bluuebunny/crossref_metadata_2025 huggingface dataset:
Maintenance and Characterization of an Epstein Barr Virus‐infected CD56‐negative T Cell Lymphoma
Authors: Maekawa Izuru , Satoh Hitoshi , Aoki Naoto , Morishita Yasuyuki , Tsukamoto Norifumi , Karasawa Masamitsu , Nonaka Yasunobu , Shiota Mami , Nojima Yoshihisa , Mori Shigeo
Published: August 2005

T cell lymphoma carrying Epstein Barr virus (EBV+TL) is very rare among Western countries while it is much more common among Japanese. Here we report an EBV+TL which has been maintained for years by the use of mice with severe combined immune deficiency (SCID) mice. Lymphoma was obtained from a 55‐year‐old male suffering from oculomotor nerve palsy and lymphadenopathy. A small piece of biopsied tumor was transplanted into SCID mice and the lymphoma has been maintained for over 3 years with passages every 2–3 weeks. The maintained lymphoma, termed as TMS24, and the original lymphoma cells showed identical phenotype and genotype, including diffuse medium‐sized cell morphology lacking granules, suppressor/cytotoxic immunophenotype and identical T cell receptor β‐chain gene rearrangement mode. Further, both were shown to carry an identical EBV clone in terms of the number of terminal repeats and the latency II‐type restricted gene expression profile. Cytogenetically, TMS24 retained two characteristic chromosomal translocations of t(l;18)(q32;q21) and t(6;12)(p21;q24). Since only one cell line with such characters has been reported previously, TMS24 should be useful for detailed analysis of EBV+TL.
Enhanced Expression of Type IV Collagen‐binding Protein (p29) in Fyn‐transfected Murine Fibrosarcoma Cells
Authors: Koike Kazuhiko , Kogawa Katsuhisa , Takayama Tetsuji , Yoshizaki Naohito , Muramatsu Hirohito , Nakamura Kiminori , Sakamaki Sumio , Niitsu Yoshiro
Published: August 2005

We investigated the mechanism of the enhancement of metastatic potential induced by transfection of the fyn gene, a member of the src family. We employed two murine fyn cDNA‐transfected clones, ML‐SN1 and ML‐SN2, which were previously established from an ML‐01 low‐metastatic clone of Meth A sarcoma of BALB/c mice and were proven to have higher metastatic ability than ML‐01 and the mock‐transfected clone ML‐MT‐neo (Takayama et al., 1993). Our present investigation revealed that the two transfectants showed higher metastatic ability and higher rates of adherence to type IV collagen than ML‐MT‐neo. However, no difference was found in in vitro or in vivo growth rates, attachment to laminin or endothelial cells or cell motility through a reconstituted basement membrane. Analysis of surface membrane proteins labeled with 125I on SDS‐PAGE showed that a 29 kD band specifically bound to type IV collagen‐coupled beads was more intense in ML‐SN2 than in ML‐MT‐neo. Genistein, a protein tyrosine kinase inhibitor, dramatically reduced protein tyrosine kinase (PTK) activity of ML‐SN2 in a dose‐dependent fashion, corresponding to the reduction of adhesiveness to type IV collagen. The expression of the type IV collagen‐binding protein (p29) of ML‐SN2 was also reduced significantly by genistein treatment. These results suggested that the fyn product in Meth A cells augments the expression of a type IV collagen‐binding protein through elevation of the PTK activity of the membrane fraction and thus facilitates the metastasis of Meth A.
Cloning of the 5’Upstream Region of the Rat p16 Gene and Its Role in Silencing
Authors: Abe Masanobu , Okochi Eriko , Kuramoto Takashi , Kaneda Atsushi , Takato Tsuyoshi , Sugimura Takashi , Ushijima Toshikazu
Published: August 2005

Hypermethylation of the 5’upstream region (5’region) of the human p16CDKN2A (p16) gene is known to cause silencing, which is involved in a wide range of human cancers. For the rat p16 gene, its 5’region has not been cloned, and it is uncertain whether surrogate use of exon la is adequate for analysis of p16 silencing. In this study, we observed that methylation analysis of exon la gave false positive results in three samples of normal rat mammary epitheliums and in two of six primary mammary carcinomas. Therefore, we determined the nucleotide sequence of the 5’region of the rat p16 gene. To confirm that methylation status of the 5’region is correlated with p16 expression, the methylation status was analyzed by bisulfite sequencing and methylation‐specific PCR in three samples of normal mammary glands, six samples of mammary carcinomas and four cell lines. The 5’region was demethylated in all of the three normal and six carcinoma samples that fully expressed p16. On the other hand, the 5’region was highly methylated in the 3Y1 cell line, which lacked p16 expression, but without deletion. These results showed that the methylation status of the 5’region was more closely correlated with p16 expression than that of the exon 1α and analysis of the methylation status is useful in examining p16 silencing in various rat tumors.
Clinical Significance of Glucose Transporter 1 (GLUT1) Expression in Human Breast Carcinoma
Authors: Kang Sung Soo , Chun Yi Kyeong , Hur Min Hee , Lee Hae Kyung , Kim Yee Jeong , Hong Sung Ran , Lee Jee Hyun , Lee Sung Gong , Park Yong Koo
Published: August 2005

Glucose uptake and glycolytic metabolism are enhanced in cancer cells compared to normal cells and tissues. Increased expression of glucose transporter 1 (GLUT1) has been reported in human malignant cells. The aim of this study is to determine the expression of the facilitative glucose transporter protein GLUT1 in human breast carcinomas and a possible correlation between GLUT1 expression and clinical outcome including disease‐free or overall survival. One hundred consecutive formalin‐fixed, paraffin‐embedded sections of invasive breast carcinomas were evaluated by means of immunohistochemical staining of GLUT1. Forty‐seven (47%) of 100 breast carcinomas showed positive staining for GLUT1. Expression of GLUT1 correlated significantly with nuclear grade (P<0.001), estrogen receptor status (P=0.002), and progesterone receptor status (P=0.001). The mean disease‐free survival periods of GLUTl‐positive and ‐negative patients were 47±2.4 months and 54.3±1.3 months, respectively (P=0.017). The mean overall survival periods of GLUTl‐positive and ‐negative patients were 48.7±2.2 and 56.1±1.3 months, respectively (P=0.043). In the multivariate analysis, disease‐free survival correlated significantly with GLUT1, tumor size, and lymph node involvement (P=0.043, P=0.014, and P=0.045, respectively). In analysis of overall survival, however, lymph node involvement, tumor size, and nuclear grade were statistically significant (P=0.024, P=0.023, and P=0.003, respectively). Our data suggest that absence of GLUT1 expression significantly increases disease‐free survival. These findings demonstrate that GLUT1 expression in breast carcinoma can be a marker of aggressive biological behavior and identifies a worse prognosis in breast carcinoma patients.
The Intraductal Carcinoma Component Is a Significant Prognostic Parameter in Patients with Invasive Ductal Carcinoma of the Pancreas
Authors: Kawahira Hiroshi , Hasebe Takahiro , Kinoshita Taira , Sasaki Satoshi , Konishi Masaru , Nakagori Toshio , Inoue Kazuto , Oda Tatsuya , Takahashi Shin‐ichirou , Ochiai Takenori , Ochiai Atsushi
Published: August 2005

We have sometimes encountered invasive ductal carcinomas (IDCs) of the pancreas containing intraductal carcinoma components in the intra‐ and/or extra‐tumor area. The purpose of this study was to investigate whether intraductal carcinoma components would be useful for predicting the outcome of IDC patients. Forty‐seven surgically treated IDCs were examined, and all histological tumor sections were stained with Elastica to accurately confirm intraductal carcinoma components. Well‐known clinicopathological parameters that exhibited a significant correlation in the univariate analyses for predicting disease‐free survival (DFS) and overall survival (OS) were entered into the Cox proportional hazard multivariate analysis. Since the lowestP‐value predicting DFS or OS periods was observed in IDCs with more than 10% intraductal carcinoma components and those with 10% or less intraductal carcinoma components (P=0.028 andP=0.019), we established the cutoff value of intraductal carcinoma components at 10%. In the multivariate analyses for DFS and OS, the presence of more than 10% intraductal carcinoma components showed a marginally significant increase in the hazard rate (HR) of tumor recurrence (P=0.067) and significantly increased the HR of mortality (P=0.040). The present study demonstrated that IDCs with more than 10% intraductal carcinoma components were associated with a significantly better patient outcome than those with 10% or less intraductal carcinoma components.
Pharmaceutical and Biomedical Differences between Micellar Doxorubicin (NK911) and Liposomal Doxorubicin (Doxil)
Authors: Tsukioka Yoshihisa , Matsumura Yasuhiro , Hamaguchi Tetsuya , Koike Hiroyo , Moriyasu Fuminori , Kakizoe Tadao
Published: August 2005

The stability and biological behavior of an in vitro system of doxorubicin (DXR) entrapped in NK911, polymer micelles, was examined and compared with those of DXR entrapped in Doxil, polyethylene‐glycol‐conjugated liposomes. The fluorescence of DXR inside micelles or liposomes in an aqueous solution is known to be strongly quenched by the outer shells of the micellar or liposomal formation. Thus, by measuring the fluorescence intensity of DXR released from NK911 or Doxil, we could determine the stability of the micellar or liposomal DXR formation. Furthermore, NK911 was found to be less stable than Doxil in saline solution. In drug distribution experiments using an in vitro solid tumor model, when spheroids formed from two human colonic cancer lines, HT‐29 and WiDr, and a human stomach cancer line, MKN28, were exposed to NK911, DXR was distributed throughout the spheroids, including their center. On the other hand, when the spheroids were exposed to Doxil, DXR was distributed only to the surface of the spheroids. It has been suggested that Doxil can deliver DXR to a solid tumor more efficiently than NK911 via the EPR (enhanced permeability and retention) effect, because Doxil may be more stable in plasma than NK911. On the other hand, DXR packed in NK911 may be distributed by diffusion to cancer cells distant from the tumor vessel, because NK911 can leak out of the tumor vessel and may be able to release free DXR more easily than Doxil. It has been suggested that drug carrier systems such as liposomes and micelles should be selected appropriately bearing in mind the characteristics of the tumor vasculature and the tumor interstitium.
Phosphorylation of Fas‐associated Death Domain Contributes to Enhancement of Etoposide‐induced Apoptosis in Prostate Cancer Cells
Authors: Shimada Keiji , Nakamura Mitsutoshi , Ishida Eiwa , Kishi Munehiro , Yonehara Shin , Konishi Noboru
Published: August 2005

Fas‐associated death domain (FADD) plays an important role as an adapter molecule in Fas (CD95/APO‐l)‐mediated apoptosis and contributes to anticancer drug‐induced cytotoxicity. We treated three human prostate cancer cell lines with etoposide, a toposiomerase II inhibitor with activity against various tumors including prostate cancer. We found that the overexpression of FADD sensitizes etoposide‐induced apoptosis through a rapid activation of c‐Jun NH2‐terminal kinase (JNK) and, subsequently, of caspase 3. In addition, phosphorylation of FADD at serine 194 coincided with this sensitization. Treatment with the caspase 3 inhibitor, N‐acetyl‐Asp‐Glu‐Val‐Asp‐aldehyde (DEVD‐CHO), or overexpression of either mitogen‐activated protein kinase kinase (MKK) 7 or Bcl‐xL canceled FADD‐mediated sensitization to etoposide‐induced apoptosis. Moreover, treatment with the caspase 8 inhibitor, benzyloxy‐carbonyl‐Val‐Ala‐Asp‐fluoromethylketone (z‐IETD‐fmk), or overexpression of viral FLICE/caspase‐8‐inhibitory protein (FLIP) from equine herpesvirus type 2 E8 also had an inhibitory effect, supporting a major involvement of a caspase 8‐dependent mitochondrial pathway. Interestingly, FADD was phosphorylated, and etoposide‐induced JNK/caspase activation and apoptosis were enhanced in the cells arrested at G2/M transition, but not in those overexpressing mutant FADD, in which 194 serine was replaced by alanine. Our results demonstrate that phosphorylated FADD‐dependent activation of the JNK/caspase pathway plays a pivotal role in sensitization to etoposide‐induced apoptosis in prostate cancer cells.
Suppression of Lung Metastasis by Aspirin but Not Indomethacin in an in vivo Model of Chemically Induced Hepatocellular Carcinoma
Authors: Futakuchi Mitsuru , Ogawa Kumiko , Sano Masashi , Tamano Seiko , Takeshita Fumitaka , Shirai Tomoyuki
Published: August 2005

To examine the effect of non‐steroidal anti‐inflammatory drugs on metastasis formation, aspirin (ASP, 0.5% in diet) and indomethacin (IM, 0.005% in drinking water) were applied to an in vivo highly metastatic rat hepatocellular carcinoma (HCC) model in F344 male rats. Administration for 8 weeks after induction of highly metastatic HCC by sequential treatment with diethylnitrosamine and N‐nitrosomorpholine did not cause any significant change in survival rate or body weight. Multiplicity of HCC in the liver increased during ASP or IM treatment without any significant histological alteration. Although absent in the rats killed at the end of the period of carcinogen exposure, lung metastasis at the end of the experiment was found in 100%, 89% and 100% of rats in the control, ASP and IM groups, respectively. Degree of metastasis was classified into three groups according to the number of metastatic nodules, i.e., slight (1–5 nodules), moderate (6–50) and severe (more than 51), which amounted to 0%, 43% and 57% in the control group. ASP significantly reduced the degree of metastasis, the incidences being 33%, 44%, and 11%, respectively, whereas IM was without significant influence. Both agents suppressed cell proliferation in HCCs, without any alteration of pan‐cadherin expression. However, expression in HCC of mRNAs for intercellular adhesion molecule‐1 and vascular cell adhesion molecule‐1, both of which are considered to play key roles in attachment of cancer cells to the endothelium, was significantly suppressed by ASP. Thus, the present study demonstrated that ASP, but not IM, has the potential to inhibit lung metastasis of rat HCC in vivo, possibly via reduced attachment of tumor cells to the vascular endothelium. Moreover, these data indicate this in vivo model for induction of rat highly metastatic HCC to be a useful tool for the assessment of the efficacy of therapeutic treatments to block metastasis formation.
Analysis of Fas Gene Mutations on Laser Capture Microdissected Specimens from Renal Cell Carcinoma
Authors: Takayama Hitoshi , Takakuwa Tetsuya , Tsujimoto Yuichi , Nonomura Norio , Okuyama Akihiko , Aozasa Katsuyuki
Published: August 2005

Renal cell carcinoma (RCC) expresses Fas antigen on the cell surface, and thus could be sensitive to apoptosis induced by the binding of Fas ligand. Fas gene mutations might be involved in the development of RCC. Fas gene mutations were examined in genomic DNA extracted from RCC lesions. With use of laser capture methods, one RCC and one non‐neoplastic lesion per case were microdissected from 15 patients with RCC. Polymerase chain reaction‐amplified products were directly sequenced. Loss of heterozygosity (LOH) was examined at four sites of known polymorphism. Mutations of the Fas gene were detected in 3 RCC lesions from 3 (20%) of 15 cases. All mutations were point mutations, 2 missense and one silent, in exons 7 and 9. Non‐neoplastic tissues never showed Fas gene mutations. Nine of 15 cases (60.0%) were heterozygous for one or more sites of the known biallelic polymorphisms, i.e., at nucleotides ‐1377, ‐670, 416, and 836. Two of these 9 cases showed LOH at promoter region ‐670. Mouse T‐cell lymphoma cells transfected with missense mutated genes were resistant to apoptosis induced by anti‐Fas antibody, indicating these to be loss‐of‐function mutations. The results of the present study suggest that Fas gene mutations play a role in the pathogenesis of RCC.
Liposome Formulations for Effective Administration of Lipophilic Malonatoplatinum(II) Complexes
Authors: Han Insook , Jun Mee Sook , Kim Moon Kyu , Kim Jung Chul , Sohn Youn Soo
Published: August 2005

For effective administration of lipophilic trans(±)‐l,2‐diaminocyclohexaneplatinum(II) complexes of malonate derivatives [(dach)PtL, L=allylmalonate (AM), diallylmalonate (DAM), allylbenzyl‐malonate (ABM), or dibenzylmalonate (DBM)] in aqueous solution, we have applied three different liposome formulations and evaluated their physical and chemical properties, along with their in vitro cytotoxicity. The liposome formulations were composed of DMPC/DMPG [DMPC=1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine, DMPG=l,2‐dimyristoyl‐sn‐glycero‐3‐(phospho‐rac‐l‐glycerol) (sodium salt)] in different molar ratios (7/3 or 3/7) or an equimolar DOTAP/DOPE formulation (DOTAP=l,2‐dioleoyl‐3‐trimethylammonium propane, DOPE=l,2‐dioleoyl‐sn‐glycero‐3‐phosphoethanolamine). Preliposomal powders of the platinum complexes were prepared by lyophilization, and reconstituted in aqueous solution to obtain the final liposomal platinum complexes. Due to the lipophilicity of the malonatoplatinum complexes, the entrapment efficiency of drugs within the liposomes was over 90% except for the AM complex, and platinum drug stability was also satisfactory (>90%) in these liposomal systems. In vitro cytotoxicity was tested in human ovarian carcinoma cells sensitive (A2780) and resistant to cisplatin (A2780/PDD). In both cell lines, the liposomal DBM complex was much more cytotoxic than the corresponding DAM and ABM complexes, which means that the more hydrophobic benzyl substituent affords higher cytotoxicity than the allyl substituent in the malonato leaving group. Furthermore, the DBM complex in DMPC/DMPG formulations was effective against both sensitive and resistant A2780 cells (resistance indexes (RI)=1.10–1.49), showing lack of cross‐resistance to cisplatin. Therefore, the liposomal DBM complex in the DMPC/DMPG formulations is a promising candidate for stable pharmaceutical liposomal platinum complexes.
Detection of Tumor DNA in Serum of Colorectal Cancer Patients
Authors: Ito Seiji , Hibi Kenji , Nakayama Hiroshi , Kodera Yasuhiro , Ito Katsuki , Akiyama Seiji , Nakao Akimasa
Published: August 2005

We previously found p16 promoter methylation in DNA in the sera of 13 colorectal cancer patients out of 44 (30%) whose tumor DNA exhibited the methylation, using methylation‐specific PCR (MSP). To examine whether the cancer detection rate could be improved by using a different tumor marker, we examined the K‐ras status in 90 colorectal cancer patients using a mismatch ligation assay. Among the 31 patients showing K‐ras gene mutations in their tumors, the same mutations were observed in serum DNA of 11 patients (35%). Among the 90 patients, 63 showed tumors positive for K‐ras mutation or p16 promoter methylation, or both, and 22 had serum DNA positive for one or both. K‐ras mutation was found even in serum DNA of patients with Dukes A cancer, suggesting that colorectal cancer might be detected even in patients without symptoms by using this ligation assay.
Hepatitis B and C Viruses Infection, Lifestyle and Genetic Polymorphisms as Risk Factors for Hepatocellular Carcinoma in Haimen, China
Authors: Yu Shun‐Zhang , Huang Xin‐En , Koide Tsuneo , Cheng Gang , Chen Gong‐Chao , Harada Ken‐ichi , Ueno Yoshio , Sueoka Eisaburo , Oda Hideaki , Tashiro Fumio , Mizokami Masashi , Ohno Tomoyoshi , Xiang Jin , Tokudome Shinkan
Published: August 2005

A case‐control study was carried out to investigate the impact of factors including virus infection, aflatoxin B1, microcystins, smoking/drinking and dietary habits as well as genetic polymorphisms of aldehyde dehydrogenase 2 (ALDH2) and cytochrome P4502E1 (CYP2E1), on susceptibility to hepatocellular carcinoma (HCC) in Haimen, China. A total of 248 patients with HCC and 248 sex‐, age‐ and residence‐matched population‐based controls were recruited into the study. Virus infection, and ALDH2 and CYP2E1 gene polymorphisms were assessed in 134 paired cases and controls. By univariate analysis, hepatitis B virus (HBV) infection (odds ratio [OR]=9.75; 95% confidence interval [CI] =4.71–20.2), history of intravenous injection (OR=1.50; 95%CI=1.02–2.22), average income (OR=0.63; 95% CI=0.43–0.92), frequent intake of foods rich in protein, e.g., egg (OR=0.6; 95% CI=0.42–0.87), chicken (OR=0.53; 95% CI=0.35–0.79), pork (OR=0.67; 95% CI=0.46–0.98) and fresh fish (OR=0.58; 95% CI=0.39–0.87) significantly differed between cases and controls. However, peanut intake (OR=0.66; 95% CI=0.43–1.01), source of drinking water, including tap (OR=1.33; 95% CI=0.81–2.20), deep well (OR=0.94; 95% CI=0.56–1.55), shallow well (OR=0.85; 95% CI=0.55‐–1.30), river (OR=0.95; 95% CI=0.65–1.38), ditch (OR=1.09; 95% CI=0.76–1.55) and pond water (OR=1.0; 95% CI=0.14–7.10) were not significantly associated with risk. Univariate analysis also indicated that the 1–1 genotype of ALDH2 (OR=1.38; 95% CI=0.86–2.23) as well as the Pst1‐ and Rsa1‐digested c1/c1 genotype of CYP2E1 (OR=1.36; 95% CI=0.81–2.28), was slightly more frequent in the case group. On multivariate analysis, HBV infection (OR=13.9; 95% CI=5.78–33.6) and history of intravenous injection (OR=2.72; 95% CI=1.24–6.00) were still associated with significantly increased risk of HCC, while frequent intake of fresh fish (OR=0.32; 95% CI=0.12–0.86) decreased this risk. These findings suggest that whereas peanut intake, water sources as well as genetic polymorphisms in ALDH2 and CYP2E1 do not significantly correlate with the risk of HCC, HBV infection is a main risk factor, and dietary items rich in protein, especially fresh fish, might protect against the risk of HCC in Haimen, China.
Primary Malignant Lymphoma of the Brain: Mutation Pattern of Rearranged Immunoglobulin Heavy Chain Gene
Authors: Endo Sumio , Zhang Shu‐Jing , Saito Takafumi , Kouno Mitsuo , Kuroiwa Toshihiko , Washiyama Kazuo , Kumanishi Toshiro
Published: August 2005

Using reverse transcription‐polymerase chain reaction (RT‐PCR), six primary brain lymphomas, pathologically diagnosed as diffuse large B‐cell lymphoma, were examined for rearranged VH‐D‐JHsequences of the immunoglobulin heavy chain gene, focusing on somatic mutations and intraclonal heterogeneity. The reliability of the isolated PCR clones was confirmed by in situ hybridization (ISH) with complementarity‐determining region (CDR) 3 oligonucleotide probes. Sequence analysis of the PCR clones revealed a high frequency of somatic mutation, ranging from 8.8 to 27.3% (mean 18.2%) in the VH gene segments in all the lymphomas. A significantly lower frequency of replacement (R) mutations than expected was also seen in their frameworks (FRs) in all cases. These findings suggested that the precursor cells were germinal center (GC)‐related cells in these lymphomas. However, despite extensive cloning experiments, intraclonal heterogeneity was not detected in any case except for one in which it could not be ruled out. Thus, it seemed likely that all of our brain lymphomas were derived from GC‐related cells and that at least most of them were from post‐GC cells.
Differential Mechanisms of Constitutive Akt/PKB Activation and Its Influence on Gene Expression in Pancreatic Cancer Cells
Authors: Matsumoto Joe , Kaneda Masako , Tada Mitsuhiro , Hamada Jun‐ichi , Okushiba Shunichi , Kondo Satoshi , Katoh Hiroyuki , Moriuchi Tetsuya
Published: August 2005

Activated Akt/protein kinase B transmits oncogenic signals leading to inhibition of apoptosis, cellular proliferation, and tolerance to hypoxia. Presently, mutational inactivation of PTEN and activation of Ras are considered to be the major causes of Akt activation. Here we report differential mechanisms of constitutive Akt activation in 4 human pancreatic cancer cell lines (KMP‐3, KMP‐4, PCI‐66, and PCI‐68). These 4 cell lines displayed phosphorylation and functional activation of Akt both in the presence and absence of serum, while three control cell lines (PCI‐79, KMP‐8, and PSN‐1) did so only in the presence of serum in culture. All the 7 cell lines harbored K‐Ras activated by mutations at codon 12 resulting in MAP kinase kinase (MEK1/2) phosphorylation, and all except one (KMP‐8) had p53 mutations, indicating that these mutations are not sufficient for constitutive Akt activation. KMP‐3 and KMP‐4 had lost PTEN function owing to loss of expression or a mutation, but PCI‐66 and PCI‐68 retained wild‐type PTEN. Phosphorylation of Akt was inhibited by the phosphatidylinositol‐3‐kinase (PI3K) inhibitor LY294002 and the tyrosine kinase inhibitor genistein in KMP‐3 and KMP‐4 cells, indicating that upstream signals are required for Akt activation in these two cell lines. In contrast, neither LY294002 nor genistein inhibited Akt activation in PCI‐66 and PCI‐68 cells, indicating the involvement of another unknown mechanism of Akt activation independent of PI3K‐mediated signaling to Akt. Irrespective of the differential mechanisms, the 4 cell lines showed similar mRNA expression patterns of 49 genes assessed by cDNA array as compared to the 3 cell lines without Akt activation, suggesting that the mechanisms have the same consequences on the downstream signaling of the constitutive Akt activation.
Costunolide Triggers Apoptosis in Human Leukemia U937 Cells by Depleting Intracellular Thiols
Authors: Choi Jung‐Hye , Ha Joohun , Park Jae‐Hoon , Lee Jae Yeol , Lee Yong Sup , Park Hee‐Juhn , Choi Jong‐Won , Masuda Yutaka , Nakaya Kazuyasu , Lee Kyung‐Tae
Published: August 2005

We have previously demonstrated that costunolide, a biologically active compound that was isolated from the stem bark of Magnolia sieboldii, induced apoptosis in human cancer cells. In the present study, we investigated the underlying mechanisms and suggest that costunolide induces apoptosis in human promonocytic leukemia U937 cells by depleting the intracellular thiols. Costunolide treatment rapidly depleted the intracellular reduced glutathione (GSH) and protein thiols, and this preceded the occurrence of apoptosis. Pretreatment with sulfhydryl compounds such as GSH, N‐acetyl‐L‐cysteine, dithiothreitol and 2‐mercaptoethanol almost completely blocked the costunolide‐induced apoptosis, highlighting the significance of the intracellular thiol level in the process. Furthermore, overexpression of Bcl‐2 also significantly attenuated the effects of costunolide. The apoptosis‐inducing activity of costunolide is likely to depend on the exomethylene moiety because derivatives in which this group was reduced, such as dihydrocostunolide and saussurea lactone, did not deplete the cellular thiols and showed no apoptotic activity. Taken together, the present study demonstrates that the costunolide‐induced apoptosis depends on intracellular thiols contents, which are modulated by Bcl‐2.
l‐β‐D‐Arabinofuranosylcytosine Is Cytotoxic in Quiescent Normal Lymphocytes Undergoing DNA Excision Repair
Authors: Yamauchi Takahiro , Kawai Yasukazu , Ueda Takanori
Published: August 2005

We have sought to clarify the potential activity of the S‐phase‐specific antileukemic agent 1‐β‐D‐arabinofuranosylcytosine (ara‐C), an inhibitor of DNA synthesis, in quiescent cells that are substantially non‐sensitive to nucleoside analogues. It was hypothesized that the combination of ara‐C with DNA damaging agents that initiate DNA repair will expand ara‐C cytotoxicity to non‐cycling cells. The repair kinetics, which included incision of damaged DNA, gap‐filling by DNA synthesis and rejoining by ligation, were evaluated using the single cell gel electrophoresis (Comet) assay and the thymidine incorporation assay. When normal lymphocytes were treated with ultraviolet C or with l,3‐bis(2‐chloroethyl)‐1‐nitrosourea (BCNU), the processes of DNA excision repair were promptly initiated and rapidly completed. When the cells were incubated with ara‐C prior to irradiation or BCNU treatment, the steps of DNA synthesis and rejoining in the repair processes were both inhibited. The ara‐C‐mediated inhibition of the repair processes was concentration‐dependent, with the effect peaking at 10 μM. The combination of ara‐C with these DNA repair initiators exerted subsequent cytotoxicity, which was proportional to the extent of the repair inhibition in the presence of ara‐C. In conclusion, ara‐C was cytotoxic in quiescent cells undergoing DNA repair. This might be attributed to unrepaired DNA damage that remained in the cells, thereby inducing lethal cytotoxicity. Alternatively, ara‐C might exert its own cytotoxicity by inhibiting DNA synthesis in the repair processes. Such a strategy may be effective against a dormant subpopulation in acute leukemia that survives chemotherapy.
Quantitative Measurement of Thymidylate Synthase and Dihydropyrimidine Dehydrogenase mRNA Level in Gastric Cancer by Real‐time RT‐PCR
Authors: Fujiwara Hisataka , Terashima Masanori , Irinoda Takashi , Takagane Akinori , Abe Kaoru , Kashiwaba Masahiro , Oyama Kennichi , Takahashi Masanori , Maesawa Chihaya , Saito Kazuyoshi , Takechi Teiji , Fukushima Masakazu
Published: August 2005

We used real‐time reverse‐transcription polymerase chain reaction (RT‐PCR) to assay expression of the mRNA of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) in gastric cancer tissue with the objective of establishing a system to measure TS and DPD in ultra‐low‐volume samples. Nude mouse xenografts of 5 human gastric cancer cell lines and 85 clinical samples were used as the specimens in this study. Sensitivity to 5‐fluorouracil (5‐FU) was determined on the basis of the relative tumor proliferation rate in mice and the results of ATP assay using serum‐free cultures of the clinical samples. mRNA expression was measured in tumor tissue by real‐tune RT‐ PCR using the ABI PRISM 7700 system. The values for expression of the mRNA for TS and DPD were corrected according to the level of glyceraldehyde‐3‐phosphate dehydrogenase mRNA expression. The xenografts yielded correlations between TS and DPD mRNA expression and the activity of the enzymes (TS: rs=0.700, DPD: rs=0.900), and an inverse correlation was noted between the mRNA levels and sensitivity to 5‐FU (TS: rs=‐0.900, DPD: rs=‐0.800). The clinical samples showed an inverse correlation between 5‐FU sensitivity and mRNA expression (TS: rs=‐0.518, DPD: rs=‐0.564). Sensitivity to 5‐FU was noted only in cases in which TS mRNA expression and DPD mRNA expression were both low. Real‐time RT‐PCR can provide a highly sensitive assessment of TS and DPD mRNA expression in gastric cancer, and it was useful for predicting 5‐FU sensitivity.
Impact of the p53 Status of the Tumor Cells on the Effect of Reactor Neutron Beam Irradiation, with Emphasis on the Response of Intratumor Quiescent Cells
Authors: Masunaga Shin‐ichiro , Ono Koji , Takahashi Akihisa , Sakurai Yoshinori , Ohnishi Ken , Kobayashi Tom , Kinashi Yuko , Takagaki Masao , Ohnishi Takeo
Published: August 2005

Human head and neck squamous cell carcinoma cells transfected with mutant p53 (SAS/mp53) or with neo vector as a control (SAS/neo) were inoculated subcutaneously into both the hind legs of Balb/cA nude mice. Tumor‐bearing mice received 5‐bromo‐2′‐deoxyuridine (BrdU) continuously to label all proliferating (P) cells in the tumors. After administration of sodium borocaptate‐10B (BSH) or p‐boronophenylalanine‐10B (BPA), the tumors were irradiated with neutron beams. The tumors not treated with 10B‐compound were irradiated with neutron beams or γ‐rays. The tumors were then excised, minced and trypsinized. The tumor cell suspensions thus obtained were incubated with a cytokinesis blocker, and the micronucleus (MN) frequency in cells without BrdU labeling (=quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. Meanwhile, 6 h after irradiation, tumor cell suspensions obtained in the same manner were used for determining the frequency of apoptosis in Q cells. The MN and apoptosis frequencies in total (P+Q) tumor cells were determined from the tumors that were not pretreated with BrdU. Without 10B‐carriers, in both tumors, the relative biological effectiveness of neutrons was greater in Q cells than in total cells, and larger for low than high cadmium ratio neutrons. With 10B‐carriers, the sensitivity was increased for each cell population, especially for total cells. BPA increased both frequencies for total cells more than BSH. Nevertheless, the sensitivity of Q cells treated with BPA was lower than that of BSH‐treated Q cells. These sensitization patterns in combination with 10B‐carriers were clearer in SAS/neo than in SAS/mp53 tumors. The p53 status of the tumor cells had the potential to affect the response to reactor neutron beam irradiation following 10B‐carrier administration.
Dietary Conjugated Linolenic Acid Inhibits Azoxymethane‐induced Colonic Aberrant Crypt Foci in Rats
Authors: Kohno Hiroyuki , Suzuki Rikako , Noguchi Ryoko , Hosokawa Masashi , Miyashita Kazuo , Tanaka Takuji
Published: August 2005

The modifying effects of dietary feeding of conjugated linolenic acid (CLN) isolated from the seeds of bitter gourd (Momordica charantia) on the development of azoxymethane (AOM)‐induced colonic aberrant crypt foci (ACF) were investigated in male F344 rats to predict its possible cancer chemopreventive efficacy. The effect of CLN on the proliferating cell nuclear antigen (PCNA) index in colonic ACF was also examined. Rats were given subcutaneous injections of AOM (20 mg/ kg body weight) once a week for 2 weeks to induce ACF. They also received the experimental diet containing 0.01%, 0.1% or 1% CLN for 5 weeks, starting one week before the first dosing of AOM. AOM exposure produced a substantial number of ACF (108±21/rat) at the end of the study (week 4). Dietary administration of CLN caused a significant reduction in the frequency of ACF: 87±14 (19.4% reduction, P<0.05) at a dose of 0.01%, 69±28 (36.1% reduction, P<0.01) at a dose of 0.1% and 40±d6 (63.0% reduction, P<0.001) at a dose of 1%. Also, CLN administration lowered the PCNA index and induced apoptosis in ACF. These findings might suggest possible chemopreventive activity of CLN in the early phase of colon tumorigenesis through modulation of cryptal cell proliferation activity and/or apoptosis.
Overexpression of Urokinase‐type Plasminogen Activator in Human Gastric Cancer Cell Line (AGS) Induces Tumorigenicity in Severe Combined Immunodeficient Mice
Authors: Choi Yang‐Kyu , Yoon Byung‐Il , Kook Yoon‐Hoh , Won Young‐Suk , Kim Jin‐Hyun , Lee Chul‐Ho , Hyun Byung‐Hwa , Oh Goo‐Taeg , Sipley John , Kim Dae‐Yong
Published: August 2005

The significance of urokinase‐type plasminogen activator (uPA) expression in gastric cancer development was tested by using a human uPA cDNA transfection approach and an in vivo severe combined immunodeficient (SCID) mouse model. The AGS gastric cancer cell line, which has urokinase‐type plasminogen‐activator receptor (uPAR) but lacks uPA, was transfected with a plasmid containing human uPA cDNA and injected into the backs of SCID mice. Compared with the parent AGS cells, uPA protein secretion in AGS‐2‐, AGS‐4‐, and AGS‐8‐transfected cells increased by 26.1‐, 34.6‐, and 4.8‐fold, respectively (Pr<0.05). mRNA expression levels of uPA in the AGS‐4 clone were much stronger than those in AGS‐2 and AGS‐8 clones. After the cancer cells (2×l06) were injected s.c. into the SCID mice, a palpable mass was observed at the injection site at around 140 days post‐injection, followed by accelerated growth of the xenograft up to 180 days post‐injection only in the high uPA‐producing clone (AGS‐4). These results suggest that continuous and high production of uPA by tumor cells is one of the important factors reflecting the malignancy of gastric cancer cells.
Expression of Hqk Encoding a KH RNA Binding Protein Is Altered in Human Glioma
Authors: Li Zheng Zhe , Kondo Tatsuya , Murata Tomoaki , Ebersole Thomas A. , Nishi Torn , Tada Kenji , Ushio Yukitaka , Yamamura Ken‐ichi , Abe Kuniya
Published: August 2005

The quaking gene family encodes single KH domain RNA‐binding proteins that play vital roles in cell differentiation, proliferation, and apoptotic processes. The human quaking gene, Hqk, maps to 6q25–q26, where cytogenetic alterations associated with a variety of human malignancies, including gliomas have been reported. To assess possible relationships of Hqk with human diseases such as glial tumors, we first isolated the Hqk gene, characterized its structure and expression pattern, and carried out mutational analysis of Hqk in primary tumor samples. The Hqk gene contains 8 exons spanning a ∼200 kb genomic region, and generating at least four alternatively spliced transcripts, Hqk–5, Hqk–6, Hqk–7 and Hqk–7B, of which Hqk–7 is abundantly expressed in brain. Analysis of primary tumors demonstrated a high incidence of expression alterations of Hqk in gliomas (30%; 6/20), but not in other tumors such as schwannomas (0/3), or meningiomas (0/8). Among the tumor samples showing expression alterations, two were devoid of all three major transcripts, one was missing only the Hqk–5 message, and only the Hqk–7 message was absent in two cases. Our results thus imply the involvement of Hqk in human glial tumor progression.
Inactivation of O6‐Methylguanine‐DNA Methyltransferase in Human Lung Adenocarcinoma Relates to High‐grade Histology and Worse Prognosis among Smokers
Authors: Hayashi Hiroyuki , Yazawa Takuya , Okudela Koji , Nagai Jun‐ichi , Ito Takaaki , Kanisawa Masayoshi , Kitamura Hitoshi
Published: August 2005

To evaluate the significance of O6‐methylguanine‐DNA methyltransferase (MGMT) activity in the development of human lung adenocarcinoma (AC), we investigated promoter hypermethylation of the MGMTx gene by methylation‐specific PCR, and the expression of MGMT protein by immuno‐histochemistry in relation to smoking history of the patients. In total, 31 of 87 AC patients (35.5%) showed hypermethylation of the MGMT gene, and no significant difference was observed between smokers (37.3%) and non‐smokers (33.3%). However, hypermethylation of the MGMT gene increased in parallel with lesser differentiation grade of tumors among smokers (well, 16.7%; moderately, 42.1%; poorly, 57.1%; P=0.022), although this trend was not observed among non‐smokers. Almost all the tumors with promoter hypermethylation of the MGMT gene showed consistently negative MGMT staining by immunohistochemistry. When the prognosis of stage‐I patients was compared among smokers, it was apparent that the prognosis of patients with inactivated MGMT was worse than that of MGMT‐positive patients (P=0.036). Such differences in the prognoses were not observed among non‐smokers. In conclusion, MGMT inactivation is related to the differentiation grade and the prognosis of lung AC patients among smokers. Although further studies are required, we speculate that smoking may induce hypermethylation, not only of the MGMT gene, but also of other important tumor suppressor genes.
Therapeutic and Analysis Model of Intrahepatic Metastasis Reflects Clinical Behavior of Hepatocellular Carcinoma
Authors: Sawada Shigeaki , Murakami Koji , Yamaura Takeshi , Mitani Noriyasu , Tsukada Kazuhiro , Saiki Ikuo
Published: August 2005

This study was designed to establish an intrahepatic metastasis model to investigate the biology and therapy of hepatocellular carcinoma (HCC) in mice. A fragment of mouse HCC tumor CBO140C12 was orthotopically implanted into the mouse liver. The number of intrahepatic metastatic colonies and the volume of the implanted tumor increased in a time‐dependent manner. At 28 days after fragment implantation, all mice showed intrahepatic metastasis. Intravenous administrations of cisplatin and doxorubicin at 7 and 21 days after the implantation significantly sup‐ pressed the growth of the primary tumor nodule, but tended to inhibit intrahepatic metastasis. However, a marked decrease of body weight was observed during the experiment. On the other hand, an inhibitor of matrix metalloproteinases (MMPs), ONO–4817, decreased the gelatinase activity of MMP–9 secreted by CBO140C12 cells, and significantly reduced the number of colonies of intrahepatic metastasis when administered orally. Our established model, which is focused on intrahepatic metastasis, is suitable for evaluating the therapeutic effect of HCC and for analyzing intrahepatic metastasis, because this model reflects the clinical features of HCC and all the steps of tumor metastasis.
Enhanced Antitumor Effect of Ultrasound in the Presence of Piroxicam in a Mouse Air Pouch Model
Authors: Okada Kyoji , Itoi Eiji , Miyakoshi Naohisa , Nakajima Mizuho , Suzuki Toshio , Nishida Jun
Published: August 2005

The antitumor effects of piroxicam, a non‐steroidal anti‐inflammatory drug, on sarcoma 180 cells under ultrasonic irradiation were examined in a mouse air pouch model. When piroxicam was added to sarcoma 180 suspension under ultrasound irradiation (2 MHz, 10 W, 120 s), the mortality rate of tumor cells immediately after the irradiation and the survival rate of mice were significantly higher than those when ultrasound alone was applied, and these effects of piroxicam were dose‐dependent. When D‐mannitol was used with piroxicam, the mortality rate of the tumors cells after the irradiation was comparable with that when piroxicam alone was applied, but when L‐ histidine was used concurrently, the antitumor effect was significantly lower than that when piroxicam alone was applied. Histological examinations one week after the ultrasound irradiation in the presence of piroxicam showed sparse tumor tissue in the air pouch and normal appearance of the air pouch and surrounding tissue. The findings suggest that piroxicam enhances the anti‐ tumor effects of ultrasound in vivo by increasing the production of singlet oxygen without damage to tissue surrounding the tumor.
Antitumor Effect of an Adeno‐associated Virus Vector Containing the Human Interferon‐β Gene on Experimental Intracranial Human Glioma
Authors: Yoshida Jun , Mizuno Masaaki , Nakahara Norimoto , Colosi Peter
Published: August 2005

We constructed an adeno‐associated virus (AAV) vector containing the human interferon‐β(HuIFN‐β) gene (AAV‐IFN‐β) and investigated its antitumor effect against human glioma cells (U251‐SP) inoculated into the brain of nude mice. Prior to this, we examined human glioma cells transduced with AAV‐IFN‐β using video‐enhanced contrast differential interference contrast (VEC‐DIC) microscopy. Infection of AAV‐IFN‐β induced apoptosis and secondary necrosis in human glioma cells. In in vivo experiments, we confirmed production of HuIFN‐β and induction of heat‐shock protein (HSP) in glioma cells transduced with AAV‐IFN‐β. Growth of the experimental gliomas was completely inhibited by six injections of AAV‐IFN‐β, starting 7 days after transplantation of glioma cells. In addition, the survival of mice treated with AAV‐IFN‐β was remarkably prolonged. These results indicate that AAV‐IFN‐β induces apoptosis of glioma cells and has a strong antitumor effect in this experimental glioma model.
Promoter Methylation of TSLC1 and Tumor Suppression by Its Gene Product in Human Prostate Cancer
Authors: Fukuhara Hiroshi , Kuramochi Masami , Fukami Takeshi , Kasahara Kohtaro , Furuhata Mutsuo , Nobukuni Takahiro , Maruyama Tomoko , Isogai Kana , Sekiya Takao , Shuin Taro , Kitamura Tadaichi , Reeves Roger H. , Murakami Yoshinori
Published: August 2005

We recently identified TSLC1, a tumor suppressor gene in human lung cancer. Gene silencing by promoter methylation has been observed frequently in adenocarcinoma of the lung, liver, and pancreas. Here, we demonstrate that TSLC1 expression is also absent or markedly reduced in 3 of 4 prostate cancer cell lines. Promoter sequences of TSLC1 were heavily methylated in PPC‐1 cells that lacked TSLC1 expression, supporting the idea that promoter methylation is strongly correlated with complete loss of gene expression. Promoter sequences of TSLC1 were also methylated significantly in 7 of 22 (32%) primary prostate cancers. Hypermethylation of the promoter occurred not only in advanced tumors, but also in relatively early‐stage tumors. Restoration of TSLC1 expression substantially suppressed tumor formation of PPC‐1 cells in nude mice. These findings indicate that alteration of TSLC1 is involved in prostate cancer.
Trends in Benign Breast Tumors in Japanese Women, 1973–1995: Experience of Hiroshima Tumor Tissue Registry
Authors: Arihiro Koji
Published: August 2005

Although some benign breast lesions such as multiple intraductal papilloma have been pointed out as a risk factor for breast cancer, there is little documentation about trends in the incidence of benign tumors of the breast in Japanese women. The author conducted an epidemiological study using data abstracted from the Hiroshima Tumor Tissue Registry file, which includes cases of malignant and benign neoplasms in Hiroshima prefecture between 1973 and 1995. A total of 17 064 cases of female breast tumor were registered in the file between 1973 and 1995. The registration rate for fibroadenoma of the female breast was 19.5 among females and peaked during the 5‐year period 1983–1987, while the fibroadenoma registration rate in Hiroshima gradually decreased between 1988 and 1995. The registration rate for intraductal papilloma has risen substantially in Hiroshima, with about a 5‐fold increase among females between 1973 and 1995. The mean proportion of fibroadenoma cases accompanied by malignant tumors of the breast was 1.85%, and there was no significant change in the mean proportion between 1973 and 1995 (P=0.17). On the other hand, the mean proportion of intraductal papilloma cases accompanied by malignant tumors of the breast gradually rose with about a 14‐fold increase among females between 1973 and 1995. The significance of intraductal papilloma as a risk factor for breast cancer may have increased.
Inhibitory Effects of Toremifene on N‐Methyl‐N‐nitrosourea and Estradiol‐17β‐induced Endometrial Carcinogenesis in Mice
Authors: Niwa Kenji , Hashimoto Midori , Lian Zenglin , Gao Jingchun , Tagami Keiko , Yokoyama Yasuhiro , Mori Hideki , Tamaya Teruhiko
Published: August 2005

Short‐ and long‐term experiments were designed to determine the effects of toremifene (TOR) on estrogen‐related endometrial carcinogenesis in mice. In the short‐term experiment, a single low dose of TOR (0.2 mg/30 g body weight) decreased expression of c‐fos, interleukin (IL)‐1α, estrogen receptor (ER)‐α mRNAs and corresponding proteins induced by estradiol‐l7β (E2), in the uteri of the ovariectomized mice. Expression of ER‐β mRNA was increased by the TOR treatment, compared with the control. In the long‐term experiment, 106 female ICR mice were given N‐methyl N‐nitrosourea (MNU) into their uterine corpora. The animals were divided into four groups as follows: group 1, E2 diet (5 ppm) plus TOR (0.2 mg/30 g body weight, subcutaneously, every four weeks); group 2, E2 diet alone; group 3, basal diet plus TOR. Group 4 served as the control. TOR treatment decreased the incidence of MNU and E2‐induced endometrial adenocarcinoma and atypical hyperplasia at the termination of the experiment (30 weeks after the start). These results suggest that TOR exerts preventive effects against estrogen‐related endometrial carcinogenesis in mice, through the suppression of c‐fos as well as IL‐1α expression induced by E2. Such suppressive effects of TOR may be related to the decreased ER‐α and increased ER‐β expressions.