 showed that one‐half of ingested UDCA‐5‐ASA was cleaved into UDCA and 5‐ASA in the colon. Thus, the two moieties may have independently affected the promotion stage of carcinogenesis.
Overexpression of Phosphatidylinositol Synthase Enhances Growth and G1 Progression in NIH3T3 cells
Authors: Deguchi Atsuko , Segawa Kaoru , Hosaka Kohei , Weinstein I. Bernard , Umezawa Kazuo
Published: August 2005

Phosphatidylinositol (PI) turnover is thought to play an important role in the regulation of cell growth. PI synthase (PIS, cytidine diphosphate (CDP)‐diacylglycerol (DG): myo‐inositol 3–phos–phatidyltransferase, EC 2.7.8.11) acts at the last step in the de novo biosynthesis of PI by catalyzing the condensation of CDP‐DG and myo‐inositol. To study the physiological role of PIS, we established murine NIH3T3 fibroblasts that stably overexpress PIS, by transfection with PIS cDNA (NIH‐PIS cells). In immunofluorescence assays, the constitutively overexpressed PIS was found to be localized in the endoplasmic reticulum, as previously reported for the native enzyme activity. NIH‐PIS cells showed an increase in PI synthesis in vitro and in vivo, as well as increased cellular levels of PI–4,5–P2 and PI–3,4,5–P3. They also displayed a decrease in their doubling tune and accelerated G1 progression. Overexpression of PIS increased cellular levels of the cyclin D1 and E proteins and Akt kinase activity in serum‐stimulated quiescent NIH3T3 cells. Moreover, PIS Overexpression potentiated the colony formation of NIH3T3 cells in soft agar. These results suggest that PIS accelerates G1 progression and stimulates growth by increasing cellular levels of cyclins D1 and E.
Not Infrequent K‐ras Mutations in Depressed‐type Early Colorectal Carcinomas Larger than 10 mm
Authors: Kazama Shinsuke , Ajioka Yoichi , Watanabe Hidenobu , Watanabe Toshiaki , Nagawa Hirokazu
Published: August 2005

The aim of this study was to elucidate whether K‐ras (codons 12 and 13) mutations occur in depressed‐type early colorectal carcinomas (DECas) larger than 10 mm in size. Thirty‐four cases of DECas including 27 larger than 10 mm were examined for K‐ras mutations by means of microdis‐section, PCR‐RFLP (polymerase chain reaction‐restriction fragment length polymorphism), and direct sequencing. Although K‐ras mutation was infrequent (1/7, 14%) in small (less than 10 mm) DECas, 16/27 (59%) and 17/27 (63%) of DECas larger than 10 mm revealed codon 12, or either codon 12 or 13 mutations, respectively. None of the evaluated pathological factors except size showed a correlation with K‐ras mutation. These data indicate that although K‐ras mutation could not be involved in the early stage of the progression of DECas, it might play a role at a later stage when the tumor size is over 10 mm.
Down‐regulation of 2‐Amino‐3,8‐dimethylimidazo[4,5‐f]quinoxaline (MeIQx)‐induced CYP1A2 Expression Is Associated with Bovine Lactoferrin Inhibition of MeIQx‐induced Liver and Colon Carcinogenesis in Rats
Authors: Fujita Ken‐ichi , Ohnishi Takamasa , Sekine Kazunori , Iigo Masaaki , Tsuda Hiroyuki
Published: August 2005

The inhibitory influence of bovine lactoferrin (bLF) on induction of preneoplastic hepatic glutathione S‐transferase placental form‐positive (GST‐P+) cell foci and colon aberrant crypt foci (ACF) by diethylnitrosamine (DEN) and 2‐amino‐3,8‐dimethylimidazo[4,5‐f]quinoxaline (MeIQx) was investigated in F344 rats. Rats were initially treated with DEN, then placed on basal diet containing MeIQx (200 ppm) alone, MeIQx plus 2% bLF, or MeIQx plus 0.2% bLF from week 2 to week 8, with partial hepatectomy performed at week 3. Concomitant administration of 2% or 0.2% bLF with MeIQx caused significant dose‐dependent decreases in both number and unit area of GST‐P+ cell foci (2% bLF, P <0.001; 0.2% bLF, P <0.01). Similar results were observed for MeIQx‐induced colon ACF in the groups without DEN treatment (2% and 0.2% bLF, P <0.05). To investigate the underlying mechanisms, we analyzed the influence of bLF on levels of cytochrome P4501A2 (CYP1A2), a metabolically activating enzyme of MeIQx in the liver. The results demonstrated that combined administration of 2% bLF significantly reduced levels of MeIQx‐induced CYP1A2 mRNA (P <0.05) and protein (P <0.05) to the normal levels, in association with reduced values for MeIQx‐DNA adducts (P <0.05), liver GST‐P+ cell foci and colon ACF. These results suggest that bLF is a chemopreventive agent for DEN alone or DEN plus MeIQx‐induced liver, and MeIQx‐induced colon carcinogenesis in rats. One possible mechanism is a normalizing down‐regulation of CYP1A2 expression by bLF, with consequent reduction of carcinogen activation and adduct formation.
Expression of Laminin‐5 Enhances Tumorigenicity of Human Fibrosarcoma Cells in Nude Mice
Authors: Mizushima Hiroto , Hirosaki Tomomi , Miyata Satoshi , Takamura Hiroyuki , Miyagi Yohei , Miyazaki Kaoru
Published: August 2005

Laminin‐5 (LN5), which consists of laminin α3, β3 and γ2 chains, is a laminin isoform produced by various kinds of normal epithelial cells and tumor cells. Strong activity of LN5 in adhesion, migration and scattering of cells in vitro and its frequent detection in human tumor tissues have suggested a possible role of LN5 in the malignant growth of tumor cells. To examine whether LN5 affects the malignant potential of tumor cells, we prepared human fibrosarcoma HT1080 cell lines producing LN5 by transfecting a cDNA of laminin α3 chain into the parent cell line, which constitutively expressed the laminin β3 and γ2 chains. The exogenous α3 chain associated with the endogenous β3 and γ2 chains to secrete the LN5 heterotrimer that has strong cell‐scattering and cell adhesion activities. The HT1080 transfectants expressing LN5 efficiently adhered to culture dishes in a serum‐free condition as compared with control HT1080 cells, which secreted the monomers and heterodimer of the β3 and γ2 chains. When injected into nude mice subcutaneously, the HT1080 transfectants expressing LN5 grew faster and formed much larger tumors than the control cells. This suggests that LN5 promotes tumor growth in vivo.
Establishment and Characterization of a New Lung Cancer Cell Line (MI‐4) Producing High Levels of Granulocyte Colony Stimulating Factor
Authors: Harada Ryoji , Uemura Yoshiki , Kobayashi Makoto , Zamecnikova Adriana , Nakata Hideshi , Taguchi Takahiro , Furihata Mutsuo , Otsuki Yuji , Taguchi Hirokuni
Published: August 2005

We established a human lung cancer cell line, MI‐4 from the pleural effusion of a 69‐year‐old male with advanced large cell undifferentiated carcinoma of the lung complicated by leukocytosis. The culture supernatant of MI‐4 contained high levels of granulocyte colony stimulating factor (G‐CSF). The intracellular localization of the G‐CSF was identified by immunocytochemistry. Reverse transcription‐polymerase chain reaction (RT‐PCR) revealed G‐CSF mRNA expression in this cell line. The cell line was successfully transplanted into nude mice. The transplanted nude mice also showed leukocytosis with a high serum G‐CSF level. Southern blot analysis did not show amplification or rearrangement of the G‐CSF gene in MI‐4 cells. Spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH) analyses revealed that this cell line has an additional chromosome 17 attached to a segment of chromosome 10 besides two intact chromosomes 17, and that each of these three chromosomes 17 has a G‐CSF gene on chromosome 17q. Inflammatory cytokines, tumor necrosis factor (TNF)‐α and interleukin (IL)‐1β, significantly enhanced G‐CSF expression at both the protein and mRNA levels in MI‐4. However, these cytokines did not stimulate the growth of MI‐4 cells, regardless of abundant G‐CSF production. TNF‐α rather suppressed it, in a dose‐dependent manner. Exogenous recombinant human G‐CSF and anti‐G‐CSF antibody did not promote or inhibit the growth of MI‐4 cells at any concentration examined. In addition, RT‐PCR analysis did not show G‐CSF receptor mRNA expression. These results suggest that this cell line does not have an autocrine growth loop for G‐CSF. This cell line should be very useful for understanding the biological activity of G‐CSF in G‐CSF‐overproducing lung cancer.
Clinical Significance of Occult Micrometastases in Axillary Lymph Nodes in “Node‐negative” Breast Cancer Patients
Authors: Umekita Yoshihisa , Ohi Yasuyo , Sagara Yoshiatsu , Yoshida Hiroki
Published: August 2005

The most important subgroup of breast cancer patients for whom reliable prognostic indicators are needed is women without axillary lymph node metastases. We evaluated the clinical significance of occult micrometastases in axillary lymph nodes in 148 consecutive “node‐negative” breast cancer patients. The median age of the patients at surgery was 52 years and the median follow‐up period after surgery was 98.5 months. Occult micrometastases were detected in 21 of 148 patients (14.2%) by means of immunohistochemical analysis using AE1/3 antibody and a single unstained section after routine histopathological examination. Log‐rank tests indicated that the 7–year disease‐free survival (DFS) and overall survival (OS) rates by Kaplan‐Meier methods were significantly better in patients without occult micrometastases than in patients with occult micrometastases [DFS, 93% versus 71% (P=0.0009); OS, 96% versus 76% (P=0.0001)]. According to Cox's multivariate analysis, the presence of occult micrometastases had the most significant effect on DFS (P=0.0053) and OS (P=0.0035). These findings suggest that the presence of occult micrometastases is an independent and significant predictor of clinical outcome, and that their immunohistochemical detection after routine histopathological examination is useful for selecting the “node‐negative” breast cancer patient subgroup at high risk for relapse and death.
Immunocytochemical Expression of Microtubule‐associated Protein‐2 (MAP‐2) in Small Cell Lung Cancer Cell Lines with Neuronal‐like Processes
Authors: Tanaka Kazuhiko , Terasaki Takeo
Published: August 2005

Five of six human small cell lung cancer (SCLC) cell lines changed morphologically into cells with neuronal‐like processes on the extracellular matrix of human lung adenocarcinoma cell line PC‐9 cells (PC‐9/ECM substrate). The features of the neuronal‐like processes of these SCLC cell lines were examined immunocytochemically using monoclonal antibodies against p‐chains of tubulin and microtubule‐associated protein‐2 (MAP‐2), which is somatodendritic MAP of neurons. It was observed that β‐chains of tubulin and MAP‐2 were expressed along the neuronal‐like processes of SCLC cell lines. These findings suggest that the β‐chains of tubulin and MAP‐2 are expressed functionally in SCLC cell lines in association with the development of dendrite‐like processes on PC‐9/ECM substrate.
Psychosocial Stress Augments Tumor Development through β‐Adrenergic Activation in Mice
Authors: Hasegawa Hideo , Saiki Ikuo
Published: August 2005

Housing conditions affect behavioral and biological responses of animals. We investigated the effect of same‐sex‐grouped (G), crowded (GC) and isolated (I) conditions on the growth of B16 melanoma or Meth A fibrosarcoma implanted in the footpad of syngeneic male C57BL/6 or BALB/c mice. Differential housing altered host resistance to tumor growth. The host responses to stress were reflected in thymic atrophy, which was lowest in the G mice, highest in the GC mice and intermediate in the I mice. The GC condition was a more stressful social environment than the I condition in both male C57BL/6 and BALB/c mice. Reflecting the extent of psychosocial stress, tumor growth was augmented in the order of GC, I and G condition, and a negative mass correlation between tumor and thymus was observed, thus clearly indicating that the host resistance to tumors was attenuated by psychosocial stress. Furthermore, the stress‐enhanced tumor growth and thymus atrophy were completely abrogated by the oral administration of the non‐selective β‐adrenergic antagonist, propranolol. On the contrary, the chronic administration of corticosterone significantly induced the atrophy of thymus and spleen without affecting tumor growth. These results suggest an interrelationship among psychosocial stress, tumor growth and β‐adrenergic activation.
Oxidative DNA Damage Induced by a Metabolite of 2–Naphthylamine, a Smoking‐related Bladder Carcinogen
Authors: Ohnishi Shiho , Murata Mariko , Kawanishi Shosuke
Published: August 2005

2‐Naphthylamine (2‐NA), a bladder carcinogen, is contained in cigarette smoke. DNA adduct formation is thought to be a major cause of DNA damage by carcinogenic aromatic amines. We have investigated whether a metabolite of 2‐NA, 2‐nitroso‐1‐naphthol (NO‐naphthol) causes oxidative DNA damage, using 32P‐labeled DNA fragments. We compared the mechanism of DNA damage induced by NO‐naphthol with that by N‐hydroxy‐4‐aminobiphenyl (4‐ABP (NHOH)), a metabolite of 4‐aminobiphenyl, another smoking‐related bladder carcinogen. NO‐naphthol caused Cu(II)‐mediated DNA damage at T>C>G residues, with non‐enzymatic reduction by NADH. Catalase and bathocuproine, a Cu(I)‐specific chelator, inhibited the DNA damage, suggesting the involvement of H2O2 and Cu(I). Some free ·OH scavengers also attenuated NO‐naphthol‐induced DNA damage, while free ·OH scavengers had no effect on the DNA damage induced by 4–ABP(NHOH). This difference suggests that the reactive species formed by NO‐naphthol has more free ·OH‐ character than that by 4–ABP(NHOH). A high‐pressure liquid chromatograph equipped with an electrochemical detector showed that NO‐naphthol induced 8–oxo–7,8–dihydro–2′–deoxyguanosine formation in the presence of NADH and Cu(II). The oxidative DNA damage by these aminoaromatic compounds may participate in smoking‐related bladder cancer, in addition to DNA adduct formation.
Enhancing Effects of β‐Estradiol 3–Benzoate but Not Methoxychlor on the Promotion/Progression Stage of Chemically‐induced Mammary Carcinogenesis in Ovariectomized Rats
Authors: Ueda Makoto , Takagi Hisayoshi , Onodera Hiroshi , Yasuhara Kazuo , Takizawa Tamotsu , Imai Toshio , Mitsumori Kunitoshi , Matsui Takane , Hirose Masao
Published: August 2005

Modifying effects of β‐estradiol 3–benzoate (EB) and methoxychlor (MXC), a pesticide which possesses weak estrogenic activity, on 7,12–dimethylbenz(a)anthracene (DMBA)‐induced mammary carcinogenesis were investigated in ovariectomized or intact female Sprague‐Dawley rats. Twenty‐eight weeks after a single DMBA (100 mg/kg body weight) initiation, when the incidence of mammary tumor‐bearing rats had reached 75%, a number of the animals were subjected to ovariectomy in order to obtain 3 groups: i) tumor‐bearing, ovariectomized group; ii) tumor‐bearing, intact group; iii) no‐tumor, ovariectomized group. Subsequently animals of each group were subjected to subcutaneous implantation of 0.5 mg EB or given diet containing 1000 ppm MXC for 13 weeks. Although the incidences, multiplicities and volumes of the palpable tumors gradually decreased after ovariectomy, EB treatment stimulated tumor growth in the tumor‐bearing, ovariectomized group thereafter. A similar effect of EB treatment was also observed in the no‐tumor, ovariectomized group. However, MXC did not show any effect in the tumor‐bearing, or no‐tumor ovariectomized groups, except that the multiplicity of tumors was significantly decreased by MXC treatment in the tumor‐bearing, intact group. The results of our study suggest that MXC has no promotion/progression effect, but rather possesses a weak inhibitory effect, whereas the strongly estrogenic substance EB clearly enhanced DMBA‐induced mammary tumorigenesis.
DNA Hypermethylation Status of Multiple Genes in Prostate Adenocarcinomas
Authors: Konishi Noboru , Nakamura Mitsutoshi , Kishi Munehiro , Nishimine Masayoshi , Ishida Eiwa , Shimada Keiji
Published: August 2005

Multiple genetic mutations and epigenetic methylation are believed to be involved in prostate carcinogenesis, but it is not known whether these events are independent or correlated in some fashion. We therefore studied 32 prostate adenocarcinomas not only for deletions and/or mutations of multiple suspect genes, but also for aberrant DNA methylation using methylation‐specific PCR (MSP). Of those genes examined, p16INK4a, O6‐MGMT, and GST‐P were found to be the most frequently methylated (66%, 25% and 75% of cases, respectively), while methylations of p14ARF, RB1, p21waf1, and p27Kip1 were far less common (3%, 6%, 6% and 6% of cases, respectively). Methylation of O6‐MGMT and GST‐P genes was defective in about 19% of the cases and there were occasional simultaneous deletions and methylations of p14ARF and p16INK4a genes (13% and 3% of cases, respectively). In p16INK4a, methylation occurred in the promoter region in 9% of samples and in exon 2 in 66% of tumors. Hypermethylation of O6‐MGMT with concurrent p53 and ras gene mutations were found in 6% and 13% of specimens, respectively; among those tumors with high Gleason scores were 2 carcinomas showing hypermethylated O6‐MGMT with G‐to‐A transitions in K‐ras. Our results demonstrate that multiple genes of a subset common in prostate carcinomas are methylated and not infrequently show concurrent deletions. Further, there is a suggestion that specific combinations of hypermethylation and mutation correlate to tumor malignancy.
Troglitazone Induces G1 Arrest by p27Kip1 Induction That Is Mediated by Inhibition of Proteasome in Human Gastric Cancer Cells
Authors: Takeuchi Shigeru , Okumura Toshikatsu , Motomura Wataru , Nagamine Miho , Takahashi Nobuhiko , Kohgo Yutaka
Published: August 2005

We examined in the present study whether human gastric cancer cells express peroxisome proliferator‐activated receptor γ (PPARγ), the effect of PPARy activation by troglitazone, a selective ligand, on cellular growth, and the mechanism of the growth arrest by troglitazone in gastric cancer cells. RT‐PCR, northern blot and western blot analysis demonstrated that all four tested human gastric cancer cell lines, MKN–28, MKN–45, MKN–74 and KATO‐III, expressed PPARγ mRNA and protein. WST–1 assay and flow cytometric analysis revealed that troglitazone inhibited the growth and induced G1 arrest in all four gastric cancer cell lines. To examine the role of p27Kip1, a cyclin‐dependent kinase inhibitor, in the G1 arrest by troglitazone, we determined p27Kip1 protein expression by western blot analysis in gastric cancer cells that had been treated with troglitazone. Troglitazone increased p27Kip1 in all four gastric cancer cell lines. Since it has been reported that the ubiquitin‐proteasome system plays a vital role in the degradation of p27Kip1 protein, we evaluated the hypothesis that inhibition of proteasome mediates the troglitazone‐induced p27Kip1 accumulation. Lactacystin, a proteasome inhibitor, inhibited cell growth and increased p27Kilp1 expression in MKN–74 cells. It was further demonstrated that troglitazone inhibited proteasome activity in a dose‐dependent manner in MKN–74 cells. All these results suggest that troglitazone inhibited proteasome activity, followed by induction of p27Kipl, which arrests cells at the Gl phase of the cell cycle in gastric cancer cells. The troglitazone‐mediated inhibition of the proteasome suggests a novel mechanism for the anti‐proliferative effect of this agent in cancer cells.