bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Continuous Hepatitis with Persistent Hepatitis C Viremia on Outcome after Resection of Hepatocellular Carcinoma
Authors: Kubo Shoji , Nishiguchi Shuhei , Shuto Taichi , Tanaka Hiromu , Tsukamoto Tadashi , Hirohashi Kazuhiro , Ikebe Takashi , Wakasa Kenichi , Kuroki Tetsuo , Kinoshita Hiroaki
Published: August 2005

The effect of persistent hepatitis C viremia on the outcome after resection of hepatocellular carcinoma (HCC) was investigated in 59 consecutive patients with a single small HCC (??3.0 cm in diameter). The presence of serum hepatitis C virus (HCV) RNA was evaluated using a reverse transcription polymerase chain reaction method as well as a branched DNA probe method. Clinicopathologic findings were compared between patients with and without viremia and the risk factors for poor outcome were evaluated. Hepatitis C virus (HCV) RNA was not detected in the sera from 7 patients (group 1), but was detected in the sera from the other 52 patients (group 2). Alanine a minotransferase (ALT) activity was significantly higher in group 2 than in group 1. The proportion of patients with active hepatitis was significantly higher in group 2. In group 2, new HCC often developed after the operation and four patients died of liver dysfunction. HCV viremia, high ALT activity, high concentration of total bilirubin, and liver cirrhosis were related to recurrence after the operation. Multivariate analysis indicated that HCV viremia and high ALT activity were independent risk factors for recurrence of HCC. Continuous hepatitis with persistent HCV viremia worsened the outcome after the resection of HCC by causing new development of HCC and deterioration of liver function. In patients with HCV‐related HCC, but without HCV viremia, satisfactory results can be expected after liver resection.
Apoptotic Changes Precede Mitochondrial Dysfunction in Red Cell‐type Pyruvate Kinase Mutant Mouse Erythroleukemia Cell Lines
Authors: Aisaki Ken‐ichi , Kanno Hitoshi , Oyaizu Naoki , Hara Yukichi , Miwa Shiro , Ikawa1 Yoji
Published: August 2005

Two erythroleukemia cell lines have been established from the splenic lesions of red blood celltype pyruvate kinase (R‐PK) activity‐deficient mice of CBA/N origin infected with a polycythemic strain of Friend leukemia virus complex (FVp). Ten to 30 % of the cells of these cell lines undergo apoptotic changes in routine passage, as shown by nuclear fragmentation, DNA laddering, DNA content (propidium iodide (PI) staining), and annexin V binding assay. In these cells, however, although adenosine 5′‐triphosphate (ATP) levels were lower than in the control cells, the mitochondrial inner transmembrane potential (Δψm), detected by rhodamine 123 (R123) and diSC3(5) staining, remained unchanged until the final stage of apoptosis. No evidence was obtained to relate this finding to R‐PK mutation due to difficulty in cloning stable, conditionally inducible R‐PK gene transfectants. However, low Δψm in the apoptotic cell population of the control T3‐K‐1 (K‐1) and T3‐Cl‐2‐0 (2‐0) Friend erythroleukemia cells supports a possible relationship, as do results obtained in two Friend erythroleukemia cells recently isolated from normal CBA/N mice. These cell lines are expected to be useful for clarifying both the primary apoptotic changes independent of mitochondrial dysfunction and the PK‐isozyme changes during erythrodifferentiation, for example, the decreased muscle type 2 (M2) PK level. Modification of growth signals in these cell lines may modulate differentiation and/or apoptosis and allow further elucidation of the signaling networks.
Activation of Caspases in p53‐induced Transactivation‐independent Apoptosis
Authors: Gao Chongfeng , Tsuchida Nobuo
Published: August 2005

Though p53‐induced apoptosis plays an important role in tumor suppression, the mechanism(s) by which p53 induces apoptosis is stillunclear. To elucidate the p53‐induced apoptotic pathway, we examined the role of p53 transactivation activity and caspase in J138V5C cells carrying a human temperature‐sensitive (ts) p53 mutant (138Ala→Val). The results showed that p53‐induced apoptosis was not blocked by cycloheximide, which effectively prevented the expression of p53 target genes, indicating that transactivation was not essential for p53‐induced apoptosis in this system. Western blotanalysis showed that PARP, CPP32 and ICH‐1 precursors were cleaved during apoptosis. The CPP32‐preferential tetrapeptide inhibitor Ac‐DEVD‐CHO blocked the cleavage of ICH‐1 and PARP precursors, suggesting that CPP32 or some other DEVD‐sensitive caspase(s) is the upstream activator of ICH‐1. We also examined the role of the Fas pathway by using Fas and Fas ligand‐neutralizing antibodies. Both antibodies failed to block p53‐induced apoptosis, suggesting that the Fas pathway was not essential for p53‐induced apoptosis in this system. Taken together, our results indicate that p53‐induced, transactivation‐independent apoptosis in Jurkat cells involves sequential activation of CPP32 or some other DEVD‐sensitive caspase(s) and ICH‐1, via a Fas‐independent pathway.
Overexpression of MDM2 in MCF‐7 Promotes Both Growth Advantage and p53 Accumulation in Response to Estradiol
Authors: Saji Shigehira , Nakashima Shigeru , Hayashi Shin‐ichi , Toi Masakazu , Saji Shigetoyo , Nozawa Yoshinori
Published: August 2005

The overexpression of the oncogene product MDM2 is often observed in human breast cancer cells, especially in estrogen receptor (ER)‐positive ones. To study the role of MDM2 protein in ER‐positive breast cancer, we have established cell lines derived from MCF‐7 which stably express increased and decreased levels of MDM2 by transfection of a mammalian expression vector containing human mdm2 cDNA in sense and antisense orientations, respectively. Interestingly, MDM2 overexpression in MCF‐7 cells afforded a remarkable growth advantage under estradiol (E2)‐sup‐plemented condition. Then, we analyzed the expression of p53, which is an important regulator of growth and the cell cycle. Unexpectedly, the p53 accumulation induced by E2 was remarkably higher in MCF‐7 cells stably overexpressing MDM2 than in the parent MCF‐7 cells. On the other hand, reduction of MDM2 suppressed the E2‐induced increase in p53 protein. Moreover, mdm2 antisense oligonucleotides prevented E2‐induced accumulation of p53. In the steady state, the cellular levels of p53 were also correlated with those of MDM2. These interactions are not consistent with the well‐known role of MDM2, which acts as a negative regulator for p53 by inhibiting its function and promoting its rapid degradation. These results suggest that MDM2 may regulate the expression of p53 in the steady state and in response to E2 in breast cancer cells, and imply a novel and important role of MDM2 during breast carcinogenesis.
Differential Induction of Apoptosis in B16 Melanoma and EL‐4 Lymphoma Cells by Cytostatin and Bactobolin
Authors: Kawada Manabu , Amemiya Masahide , Ishizuka Masaaki , Takeuchi Tomio
Published: August 2005

Most solid tumor cells are less sensitive to apoptosis induced by anticancer drugs than hematopoietic cancer cells. However, the mechanisms of the different responses to apoptosis in these cell types remain unknown. To explore this question, we used B16 melanoma and EL‐4 lymphoma cells as solid tumor‐ and hematopoietic cancer‐derived cell lines, and examined the effects of two apoptosis inducers, cytostatin and bactobolin, on both cell lines. Apoptosis in B16 cells was induced strongly by bactobolin, but weakly by cytostatin. In contrast, apoptosis in EL‐4 cells was induced strongly by cytostatin, but weakly by bactobolin. While caspase‐3 was activated upon induction of apoptosis in both cell lines, Ac‐DEVD‐CHO, a specific inhibitor of caspase‐3, suppressed only the apoptosis in B16 cells. In B16 cells, cyclins E, A, and B1 were decreased by strongly apoptosis‐inducing bactobolin prior to apoptosis commitment, but cyclin E was not decreased by weakly apoptosis‐inducing cytostatin. On the other hand, in EL‐4 cells cyclins D1, E, A, and B1 were decreased by strongly apoptosis‐inducing cytostatin prior to apoptosis commitment, but neither cyclin A nor B1 was decreased by weakly apoptosis‐inducing bactobolin. These results indicate that the dependency of apoptosis induction on caspase activity is different between the two cell lines. Furthermore, there may be an inverse correlation between specific cyclins and apoptosis induction in the two cell lines.
k‐ras Point Mutations in the Supernatants of Pancreatic Juice and Bile Are Reliable for Diagnosis of Pancreas and Biliary Tract Carcinomas Complementary to Cytologic Examination
Authors: Yamashita Kazuya , Kida Yoshiki , Shinoda Hiroshi , Kida Mituhiro , Okayasu Isao
Published: August 2005

In order to clarify whether DNA analysis for K‐ras mutation can be used to diagnose cancers in supernatants of pancreatic juice and bile, samples from 29 cases of pancreatic, biliary tract, gastric, and neuroendocrine carcinomas, 1 malignant lymphoma case, 2 cases of pancreatic adenoma, 9 cases of chronic pancreatitis and 21 other non‐cancer cases were examined. Polymerase chain reaction (PCR) products for K‐ras gene codons 2 to 97 of exons 1 and 2 were generated with 33/33 (100%) pancreatic juice and 41/41 (100%) bile samples. By the single strand conformation polymorphism (SSCP) method, point mutations were detected in the pancreatic juice or bile supernatants of 13/13 (100%) pancreas cancer cases, 5/14 (35.7%) biliary tract cancer cases, 1/2 (50.0%) pancreatic adenoma cases and 3/9 (33.3%) chronic pancreatitis cases. Direct sequencing confirmed identical point mutations in the supernatants, malignant cells of cytologic smears of pancreatic juice or bile and cancer tissues. The DNA analysis demonstrated the presence of K‐ras point mutations in 3 cases of pancreatic carcinomas with false‐negative cytologic diagnoses. This novel method allows simultaneous testing for genetic abnormalities in supernatants of pancreatic juice and bile, after removing cells for cytologic diagnosis and screening for pancreatic and biliary tract tumors.
Prognostic Factors for Short‐term Survival in Patients with Stage IV Non‐small Cell Lung Cancer
Authors: Ando Masahiko , Ando Yuichi , Sugiura Seiji , Minami Hironobu , Saka Hideo , Sakai Shuzo , Shimokata Kaoru , Hasegawa Yoshinori
Published: August 2005

Prognostic factors which can forecast short‐term survival in patients with stage IV non‐small cell lung cancer have not been well evaluated. Characteristics of such factors may be different from those for overall survival, and would be an important eligibility criterion for clinical trials of chemotherapy. We retrospectively analyzed the data of 158 patients with stage IV non‐small cell lung cancer whose performance status was 0, 1 or 2. Univariate and multivariate logistic regression models revealed demographic variables which significantly correlated with the survival at 8 or 12 weeks. The univariate model showed the following significant variables: T factor, N factor, number of organs with metastases, grade of performance status, weight loss within 6 months, evidence of metastasis either at bone or lymph node, and lactate dehydrogenase level. The subsequent multivariate model demonstrated that both grade of performance status under 2 and number of metastasized organs less than 3 are important factors for 8‐ or 12‐week survival. The survival rate in patients meeting the two criteria (grade of performance status under 2 and number of metastasized organs less than 3) and in those meeting only one of them was 93% versus 80% at 8 weeks (P=0.030) and 88% versus 62% at 12 weeks (P<0.001), respectively. Grade of performance status and number of organs with metastases appear to be important prognostic factors for short‐term survival in patients with stage IV non‐small cell lung cancer.
Comparative Case‐referent Study of Risk Factors among Hormone‐related Female Cancers in Japan
Authors: Hirose Kaoru , Tajima Kazuo , Hamajima Nobuyuki , Kuroishi Tetsuo , Kuzuya Kazuo , Miura Shigeto , Tokudome Shinkan
Published: August 2005

To assess the impact of reproductive and anthropometric factors as a risk indicator for female cancers in hormone‐related organs, i.e., the breast, endometrium and ovary, we conducted a comparative case‐referent study using data from the Hospital‐based Epidemiologic Research Program at Aichi Cancer Center (HERPACC), Japan. The case group consisted of 1,465, 133 and 99 women who had first been diagnosed as having breast, endometrial and ovarian cancer, respectively. The referents were 25,488 female first‐visit outpatients who had not previously been diagnosed with any type of cancer. The odds ratios (ORs) and their 95% confidence intervals (95%CI) were estimated using an unconditional logistic regression model. An inverse association with experience of delivery and a positive association with body mass index (BMI) and with change of BMI after 20 years of age, were observed consistently for all three cancer sites. We observed similar risk and protective factors for breast and endometrial cancer, but the effect of reproduction and overweight condition (BMI≥25) were more prominent in endometrial cancer. Although the present study failed to find site‐specific risk factors for ovarian cancer, the results provided evidence that being overweight and/or weight gain in adult life is a common risk factor for all three cancer sites. The results obtained from this study suggested that avoidance of weight gain may reduce the risk of female hormone‐related cancers.
Mechanism of Oxidative DNA Damage Induced by a Heterocyclic Amine, 2‐Amino‐3,8‐dimethylimidazo[4,5‐f]quinoxaline
Authors: Murata Mariko , Kobayashi Mikiko , Kawanishi Shosuke
Published: August 2005

Adduct formation has been considered to be a major causal factor of DNA damage by carcinogenic heterocyclic amines. By means of experiments with 32P‐labeled DNA fragments and an electrochemical detector coupled to a high‐pressure liquid chromatograph, we investigated whether the N‐hydroxy metabolite of 2‐amino‐3,8‐dimethylimidazo[4,5‐f]quinoxaline (MeIQx) can cause oxidative DNA damage or not. This metabolite [MeIQx(NHOH)] was found to cause Cu(II)‐mediated DNA damage, including 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine formation. When an endogenous reductant, β‐nicotinamide adenine dinucleotide (NADH), was added, the DNA damage was greatly enhanced. Catalase and bathocuproine, a Cu(I)‐specific chelator, inhibited the DNA damage, suggesting the involvement of H2O2 and Cu(I). MeIQx(NHOH) frequently induced DNA cleavage at thymine and cytosine residues in the presence of NADH and Cu(II). A UV‐visible spectroscopic study showed that little decomposition of MeIQx(NHOH) occurred in the absence of Cu(II), whilst rapid spectral change was observed in the presence of Cu(II), suggesting that Cu(II) catalyzes the autoxidation. The addition of NADH reduced the oxidized product back to MeIQx(NHOH). These results suggest that a copper‐peroxo intermediate, derived from the reaction of Cu(I) with H2O2, participates in Cu(II)‐dependent DNA damage by MeIQx(NHOH), and NADH enhances the DNA damage via a redox cycle. We conclude that in addition to DNA adduct formation, oxidative DNA damage plays an important role in the carcinogenic process of MeIQx.
Polymerase Chain Reaction‐based Enzyme Immunoassay for Quantitation of Telomerase Activity: Application to Colorectal Cancers†
Authors: Cheng Ann‐Joy , Tang Reiping , Wang Jeng‐Yi , Chang Joseph T. , Wang Tzu‐Chien V.
Published: August 2005

Telomerase is a specialized reverse transcriptase that synthesizes telomeric sequences onto human chromosomal ends. It appears to be present in the majority of primary human cancer tissues, and may have potential as a universal tumor marker. In this report, we describe a sensitive, non‐radioactive, polymerase chain reaction (PCR)‐based enzyme immunoassay (EIA) for the quantitation of telomerase activity in human cells. This PCR‐EIA is convenient and can be easily completed within 3 h. The correlation coefficient between the results of PCR‐EIA and the conventional telomeric repeat amplification protocol (TRAP) method, as measured on 4 different cell lines, was over 0.98. Evaluation of this method for clinical application was conducted with tissues obtained from patients with colorectal cancers and the results were compared with those of the conventional TRAP method. Our data indicate that telomerase activities measured by conventional TRAP and PCR‐EIA are highly correlated, and we suggest that the PCR‐EIA method can substitute for conventional TRAP.
Histological Factors Associated with Initial Bone Metastasis of Invasive Ductal Carcinoma of the Breast
Authors: Koyama Tadaaki , Hasebe Takahiro , Tsuda Hitoshi , Hirohashi Setsuo , Sasaki Satoshi , Fukutomi Takashi , Imoto Shigeru , Umeda Toru , Mukai Kiyoshi
Published: August 2005

Bone is one of the most common sites of recurrence of breast cancer. Therefore, it would be clinically very useful if breast cancers with a high probability of bone metastasis (BM) could be identified by histopathological examination of the primary lesions. To elucidate histological characteristics associated with predisposition to initial BM, we examined nine histopathological parameters in the primary lesions of 110 invasive ductal carcinomas (IDCs) of the breast with 0 to 3 regional node metastases. These cases had recurrence between 4 months and 10.1 years after the initial radical surgery. The first metastatic site was bone in 24 cases, whereas other sites were involved in 86 cases. IDCs growing in a strand growth pattern or with fibrotic focus (FF) had a significantly higher frequency of initial BM than those growing in a non‐strand growth pattern or without FF, respectively. Strand growth pattern was a significant predictor of the initial BM in multivariate analysis. In all 54 IDCs that developed BM during the follow‐up period, osteolytic metastasis was significantly more frequent in the group with FF than in that without FF. This study demonstrated that strand growth pattern and the presence of FF are significant histopathological factors associated with initial BM. The combination of those predictive factors along with prognostic factors may provide a useful approach to identify patients at high risk for initial BM, enabling early treatment for the recurrent cancer.
Relation between the Serum E‐Selectin Level and the Survival Rate of Patients with Resected Non‐small Cell Lung Cancers
Authors: Tsumatori Gentaro , Ozeki Yuichi , Takagi Keigo , Ogata Toshiro , Tanaka Susumu
Published: August 2005

E‐Selectin is an inducible adhesion molecule, which is expressed on cytokine‐activated endothelial cells and is thought to interact with cancer cells to initiate metastases. The relationship between serum E‐selectin levels and prognoses in 101 patients with resected non‐small cell lung cancers (NSCLCs) was studied, and survival curves were compared in relation to E‐selectin levels and expression of two carbohydrate antigens, Sialyl Lewisx (SLX) and Sialyl Lewisa (CA19‐9), which were immunohistochemically detected in resected specimens in 65 of the 101 cases. The serum Eselectin level on admission was 48.9±25.7 ng/ml (mean±SD, n=101), and the E‐selectin‐positive rate was 22.7%, being correlated with the progression of T‐factor. The high E‐selectin group showed a significantly worse survival rate than the normal E‐selectin group. Multivariate analysis confirmed the significant prognostic value of E‐selectin. The mean postoperative E‐selectin level in 52 cases (36.93 ng/ml) was significantly lower than the preoperative E‐selectin level (43.57 ng/ml), indicating that certain NSCLCs might induce the expression of E‐selectin. In cases expressing carbohydrate antigens (SLX, CA19‐9), the high E‐selectin group showed a significantly worse survival curve than the normal E‐selectin group. On the other hand, there was no significant difference in the survival curve between the high and normal E‐selectin groups when carbohydrate antigens were negative. These results suggest that patients who have high serum E‐selectin levels, especially with carbohydrate antigen‐positive NSCLC, might be expected to have poor prognoses.
Expression of Fibronectin Isoforms in Human Breast Tissue: Production of Extra Domain A+/Extra Domain B+ by Cancer Cells and Extra Domain A+ by Stromal Cells
Authors: Matsumoto Ei‐ichi , Yoshida Toshimichi , Kawarada Yoshifumi , Sakakura Teruyo
Published: August 2005

The expression of fibronectin (FN) isoforms including extra domain A (EDA) and extra domain B (EDB) segments, was investigated in 36 invasive ductal carcinomas and 13 benign tumors of human breast tissues by in situ hybridization using probes specific to alternative splicing sites. Signals for the constant region of FN mRNA in cancer cells were found in 53% of the invasive ductal carcinomas. The EDA+ and EDB+ mRNA signals were found in 47% and 33%, respectively. Stromal cells expressing FN, EDA+ and EDB+ mRNA signals were present in 100%, 69% and 14% of cases, respectively. Expression of FN mRNAs by cancer cells was most frequent in intraductal lesions or large cancer nests, and that by stromal cells was associated with desmoplastic areas. In representative cases, proportions of FN mRNA‐positive cancer cells expressing EDA and EDB segments were 45% and 39%, respectively, signals for both being frequently found in the same cells. EDA+ and EDB+ mRNA were labeled in 25% and 6% of the FN mRNA‐positive stromal cells, a large proportion thus being EDA‐/EDB‐ FN. In conclusion, the splicing pattern of FN pre‐mRNA is dependent on the cell type and histology of breast cancer tissues. The observed lack of expression in fibroadenomas and other benign conditions suggests a link with tumor progression.
Radioimmunotherapy for Liver Micrometastases in Mice: Pharmacokinetics, Dose Estimation, and Long‐term Effect
Authors: Saga Tsuneo , Sakahara Harumi , Nakamoto Yuji , Sato Noriko , Zhao Songji , Iida Yasuhiko , Kuroki Masahide , Endo Keigo , Konishi Junji
Published: August 2005

The pharmacokinetics of a therapeutic dose of 131I‐labeled antibody and the absorbed dose in liver micrometastases of human colon cancer LS174T in female BALB/c nu/nu mice were investigated, along with the long‐term therapeutic effect. Mice with liver micrometastases were given an intravenous injection of 131I‐labeled anti‐carcinoembryonic antigen (CEA) antibody F33‐104 (8.88 MBq/40 μg). The biodistribution of the antibody was determined 1, 2, 4, 6, and 10 days later. The absorbed dose was estimated for three hypothetical tumor diameters; 1,000, 500, and 300 μm. Autoradiography showed a homogeneous distribution of radioactivity in the micrometastases, and a high uptake was maintained until day 6 (24.0 % injected dose (ID)/g on day 1 to 17.8 %ID/g on day 6), but decreased thereafter. The absorbed doses in the 1,000‐, 500‐, and 300‐μm tumors were calculated to be 19.1, 12.0, and 8.2 Gy, respectively. The intravenous injection of the 131I‐labeled antibody also showed a dose‐dependent therapeutic effect (all mice of the nontreated group died, with a mean survival period of 4 weeks; 3 of the 8 mice that received 9.25 MBq survived up to 120 days with no sign of liver metastasis). These data give further evidence that micrometastasis is a good target of radioimmunotherapy, and that an absorbed dose of less than 20 Gy can effectively control small metastatic lesions.
Somatic Mutation of the APC Gene in Thyroid Carcinoma Associated with Familial Adenomatous Polyposis
Authors: Iwama Takeo , Konishi Motoko , Iijima Takeru , Yoshinaga Keigo , Tominaga Takeshi , Koike Morio , Miyaki Michiko
Published: August 2005

We report the existence of both germline and somatic mutations of the APC gene in thyroid carcinomas from familial adenomatous polyposis (FAP) patients. One papillary thyroid carcinoma from a 20‐year‐old woman, with germline mutation of the APC gene (TCA to TGA at codon 1110), showed a somatic mutation of AAAAC deletion between codons 1060 and 1063. Another somatic mutation of CAG to TAG at codon 886 was also found in one of multiple thyroid carcinomas from a 26‐year‐old woman with attenuated FAP and germline mutation at codon 175 (C deletion). This is the first evidence that total absence of the normal function of the APC gene is involved in development of thyroid carcinomas in FAP.
Effects of a Soybean Isoflavone Mixture on Carcinogenesis in Prostate and Seminal Vesicles of F344 Rats
Authors: Onozawa Mizuki , Kawamori Toshihiko , Baba Masaki , Fukuda Kazunori , Toda Toshiya , Sato Hidetaka , Ohtani Mikinobu , Akaza Hideyuki , Sugimura Takashi , Wakabayashi Keiji
Published: August 2005

Several epidemiological studies have suggested an inverse association between the risk of prostate cancer and intake of soybeans and their products. In vitro data pointing to possible anti‐carcinogenic properties of the soybean isoflavone, genistein, led us to investigate the chemopreventive potential of soybean isoflavones in a rat carcinogenesis model induced by 3,2′‐dimethyl‐4‐aminobi‐phenyl (DMAB) and testosterone propionate (TP). Animals received DMAB s.c. injections at 2‐week intervals for the first 20 weeks and implanted silicon tubes containing 40 mg of TP, replaced at 6‐week intervals throughout the experiment. The soybean isoflavone mixture consisting of 74% genistein and 21% daidzein was mixed in basal diet (AIN‐76A) at concentrations of 100 and 400 ppm and fed to F344 male rats throughout the experiment. Rats treated with carcinogens and administered isoflavone mixture at 100 and 400 ppm developed adenocarcinomas at incidences of 35% and 29%, respectively, in the prostate and seminal vesicles, whereas the figure was 60% for those maintained on control diet. Feeding of the isoflavone mixture at 100 and 400 ppm significantly inhibited the number of argyrophilic nucleolar organizer regions (AgNORs) in adenocarcinomas of the accessory sex glands as compared to those of rats fed control diet. No influence on the development of neoplastic lesions originating in other organs was noted. The results of this study provide evidence that soybean isoflavones may have potential as chemopreventive agents against carcinogenesis in the prostate.
PTEN/MMAC1 Mutations in Hepatocellular Carcinomas: Somatic Inactivation of Both Alleles in Tumors
Authors: Kawamura Naoki , Nagai Hisaki , Bando Koichi , Koyama Masaaki , Matsumoto Satoshi , Tajiri Takashi , Onda Masahiko , Fujimoto Jiro , Ueki Takahiro , Konishi Noboru , Shiba Tadayoshi , Emi Mitsuru
Published: August 2005

Allelic loss of loci on chromosome 10q occurs frequently in hepatocellular carcinomas. Somatic mutations of the PTEN/MMAC1 gene on this chromosome at 10q23 were recently identified in sporadic cancers of the uterus, brain, prostate and breast. To investigate the potential role of PTEN/MMAC1 gene in the genesis of hepatocellular carcinomas, we examined 96 tumors for allelic loss on 10q and also for subtle mutations anywhere within the coding region of PTEN/MMAC1 gene. Allelic loss was identified in 25 of the 89 (27%) tumors that were informative for polymorphic markers in the region. Somatic mutations were identified in five of those tumors: three frameshift mutations, a 1‐bp insertion at codon 83–84 in exon 4 and two 4‐bp deletions, both at codon 318–319 in exon 8; two C‐to‐G transversion mutation, both at ‐9 bp from the initiation codon in the 5’non‐coding region of exon 1. No missense mutation was observed in this panel of tumors. In most of the informative tumors carrying intragenic mutations of one allele, we were able to detect loss of heterozygosity as well. These findings suggest that two alleles of the PTEN/MMAC1 gene may be inactivated by a combination of intragenic point mutation on one allele and loss of chromosomal material on the other allele in some of these tumors.
Therapeutic Effect of 1 M Tegafur‐0.4 M 5‐Chloro‐2,4‐dihydroxypyridine‐1 M Potassium Oxonate (S‐1) on Liver Metastasis of Xenotransplanted Human Colon Carcinoma
Authors: Konno Hiroyuki , Tanaka Tatsuo , Baba Megumi , Kanai Toshikazu , Matsumoto Keigo , Kamiya Kinji , Nakamura Satoshi
Published: August 2005

S‐1 [1 M tegafur (FT)‐0.4 M 5‐chloro‐2,4‐dihydroxypyridine (CDHP)‐1 M potassium oxonate (Oxo)], was developed as a new oral antineoplastic agent based on biochemical modulation of fluorouracil (5‐FU) by CDHP and Oxo. The therapeutic effect of S‐1 on human colon cancer xenografts (TK‐13) with high metastatic potential to the liver was evaluated. Small pieces of TK‐13 were sutured into the cecal wall of 52 nude mice, and the animals were randomly divided into 3 groups [control (n=17), UFT (combination of 1 M FT and 4 M uracil) (n=18) and S‐1 (n=17)]. S‐1 or UFT was administered orally at an equitoxic dose (S‐1, 7.5 mg/kg; UFT, 17.5 mg/kg as FT) for 37 consecutive days beginning 10 days after the transplantation. S‐1 showed higher tumor growth inhibition than UFT (P < 0.05) and also showed a significant anti‐metastatic effect on liver metastasis, while UFT did not. Liver metastasis developed in only 2 of the 17 mice (12%) in the S‐1 group, whereas it developed in 9 of the 17 (53%) and 7 of the 18 (39%) in the control and UFT group, respectively. Analysis of AUC (area under the curve) revealed that S‐1 yielded higher 5‐FU levels in both tumor tissue (1.6 times) and plasma (2.5 times) than UFT. These results suggest that S‐1 will show a higher clinical therapeutic effect against human colorectal cancer than UFT.
Enhancement of the Anti‐tumor Effect of 5′‐Deoxy‐5‐fluorouridine by Transfection of Thymidine Phosphorylase Gene into Human Colon Cancer Cells
Authors: Kanyama Hiroki , Tomita Naohiro , Yamano Tomoki , Miyoshi Yasuo , Ohue Masayuki , Fujiwara Yoshiyuki , Sekimoto Mitsugu , Sakita Isao , Tamaki Yasuhiro , Monden Morito
Published: August 2005

Thymidine phosphorylase (dThdPase) is an enzyme that converts 5′‐deoxy‐5‐fluorouridine (5′DFUR) to the toxic substance 5‐fluorouracil (5‐FU); it is also known to be a platelet‐derived endothelial cell growth factor. In order to investigate the feasibility of suicide gene therapy against colorectal cancer by means of the combination of 5′DFUR and the converting enzyme dThdPase, we transfected the dThdPase gene into the human colon cancer cell line SW480 and analyzed the growth pattern as well as the sensitivity to 5‐FU or 5′DFUR of the dThdPase‐transfected cells. The 50% inhibition (IC50) values of 5‐FU against the SW480 parental cells, control vector‐transfected cells SW480/V1, and dThdPase‐transfected cells SW480/dThdPase were approximately 4.9, 6.3, and 2.9 μM, respectively. The IC50 of SW480/dThdPase was lower than that of SW480 or SW480/V1, although the differences were not statistically significant. The IC50 values of 5′DFUR for SW480, SW480/V1, and SW480/dThdPase were approximately 300, 330, and 3.2 αM, respectively. The sensitivity to 5′DFUR of SW480/dThdPase was increased by about 100‐fold compared with that of SW480 or SW480/V1. With only 10% transfection efficacy, a high enough sensitivity to 5′DFUR was obtained to suppress the cell growth, indicating that a strong bystander effect was induced by this system. The in vivo growth of the s.c. transplanted SW480/dThdPase tumor in nude mice was significantly suppressed by i.p. injection of 5′DFUR compared with that in control mice that received phosphate‐buffered saline (PBS) treatment. These results suggest that gene therapy using the combination of 5′DFUR and the dThdPase gene may be a useful approach for treatment of colon cancer.
Frequent Expression of Midkine Gene in Esophageal Cancer Suggests a Potential Usage of Its Promoter for Suicide Gene Therapy
Authors: Miyauchi Motohiro , Shimada Hideaki , Kadomatsu Kenji , Muramatsu Takashi , Matsubara Shuichiro , Ikematsu Shinya , Takenaga Keizo , Asano Takehide , Ochiai Takenori , Sakiyama Shigeru , Tagawa Masatoshi
Published: August 2005

We have examined the expression of midkine (MK), a neurotrophic factor with heparin‐binding activity, in human esophageal cancer cells. Seven esophageal cell lines tested expressed the transcript and 8 out of 14 human esophageal tumor specimens were positively stained with anti‐MK antibody, while surrounding normal esophageal tissues in these specimens were not stained. The 5′‐flanking, 2.3 kb genomic region of the MK gene was shown to drive the transcription of a reporter gene in the esophageal cell lines in a cis acting manner. Forced expression in esophageal cancer cells of herpes simplex virus‐thymidine kinase gene mediated by the flanking region of the MK gene conferred sensitivity to a prodrug, ganciclovir. The 5′‐upstream region of the MK gene thus possesses putative promoter activity which can be used for suicide gene‐based gene therapy for esophageal cancer.
Efficient Production of Adeno‐associated Virus Vectors Using Split‐type Helper Plasmids
Authors: Ogasawara Yoji , Urabe Masashi , Kogure Katsuhiro , Kume Akihiro , Colosi Peter , Kurtzman Gary J. , Ozawa Keiya
Published: August 2005

Adeno‐associated virus (AAV) vectors are potentially useful vehicles for the delivery of therapeutic genes into human cells. To determine the optimal expression pattern of AAV proteins (Rep78, Rep68, Rep52, Rep40, and Cap proteins) for packaging the recombinant AAV genome, helper plasmids were split into two portions. In this study, two sets of split‐type helper plasmids were prepared; i.e., 1) a Rep expression plasmid (pRep) and Cap expression plasmid (pCap), and 2) a large Rep expression plasmid (pR78/68) and small Rep plus Cap expression plasmid (pR52/40Cap). When AAV vectors were produced using these sets of split‐type helper plasmids at various ratios, the optimal ratio of (large) Rep expression plasmid and Cap expression plasmid was 1 to 9 for both sets. More importantly, the titers were comparable to or even higher than that of a conventional helper plasmid (pIM45) (4.9±2.1×1011 vector particles/10 cm dish for pRep and pCap; 2.9±1.6×1011 vector particles/10 cm dish for pR78/68 and pR52/40Cap; and 1.8±0.16×1011 particles/10 cm dish for pIM45). Western analysis of AAV proteins suggests that the expression of a relatively small amount of large Rep and a large amount of Cap is important for optimal vector production. The present study shows that the AAV helper plasmid can be split without losing the ability to package the recombinant AAV genome, and provides us with valuable basic information for the development of efficient AAV packaging cell lines.
Manganese Superoxide Dismutase Negatively Regulates the Induction of Apoptosis by 5‐Fluorouracil, Peplomycin and γ‐Rays in Squamous Cell Carcinoma Cells
Authors: Ueta Eisaku , Yoneda Kazunori , Yamamoto Tetsuya , Osaki Tokio
Published: August 2005

We investigated the relationship between manganese superoxide dismutase (Mn‐SOD) activity and apoptosis induced by anticancer drugs and radiation. Although the activity of copper, zinc‐SOD did not differ greatly among 9 squamous cell carcinoma (SCC) cell lines (OSC‐1 to OSC‐9), the Mn‐SOD activity did differ among the cell lines. The Mn‐SOD activity was increased by treatments with 5‐fluorouracil (5‐FU), peplomycin and 137Cs, reaching plateau levels at 12 h after treatment and then decreasing gradually. When OSC‐1 and OSC‐3, and OSC‐2 and OSC‐4 were examined as representative cell lines with low and high Mn‐SOD activity, respectively, the decrease was more prominent in OSC‐1 and OSC‐3 than in OSC‐2 and OSC‐4. The intracellular levels of superoxide and hydrogen peroxide (H2O2) were increased after treatment with the anti‐cancer agents, and the increases were larger in OSC‐1 and OSC‐3 than in OSC‐2 and OSC‐4. The decrease of mitochondrial membrane potential (Δψm) by the anticancer agents was marked in OSC‐1 and OSC‐3. Correspondingly, the release of cytochrome c, the activation of caspase‐3 and the cleavage of poly(ADP‐ribose)polymerase were stronger in OSC‐3 than in OSC‐4. In addition, apoptosis induced by the anticancer agents was prominent in OSC‐3, exhibiting a close relationship with the Δ7psi;m and the H2O2 level. These results indicate that Mn‐SOD in SCC cells modulates apoptosis induction and the inactivation of Mn‐SOD might be a promising strategy for SCC treatment.
Regulatory Network of Mitomycin C Action in Human Colon Cancer Cells
Authors: Suzuki Katsuyuki , Yamamoto Wataru , Park Ji‐Seon , Hanaoka Hohken , Okamoto Ryo , Kirihara Yoshimasa , Yorishima Takashi , Okamura Tatsunori , Kumazaki Tsutomu , Nishiyama Masahiko
Published: August 2005

A network composed of activation and inactivation pathways to regulate mitomycin C (MMC) action is suggested to exist in human cancer cells. COLO201 colon cancer cells were stably transfected with human NQO1 cDNA that encodes NAD(P)H: quinone oxidoreductase (DT‐diaphorase, DTD), and a clonal cell line with about 57‐fold elevated DTD activity was obtained. Northern analysis revealed that expression of the NADPH:cytochrome P450 reductase (P450 reductase) gene was decreased in the transfectant, COLO201/NQO1, associated with the increase of NQO1 expression. Biochemical characterization of the cells showed a significant increase of the glutathione (GSH) content concomitantly with the decrease of the P450 reductase activity. As a result of these coordinated modulations, sensitivity of COLO201/NQO1 to MMC was not increased as compared to the parent cells. Analyses of inhibition by specific inhibitors of DTD, P450 reductase and glutathione S‐transferase (GST) in 5 human colon cancer cell lines including the transfectant showed that DTD and P450 reductase play significant roles in MMC activation in cells with sufficiently high DTD activity and with marginal DTD activity, respectively. In contrast, GST appeared to participate in MMC inactivation in cells with a high level of GST activity. These results indicated that DTD, P450 reductase, GSH and GST may act together compensatively or competitively, depending on their levels in cells, to determine the cellular sensitivity to MMC.
In vitro Antitumor Activity of TAS‐103, a Novel Quinoline Derivative That Targets Topoisomerases I and II
Authors: Aoyagi Yoshimi , Kobunai Takashi , Utsugi Teruhiro , Oh‐hara Tomoko , Yamada Yuji
Published: August 2005

TAS‐103 is a novel anticancer agent targeting both topoisomerase (Topo) I and Topo II, that stabilizes cleavable complexes of Topo‐DNA at the cellular level. In this study, the in vitro antitumor effects of TAS‐103 were compared with those of other known Topo I and Topo II inhibitors. TAS‐103 inhibited DNA synthesis more strongly than RNA and protein synthesis, and induced an increase of cell population in the S‐G2/M phase. The cytotoxicity of TAS‐103 was strongest against S‐phase cells, but its cell cycle phase specificity was not clear, and depended on drug concentration and exposure time. The cytotoxicity of TAS‐103 (IC50: 0.0030–0.23 μM) against various tumor cell lines was much stronger than that of VP‐16 and comparable to that of SN‐38. The cytotoxicity of TAS‐103 seemed to be more related to the amount of protein‐DNA complexes than to the accumulation of TAS‐103 in the cells. P‐Glycoprotein (P‐gp)‐mediated MDR, CDDP‐resistant and 5‐FU‐resistant cell lines did not show cross‐resistance to TAS‐103. Although PC‐7/CPT cells bearing a Topo I gene mutation showed cross‐resistance to TAS‐103, the sensitivity of P388/CPT, HT‐29/ CPT and St‐4/CPT cells, showing decreased Topo I expression, was not changed. KB/VM4 and HT‐29/Etp cells, showing decreased Topo II expression, were slightly cross‐resistant to TAS‐103. These results suggest that TAS‐103 may act as an inhibitor of both Topo I and Topo II at the cellular level. This property may be responsible for its strong antitumor effect and broad‐spectrum, growth‐inhibitory effect on drug‐resistant cell lines.
Risk of Breast Cancer in Japanese Women with Benign Breast Disease
Authors: Minami Yuko , Ohuchi Noriaki , Taeda Yoshinori , Takano Akira , Fukao Akira , Satomi Susumu , Hisamichi Shigeru
Published: August 2005

To investigate the risk of breast cancer development in women with benign breast disease (BBD), 387 screen‐detected BBD women and 1,489 normal women, taken from participants in the breast cancer screening program during 1978–1986, were followed through 1991. While 2,811 person‐years in the BBD group and 11,018 person‐years in the normal group were accumulated, 5 women in the BBD group and 6 women in the normal group developed breast cancer. Using the Mantel‐Haenszel method, relative risks (RR) were estimated for all women with BBD and women in some BBD types. Significantly elevated risk of breast cancer was observed in all women with BBD (RR=3.26, 95% confidence interval (CI) 1.08−9.83). Women with proliferative BBD were at high risk of breast cancer (RR=8.48, 95%CI 2.99−24.10), but no increased risk was observed for women with non‐proliferative BBD (RR=0.93, 95%CI 0.11−7.66). These results are consistent with those in high‐risk countries for breast cancer. In the management of women with BBD, histopathological diagnosis of the breast lesion is essential and women with proliferative BBD should be followed up carefully.
Lack of Carcinogenicity of 2‐Amino‐3,8‐dimethylimidazo[4,5‐f]quinoxaline (MeIQx) in Cynomolgus Monkeys
Authors: Ogawa Kumiko , Tsuda Hiroyuki , Shirai Tomoyuki , Ogiso Tadashi , Wakabayashi Keiji , Dalgard Dan W. , Thorgeirsson Unnur P. , Thorgeirsson Snorri S. , Adamson Richard H. , Sugimura Takashi
Published: August 2005

The carcinogenic potential of 2‐amino‐3,8‐dimethylimidazo[4,5‐f]quinoxaline (MeIQx) was evaluated in cynomolgus monkeys. The animals received MeIQx, beginning at the age of one year, at doses of 10 or 20 mg/kg body weight by gavage five times a week for 84 months and were autopsied 8 months thereafter. Although sporadic development of aberrant crypt foci in the colon and glutathione S‐transferase π‐positive foci in the liver as well as hyper plastic changes of the lymphatic tissue in the lung and gastro‐intestinal tract were observed in several monkeys, this was not treatment‐related. No neoplastic or preneoplastic lesions were found in other organs. Serum chemistry data and organ weights were also within the normal ranges. From these data, it is concluded that MeIQx is not carcinogenic in the cynomolgus monkey under the conditions examined. This lack of carcinogenicity is probably related to the poor activation of MeIQx due to the lack of constitutive expression of CYP1A2 as well as an inability of other cytochrome P450s to catalyze N‐ hydroxylation of MeIQx in the cynomolgus monkey.
Frameshift Mutation of the STK11 Gene in a Sporadic Gastrointestinal Cancer with Microsatellite Instability
Authors: Nakagawa Hidewaki , Koyama Kumiko , Nakamori Shoji , Kameyama Masao , Imaoka Shingi , Monden Morito , Nakamura Yusuke
Published: August 2005

Germline mutations of the STK11 gene lead to emergence of hamartomas in the gastrointestinal tract of patients with Peutz‐Jeghers syndrome, who bear an increased risk of malignancies of the gastrointestinal tract, genital tract, and other organs. We analyzed 80 sporadic colorectal cancers, six small‐intestinal cancers, and 40 gastric cancers for somatic mutations of STK11 by SSCP methods. Among them only one colorectal cancer, which showed a phenotype of microsatellite instability, was found to possess a deleterious mutation in this gene, a frameshift involving deletion of one base at codons 279–281. This region of the gene contains a mononucleotide‐repeat sequence, CCCCCC. The other allele of STK11 had been lost in this tumor. If the STK11 gene is one of the mutational targets of microsatellite instability, its inactivation may be associated with tumor development in a small proportion of colorectal cancers.
Disaccharide Esters Screened for Inhibition of Tumor Necrosis Factor‐α Release Are New Anti‐cancer Agents
Authors: Okabe Sachiko , Suganuma Masami , Tada Yukiko , Ochiai Yumiko , Sueoka Eisaburo , Kohya Hidehiko , Shibata Akihiro , Takahashi Masami , Mizutani Masashi , Matsuzaki Toshiake , Fujiki Hirota
Published: August 2005

Tumor necrosis factor‐α (TNF‐α) is a proinflammatory cytokine playing a part in various pathological states. Non‐toxic inhibitors of TNF‐α release are thought to be promising agents for cancer prevention. We found that the acetone fraction of the tobacco leaf surface lipid containing glucose esters and sucrose esters inhibited both TNF‐α release from BALB/3T3 and KATO III cells induced by okadaic acid and tumor promotion by okadaic acid on mouse skin initiated with 7,12‐dimethylbenz(a)anthracene (DMBA). Next, we investigated the inhibition of TNF‐α release with synthetic disaccharide esters, such as 6,6′‐di‐0‐alkanoyl‐α,α‐trehaloses (6,6′‐diester‐trehaloses), 4,4′‐di‐0‐alkanoyl‐α,α‐trehaloses (4,4′‐diester‐trehaloses) and 6,6′‐diamino‐6,6′‐dideoxy‐N, N′‐dial‐kanoyl‐α,α‐trehaloses (6,6′‐diamide‐trehaloses) bearing fatty acids of various chain lengths, and n‐ dodecyl‐β‐D‐maltoside as a disaccharide monoester. 6,6′‐Diester‐trehaloses and 4,4′‐diester‐treha‐loses of C8 to C14 fatty acids, 6,6′‐diamide‐trehaloses of C8 to C14 fatty acids, and n‐dodecyl‐β‐D‐maltoside all inhibited TNF‐α release in a dose‐dependent manner. The IC50 values are 7.4‐14.8 μM for 6,6′‐diester‐trehaloses (C8 to C12), 14.6‐21.6 μM 4,4′‐diester‐trehaloses (C8 to C12), 2.9‐15.0 μM for 6,6′‐diamide‐trehaloses (C8 to C14) and 23 μM for dodecyl‐β‐D‐maltoside. Both 6,6′‐di‐O‐octanoyl‐α,α‐trehalose (C8, designated as SS555) and n‐dodecyl‐β‐D‐maltoside (C12) inhibited tumor promotion by okadaic acid on mouse skin initiated with DMBA. Percentages of tumor‐bearing mice in week 15 of tumor promotion were reduced from 60.0 to 13.3 with SS555, and to 46.7 with n‐dodecyl‐β‐D‐maltoside. Moreover, SS555 inhibited TNF‐α gene expression mediated through inhibition of AP‐1 activation, but not NF‐αB activation. This paper reports that diester‐trehaloses of C8 to C12 fatty acids and mimics of disaccharide monoesters such as n‐dodecyl‐β‐D‐maltoside appear to be potential cancer‐preventive agents of a new type.
Effect of 5‐Fluorouracil on Cell Cycle Regulatory Proteins in Human Colon Cancer Cell Line
Authors: Takeda Haruo , Haisa Minoru , Naomoto Yoshio , Kawashima Ryuichi , Satomoto Kazutaka , Yamatuji Tomoki , Tanaka Noriaki
Published: August 2005

We investigated the effects of 5‐fluorouracil (5‐FU) on cell cycle‐regulating proteins in RPMI 4788 cells. 5‐FU inhibited cell growth dose‐dependently and this growth inhibition was accompanied with cell cycle accumulation in early S phase and increased expression of cyclin A. When cells were released from short‐term treatment (3 or 24 h) with 5‐FU, the cell cycle started to progress again and cyclin A protein levels decreased. Cyclin A‐associated kinase activity assay showed that cyclin A‐cyclin‐dependent kinase (Cdk) 2 kinase activity was altered by 5‐FU treatment concomitantly with the changes in cell cycle state seen in flow cytometric analysis. Furthermore, the elevation of cyclin A protein level by 5‐FU treatment was observed in three other human cancer cell lines, DLD‐1, H226Br and T.Tn. These results suggest that cyclin A protein levels in cancer cells are increased by 5‐FU, and the cyclin A function and degradation mechanism remain normal.