bluuebunny/crossref_metadata_2025 huggingface dataset:
Preferential Activity of Wild‐type and Mutant Tumor Necrosis Factor‐α against Tumor‐derived Endothelial‐like Cells
Authors: Kuroda Kensei , Miyata Keizo , Tsutsumi Yasuo , Tsunoda Shinichi , Nishimura Koji , Mitsuishi Yachiyo , Nakagawa Shinsaku , Mayumi and Tadanori
Published: August 2005

Tumor‐derived endothelial‐like cells (tEC) were prepared by culturing human umbilical vein endothelial cells (HUVEC) in the presence of HT1080 human fibrosarcoma‐conditioned medium. tEC showed higher permeability and less cell‐adhesion activity than normal HUVEC (nEC). Tumor necrosis factor‐α (TNF) is known to have tumor‐vasculature disrupting activity. tEC showed higher cytotoxicity to recombinant human TNF (rhTNF) than nEC, and was not observed using HUVEC cultured with WI38 human diploid cell‐conditioned medium as a medium‐control. These results demonstrate that tEC acquire physiological properties of tumor‐associated vasculature, and may be a useful model system for the study of the mechanisms of TNF antitumor action. The TNF‐mutant RGD‐V29 (code No. F4614), which has an inserted 4Arg‐Gly‐Asp sequence and an 29Arg→Val replacement, was found to induce greater preferential destruction of tEC compared to rhTNF. When the preferential activities were evaluated in terms of 30% cytotoxicity (IC30) ratio (nEC/tEC), the ratio was 460 for RGD‐V29 compared to 4.2 for rhTNF. RGD‐V29 also exhibited cell‐adhesive function and bound preferentially to the p55 TNF‐receptor. Both these properties of RGD‐V29 contributed to the tEC selective cytotoxicity, indicating that the RGD ligands and selective p55 receptor binding on the cells, although uncharacterized, are involved in tEC targeting. Therefore, the TNF mutant RGD‐V29 may show greater selectivity toward tumor vasculature than wild‐type TNF.
Vitamin K Contents in Liver Tissue of Hepatocellular Carcinoma Patients
Authors: Miyakawa Takayuki , Kajiwara Yasuhiro , Shirahata Akira , Okamoto Kohji , Itoh Hideaki , Ohsato Keiichi
Published: August 2005

Serum protein induced in vitamin K absence‐II (PIVKA‐II) is used as a tumor marker because it increases at a notably higher rate in patients with hepatocellular carcinoma. To clarify the mechanism causing the elevation of serum PIVKA‐II, we measured the contents of vitamins K1 (phylloquinone, PK) and K2 (menaquinone, MK) (MK‐4, MK‐5, MK‐6, MK‐7, MK‐8, MK‐9, MK‐10) in liver tissue resected from 21 hepatic cancer patients (12 patients with hepatocellular carcinoma and 9 patients with metastatic hepatic cancer), using HPLC combined with coulometric reduction and fluorometric detection. In the cancerous tissue of hepatocellular carcinoma patients, PK, MK‐7, MK‐8, and MK‐10 in the cancerous tissue of hepatocellular carcinoma patients were significantly lower than that in the cancerous tissue of metastatic hepatic cancer patients. These data suggested that one of the mechanisms of the elevation of serum PIVKA‐II levels in hepatocellular carcinoma patients is a vitamin K deficiency in the local cancerous tissue.
Annexin VII as a Novel Marker for Invasive Phenotype of Malignant Melanoma
Authors: Kataoka Tatsuki R. , Ito Akihiko , Asada Hideo , Watabe Kenji , Nishiyama Kazutaka , Nakamoto Ken'i , Itami Satoshi , Yoshikawa Kunihiko , Ito Masaki , Nojima Hiroshi , Kitamura Yukihiko
Published: August 2005

Both F10 and BL6 sublines of B16 mouse melanoma cells are metastatic after intravenous injection, but only BL6 cells are metastatic after subcutaneous injection. While examining the genetic difference between the two sublines, we found a marked reduction of annexin VII expression in BL6 cells. In addition, fusion cell clones of both sublines, were as poorly metastatic as F10 cells after subcutaneous injection, and contained the annexin VII message as abundantly as F10 cells. Hence, we examined whether the annexin VII expression was correlated with the less malignant phenotype of clinical cases by immunohistochemistry. Immunoreactivities to anti‐annexin VII antibody in melanoma cells were evaluated quantitatively by using skin mast cells as an internal positive control. Eighteen patients with malignant melanoma were divided into two groups: lymph node metastasis‐negative and positive groups. The ratio of numbers of patients positive versus negative to the antibody was significantly larger in the former than in the latter group. These results not only indicated that annexin VII serves as a marker for less invasive phenotype of malignant melanoma, but also suggested a possible role of annexin VII in tumor suppression.
In vitro Antitumor Activity, Intracellular Accumulation, and DNA Adduct Formation of cis‐[((1R, 2R)‐1,2‐Cyclohexanediamine‐N, N')bis(myristato)] Platinum (II) Suspended in Lipiodol
Authors: Kishimoto Shuichi , Miyazawa Kenji , Fukushima Shoji , Takeuchi Yoshikazu
Published: August 2005

SM‐11355, cis‐[((1R,2R)‐1,2‐cyclohexanediamine‐N,N')bis(myristato)] platinum (II), is a lipophilic platinum complex under clinical development that targets primary hepatocellular carcinoma using Lipiodol as a carrier. SM‐11355 was compared with cisplatin (CDDP) using an in vitro evaluation system capable of examining the release characteristics and the cytotoxicity of drugs suspended in Lipiodol. SM‐11355 suspended in Lipiodol (SM‐11355/Lipiodol) and CDDP suspended in Lipiodol (CDDP/Lipiodol) showed cytotoxic activity against rat ascites hepatoma AH‐109A cells in a dosedependent manner. Their IC50 values following 7‐day exposure were 22.3 and 0.40 μg/ml, respectively. Following the subsequent 7‐day exposure, from day 7 to day 14 after preparation of the suspension, SM‐11355/Lipiodol showed an almost equivalent activity, but CDDP/Lipiodol did not show any activity at all. SM‐11355/Lipiodol showed a sustained release into the culture medium over the course of a 14‐day exposure. Following the exposure to CDDP/Lipiodol, the platinum concentration in the medium was at its maximum on the first day and remained constant thereafter. Intracellular platinum uptake and formation of platinum‐DNA adducts were dependent on the release characteristics of each drug suspension. For SM‐11355/Lipiodol, the drug release, intracellular drug uptake, and formation of platinum‐DNA adducts over the course of the subsequent 7‐day exposure were similar to those observed during the first 7 days. DPC, one of the compounds released from SM‐11355/Lipiodol, was taken up by cells and showed formation of platinum‐DNA adducts. Thus, this study suggests that SM‐11355/Lipiodol may release active platinum compound(s) that bind to nuclear DNA and mediate the cytotoxic activity of SM‐11355/Lipiodol.
Apoptosis in Human Hepatoma Cell Line SMMC‐7721 Induced by Water‐soluble Macromolecular Components of Artemisia capillaris Thunberg
Authors: Hu Yi Qiao , Tan Ren Xiang , Chu Ming Yian , Zhou Jing
Published: August 2005

The aim of this study was to investigate the effect of water‐soluble macromolecular components of Artemisia capillaris Thunberg (ACT) on human hepatoma cell line SMMC‐7721 (SMMC‐7721). The morphological changes of SMMC‐7721 were observed under a light microscope and an electron microscope. Inhibition of proliferation was measured with a colorimetric MTT assay. It was discovered that ACT extract‐treated cells exhibit morphological changes typical of apoptosis, including condensed chromatin and a reduction in volume. ACT extract at 25–200 μg/ml dosedependently inhibited the proliferation of SMMC‐7721. The 50% effective dose, evaluated on day 3 of exposure to the extract, was 64.52±3.53 μg/ml. Upon gel electrophoresis, the fragmented DNA showed a characteristic ladder pattern. Cell cycle analyses revealed that ACT induced cell cycle arrest at the G0/G1 phase.
Targeting Chemotherapy to Solid Tumors with Long‐circulating Thermosensitive Liposomes and Local Hyperthermia
Authors: Ishida Osamu , Maruyama Kazuo , Yanagie Hironobu , Eriguchi Masazumi , Iwatsuru Motoharu
Published: August 2005

The effectiveness of the combination of long‐circulating, thermosensitive liposomes and hyperthermia is described. Small‐sized, thermosensitive liposomes that encapsulate doxorubicin (DXR‐PEGTSL (SUV)) have a prolonged circulation time and are extravasated to targeted solid tumors in vivo, where they preferentially release the agent in an anatomical site subjected to local hyperthermia. Liposomes were prepared by the incorporation of amphipathic polyethyleneglycol (PEG) to prolong their circulation time. DXR‐PEG‐TSL (SUV) was retained longest and was accumulated most efficiently in solid tumors in Balb/c mice. The combination of DXR‐PEG‐TSL (SUV) and hyperthermia at the tumor sites 3 h after injection, gave high concentrations of doxorubicin in tumor tissue and resulted in more effective tumor retardation and increased survival time. A large amount of DXR‐PEG‐TSL (SUV) was extravasated into the tumors during circulation for 3 h after injection, suggesting that the encapsulated drug was released into the interstitial spaces of the lesions by local hyperthermia. This system is expected to be clinically valuable for the delivery of a wide range of chemotherapeutic agents in the treatment of solid tumors.
Detection of Apoptosis and Expression of Apoptosis‐associated Proteins as Early Predictors of Prognosis after Irradiation Therapy in Stage IIIb Uterine Cervical Cancer
Authors: Yuki Hiroyoshi , Fujimura Masaki , Yamakawa Yoshihiro , Hidaka Takao , Saito Shigeru
Published: August 2005

We investigated the proportion of apoptotic cells and the expression of apoptosis‐associated proteins after the delivery of the first week of irradiation for stage IIIb uterine cervical cancer. Thirty patients with stage IIIb squamous cell carcinoma of the uterine cervix who received only irradiation therapy were registered in this study. Specimens were obtained before irradiation therapy and at the end of the first week of irradiation. The apoptotic index (AI) of each tissue specimen was calculated by counting the apoptotic cells and expressed as a percentage. Immunohistochemical evaluation for apoptosis‐related proteins, p53, Bcl‐2, Bax, caspase‐1 and caspase‐3 was also performed. The AI was 0.8±0.9% (mean±SD) before irradiation and 1.7±1.3% at the end of the first week of irradiation. We observed that the patients who survived more than 5 years had AI levels of 2.1±1.3% at the end of their first week of therapy. This rate was significantly higher than the rate of 1.1±0.8% (P=0.02) of the patients who died within 5 years. When the cut‐off value of the AI was set at 1.7%, the sensitivity, specificity, positive predictive value, and negative predictive value for the prediction of patients' prognosis after irradiation therapy were 73.4%, 72.4%, 82.4%, and 61.5%, respectively. In 17 of the AI‐positive cases, expressions of Bax (P=0.006), caspase‐1 (P=0.045), and caspase‐3 (P=0.013) at the end of the first week were significantly higher than before irradiation. The proportion of apoptotic cells and the expression of apoptosis‐associated proteins, Bax, caspase‐1, and caspase‐3, at the end of the first week of irradiation could be useful predictors of the prognosis in stage IIIb squamous cell carcinoma of the uterine cervix treated by irradiation therapy.
Isolation of Differentiated Squamous and Undifferentiated Spindle Carcinoma Cell Lines with Differing Metastatic Potential from a 4‐Nitroquinoline N‐Oxide‐induced Tongue Carcinoma in a F344 Rat
Authors: Takeuchi Shinichi , Nakanishi Hayao , Yoshida Kenji , Yamamoto Shinji , Tonoki Hidefumi , Tsukamoto Takahisa , Fukushima Shoji , Moriuchi Tetsuya , Kurita Kenichi , Tatematsu Masae
Published: August 2005

One differentiated squamous cell carcinoma (SCC) cell line (RSC3‐E2) and two undifferentiated tumor cell lines (RSC3‐LM and RSC3‐E2R) with different metastatic potential were established from a 4‐nitroquinoline N‐oxide (4NQO)‐induced differentiated SCC in F344 rat tongue. The RSC3‐E2 subline was isolated from a parental cell line (RSC3‐P) by single cell cloning in vitro, whereas the RSC3‐LM subline was isolated from a lung metastatic focus after subcutaneous (s.c.) injection of RSC3‐P cells. The RSC3‐E2R cell line was isolated from a lung metastatic focus following s.c. injection of RSC3‐E2 cells after X‐irradiation in vitro. The RSC3‐E2 cell line is keratinpositive and grows as a keratinizing tumor in nude mice, whereas RSC3‐LM and RSC3‐E2R cells are keratin‐negative, vimentin‐positive and form undifferentiated tumors. When s.c. injected into nude mice, the RSC3‐E2 cell line proved to be non‐metastatic, while the RSC3‐LM cell line was metastatic by both hematogenous and lymphogenous routes, and the RSC3‐E2R cell line was metastatic only hematogenously. In vitro relative growth rates and in vitro invasion activity of these cell lines were in the order RSC3‐LM>RSC3‐E2R>RSC3‐E2. Chromosome analysis revealed two peaks with modal chromosome numbers of 83 and 78 for RSC3‐P cells and single peaks at 83, 78 and 56 for RSC3‐LM, RSC3‐E2 and RSC3‐E2R cell lines, respectively. Common structural abnormalities on chromosome 11 were shared by all cell lines. Mutation analysis of the p53 gene using a yeast functional assay demonstrated RSC3‐LM cell line to have a point mutation at codon 269, whereas RSC3‐E2 and RSC3‐E2R had double mutations at codons 106 and 170 on each allele. These results suggest that the two undifferentiated RSC3‐LM and RSC3‐E2R tumor cell lines with different metastatic potential were generated from differentiated SCC cells via different genetic pathways as a consequence of tumor progression in vivo and in vitro, respectively. These cell lines should provide a useful model for understanding mechanisms of hematogenous and lymphogenous metastasis, as well as tumor progression of oral SCCs.
Critical Role of Extracellular Signal‐regulated Kinase (ERK) Phosphorylation in Novel Vitamin K Analog‐induced Cell Death
Authors: Osada Shinji , Carr Brian I.
Published: August 2005

In the present study, we show that 2‐(2‐hydroxyethylsulfaryl)‐3‐methyl‐1,4‐naphthoquinone, or CPD 5, is a potent growth inhibitor for pancreas cancer cell lines (ID50: 21.4±3.8, 31.8±2.7 and 55.2±4.5 μM for MiaPaCa, Panc‐1 and BxPc3, respectively). It induced protein tyrosine phosphorylation of hepatocyte growth factor (HGF) receptor (c‐Met) or epidermal growth factor receptor (EGFR), which increased progressively to a maximum level at 30 min in Panc‐1 cells. The receptor phosphorylation by CPD 5 was indicated to be functional, since these receptors were found to bind with Grb2 or SOS1 protein. CPD 5 was also suggested to induce phosphorylation of external signal‐regulated kinase (ERK). EGF induced cell proliferation through ERK phosphorylation, since U0126, which is an inhibitor of ERK phosphorylation, abrogated the increase of cyclin D1 by EGF. HGF increased the amount of p27 protein, suggesting that it is associated with cell differentiation. By contrast, U0126 reduced CPD 5‐induced cell death. On two‐dimensional electrophoresis, we found an extra type of phospho‐ERK, and this was completely and selectively abolished by U0126. These results suggest that ERK phosphorylation, especially the extra spot on two‐dimensional gel, is critically associated with CPD 5‐mediated cell death.
Mitochondrial Lipid Peroxides and Antioxidant Enzymes in Colorectal Adenocarcinoma Tissues
Authors: Kanbağh Öznur , Özdemirler Gül , Bulut Türker , Yamaner Sümer , Aykaç‐Toker Gülçin , Uysal Müjdat
Published: August 2005

Lipid peroxide levels and superoxide dismutase (SOD), glutathione peroxidase (GSH‐Px), and glutathione transferase (GST) activities were investigated in mitochondrial fractions obtained from tumorous and nontumorous colorectal tissues of fourteen patients with colon and rectum cancer. Histopathological evaluations, including type, stage, necrosis and lymphocyte infiltration were also performed for each patient. The activities of SOD, GSH‐Px and GST were increased significantly, but lipid peroxide levels remained unchanged in mitochondria obtained from tumors compared to adjacent normal tissues of subjects with colorectal cancer. When the patients were grouped according to their histopathological evaluation, such as type, stage, necrosis and lymphocyte infiltration, no relationship was observed between the histopathological results and the mitochondrial lipid peroxidation or antioxidant enzyme activities.
Bax Induction Activates Apoptotic Cascade via Mitochondrial Cytochrome c Release and Bax Overexpression Enhances Apoptosis Induced by Chemotherapeutic Agents in DLD‐1 Colon Cancer Cells
Authors: Kobayashi Tohru , Sawa Hidehiko , Morikawa Jun , Zhang Wei , Shiku Hiroshi
Published: August 2005

Cancer cells express different levels of apoptosis‐promoting Bax protein. The present study evaluated whether induction of Bax initiates apoptosis and whether Bax overexpression enhances apoptosis induced by several chemotherapeutic agents in DLD‐1 colon cancer cells, which originally express a high level of endogenous Bax protein and a low level of Bcl‐2 protein. To investigate these two points, parental DLD‐1 cells were transfected with the Tet‐On Bax induction system (pTet‐On and pTRE‐Bax plasmids), and stable transduced cells were obtained. Induction of Bax by the Tet‐On system initiated cytochrome c release from mitochondria, caspase‐3 activation, and apoptosis to some extent in DLD‐1 cells. Apoptosis induced by a chemotherapeutic agent, 5‐fluorouracil, mitomycin C, paclitaxel, doxorubicin, or cisplatin, was enhanced by Bax overexpression. These findings suggest that Bax‐overexpression‐based gene therapy combined with chemotherapy would be effective in the treatment of colon cancer.
Abnormal Intracellular Localization of Bax with a Normal Membrane Anchor Domain in Human Lung Cancer Cell Lines
Authors: Salah‐eldin Alaa‐eldin , Inoue Shoichi , Tsuda Masumi , Matsuura Akihiro
Published: August 2005

Proapoptotic Bax is a member of the Bcl‐2 family proteins, which have a key role in regulating programmed cell death. The intracellular localization and redistribution of Bax are important in promoting apoptosis. Bax contains a BH3 domain heterodimerizing with Bcl‐2 and a hydrophobic transmembrane segment to be inserted in specified organelle membranes. In this study, Bcl‐2 showed cytoplasmic localization in all of ten human lung cancer cell lines tested. Interestingly, Bax was localized in the nucleus in 7 cell lines, although Bax lacks nuclear import signals. This may allow cancer cells to escape from apoptosis. Why Bax is able to exist in the nucleus is still unclear. We hypothesized that mutation in the BH3 domain and/or transmembrane segment of Bax possibly causes intracellular Bax distribution. We analyzed the sequence of the bax gene in these cell lines and found only a silent point mutation at codon 184 (TCG → TCA) in the transmembrane segment in all cell lines. This finding indicates that changes in cellular localization of Bax in lung cancer cell lines do not depend on bax mutation and that Bax is possibly translocated into the nucleus without any mutation. This is the first report showing that Bax with the normal amino acid sequence can be localized in the nucleus in established lung cancer cell lines without any treatment of the cells.
Oncolytic Viral Therapy for Human Prostate Cancer by Conditionally Replicating Herpes Simplex Virus 1 Vector G207
Authors: Oyama Masafumi , Ohigashi Takashi , Hoshi Michio , Murai Masaru , Uyemura Keiichi , Yazaki Takahito
Published: August 2005

Over the last few years, a conditionally replicating herpes simplex virus 1 (HSV‐1) vector, G207 has been used for the treatment of several malignant tumors. In this article we evaluate the antitumoral effect of G207 against prostate cancer in vitro and in vivo. The susceptibility of the human prostate cancer cell lines, DU145 and PC3 to G207 at a multiplicity of infection (MOI) of 0.1 was examined. In addition, the growth characteristics of G207 were assessed. Athymic mice with s.c. tumors were inoculated in vivo intraneoplastically with 1x107 plaque‐forming units (PFU) of G207. For the pathological analyses, s.c. tumors were stained with X‐gal. DU145 and PC3 were efficiently destroyed by G207 within 7 days. The viral yields of G207 increased time‐dependently. In vivo, the intraneoplastic inoculation of G207 induced a significant inhibition of the tumor growth. The mean tumor growth ratio was significantly inhibited in the G207‐treated tumors (DU145, P< 0.0001; PC3, P < 0.001 versus controls). In a pathological study, many lacZ‐positive cells were diffusely present in the G207‐treated tumors. G207 showed a significant antitumoral effect against human prostate cancer cell lines, and thus may be considered a useful agent for the treatment of prostate cancer.
Atypical Osteosarcomas in Werner Syndrome (Adult Progeria)
Authors: Ishikawa Yuichi , Miller Robert W. , Machinami Rikuo , Sugano Haruo , Goto Makoto
Published: August 2005

Werner syndrome (WS), adult progeria, is more common in Japan than elsewhere. It predisposes to osteosarcoma (OS) and five other rare tumors. To determine if and how OS is atypical in this genetic disorder, we studied the characteristics of ten Japanese cases with respect to clinical features, pathology, and radiographs, and compared them with a hospital series of 36 skeletal OS with the same atypical age‐range, 35–57 years. The anatomic sites were also atypical: seven ankle/foot, two radius and one patella compared with only one at the ankle in the hospital series. The osteoblastic cell‐type was about equally frequent in both series, but, among others than the three major subtypes, there was only one in WS as compared with 14 (39%) in the hospital series. The types of mutations were sought in five WS cases with OS. One showed no mutation at any of the ten known loci for Japanese, two were of type 4/4 and two of type 6/6. The mutations 4 and 6 have been found in 66% of alleles of WS cases in Japan. The increased frequency and unusual age and site distributions of OS in WS may be due to increased susceptibility, related to later‐life leg ulcers, and weight‐bearing on spindly ankles weakened by severe loss of lower limb subcutaneous tissue.
p53‐independent Induction of p21 (WAF1/CIP1), Reduction of Cyclin B1 and G2/M Arrest by the Isoflavone Genistein in Human Prostate Carcinoma Cells
Authors: Choi Yung Hyun , Lee Won Ho , Park Kun‐Young , Zhang Lijuan
Published: August 2005

Genistein, a natural isoflavonoid phytoestrogen, is a strong inhibitor of protein tyrosine kinase and DNA topoisomerase II activities. Genistein has been shown to have anticancer proliferation, differentiation and chemopreventive effects. In the present study, we have addressed the mechanism of action by which genistein suppressed the proliferation of p53‐null human prostate carcinoma cells. Genistein significantly inhibited the cell growth, which effect was reversible, and induced dendritelike structure. The inhibitory effects of genistein on cell growth proliferation were associated with a G2/M arrest in cell cycle progression concomitant with a marked inhibition of cyclin B1 and an induction of Cdk inhibitor p21 (WAF1/CIP1) in a p53‐independent manner. Following genistein treatment of cells, an increased binding of p21 with Cdk2 and Cdc2 paralleled a significant decrease in Cdc2 and Cdk2 kinase activity with no change in Cdk2 and Cdc2 expression. Genistein also induced the activation of a p21 promoter reporter construct, utilizing a sequence distinct from the p53‐binding site. Analysis of deletion constructs of the p21 promoter indicated that the response to genistein could be localized to the 300 base pairs proximal to the transcription start site. These data suggest that genistein may exert a strong anticarcinogenic effect, and that this effect possibly involves an induction of p21, which inhibits the threshold kinase activities of Cdks and associated cyclins, leading to a G2/M arrest in the cell cycle progression.
Adenovirus‐mediated Transfer of p53‐related Genes Induces Apoptosis of Human Cancer Cells
Authors: Ishida Setsuko , Yamashita Toshiharu , Nakaya Uichiro , Tokino Takashi
Published: August 2005

Two p53‐related genes, p73 and p51, were recently identified as structural homologues of the p53 tumor suppressor gene, suggesting that the roles of these two genes may be similar to those of p53, including growth suppression and induction of apoptosis. Here we show that introduction of p73 or p51 cDNAs into cultured human cancer cells suppressed colony formation in the presence of G418. We then examined the ability of various isoforms of p73 and p51 to activate transcription of a reporter gene. This assay showed that p73 β and p51A activated transcription through a consensus p53 binding sequence, while p73 α and p51B isoforms minimally transactivated the p53 reporter gene. To characterize further the biological functions of the p53‐related genes, we constructed recombinant adenoviruses containing the p73 and p51 cDNAs. Ad‐p73 β and Ad‐p51A induced endogenous p21 gene expression more effectively than Ad‐p73 β and Ad‐p51B, respectively. To evaluate the mode of cell death induced by p53‐related genes, Ad‐p73 and Ad‐p51 were used to infect human cancer cells. Infection of Ad‐p73 β, Ad‐p51A or Ad‐p51B resulted in DNA fragmentation in a subset of cancer cell lines more efficiently than did infection of Ad‐p53. We then examined the combined effect of each p53‐related gene and the E1A oncogene in the induction of apoptosis. The E1A oncogene cooperated with p51 as well as p53 to induce apoptosis, while p73 resulted in a weak induction of apoptosis by E1A. Overall, apoptosis induction by p51B and p73 α isoforms may be due to mechanisms other than transcriptional activation of p53‐target genes. Our results suggest that p53‐related genes are both similar to and different from p53 in their pathways leading to growth suppression.
Mouse Homologue of the Human SART3 Gene Encoding Tumor‐rejection Antigen
Authors: Harada Kenji , Yamada Akira , Mine Takashi , Kawagoe Nobutoshi , Takasu Hideo , Itoh Kyogo
Published: August 2005

We recently isolated a human SART3 (hSART3) gene encoding a tumor‐rejection antigen recognized by HLA‐A2402‐restricted cytotoxic T lymphocytes (CTLs). The hSART3 was also found to exist as an RNA‐binding nuclear protein of unknown biological function. In this study, we cloned and analyzed the homologous mouse SART3 (mSART3) gene in order to understand better the function of hSART3, and to aid in establishing animal models of specific immunotherapy. The cloned 3586‐bp cDNA encoded a 962‐amino acid polypeptide with high homology to hSART3 (80% or 86% identity at the nucleotide or protein level, respectively). Nonapeptides recognized by the HLA‐A2402‐restricted CTLs and all of the RNA‐binding motifs were conserved between hSART3 and mSART3. The mSART3 mRNA was ubiquitously expressed in normal tissues, with low level expression in the liver, heart, and skeletal muscle. It was widely expressed in various organs from as early as day 7 of gestation. mSART3 was mapped to chromosome 5, a syntenic region for human chromosome 12q23–24, and its genomic DNA extended over 28‐kb and consisted of 19 exons. This information should be important for studies of the biological functions of the SART3 protein and for the establishment of animal models of specific cancer immunotherapy.
The Novel Anticancer Drug KRN5500 Interacts with, but is Hardly Transported by, Human P‐Glycoprotein
Authors: Takara Kohji , Tanigawara Yusuke , Komada Fusao , Nishiguchi Kohshi , Sakaeda Toshiyuki , Okumura Katsuhiko
Published: August 2005

The interaction of the novel anticancer drug KRN5500, a spicamycin derivative, with human Pglycoprotein (P‐gp) was analyzed from the viewpoint of cellular pharmacokinetics, i.e. by means of [3H]azidopine photoaffinity labeling, cellular accumulation and transcellular transport experiments. In this study, P‐gp‐overexpressing LLC‐GA5‐COL150 cells, porcine kidney epithelial LLC‐PK1 cells transformed with human MDR1 cDNA, were used, since this cell line constructs monolayers with tight junctions, and would provide sufficient information for analyzing the cellular pharmacokinetics. 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay revealed that the growth‐inhibitory effect of KRN5500 in LLC‐GA5‐COL150 cells was comparable to that in LLC‐PK1 cells (IC50= 79.4 and 72.7 nM, respectively), but the inhibition of [3H]azidopine binding by KRN5500 was concentration‐dependent in the membrane fraction of LLC‐GA5‐COL150 cells. The cellular accumulation of [14C]KRN5500 after its basal application in LLC‐GA5‐COL150 cells was slightly lower than that in LLC‐PK1 cells, and was restored by the multidrug resistance (MDR) modulator SDZ PSC 833. The basal‐to‐apical transport of [14C]KRN5500 in LLC‐GA5‐COL150 cells was also slightly higher than that in LLC‐PK1 cells, and was inhibited by SDZ PSC 833. However, the basal‐to‐apical transport of [14C]KRN5500 in LLC‐GA5‐COL150 cells was only a little higher than the apical‐to‐basal transport. Consequently, these results demonstrated that KRN5500 interacted with, but was hardly transported via, P‐gp. These observations suggested that KRN5500 may be useful even for the treatment of tumors exhibiting P‐gp‐mediated MDR.
Sonodynamically Induced Antitumor Effect of 4‐Formyloximethylidene‐3‐Hydroxy‐2‐vinyl‐deuterio‐porphynyl(IX)‐6,7‐diaspartic Acid (ATX‐S10)
Authors: Yumita Nagahiko , Nishigaki Ryuichiro , Sakata Isao , Nakajima Susumu , Umemura Shin‐ichiro
Published: August 2005

The sonodynamically induced antitumor effect of 4‐formyloximethylidene‐3‐hydroxy‐2‐vinyl‐deuterio‐porphynyl(IX)‐6,7‐diaspartic acid (ATX‐S10) was investigated. Both in vitro and in vivo antitumor effects were tested in combination with ultrasound at 2 MHz. The rate of ultrasonically induced damage to isolated sarcoma 180 cells in air‐saturated suspension was enhanced two‐fold with 80 μM ATX‐S10. This enhancement was significantly inhibited by histidine, which may suggest that it was mediated by ultrasonically induced oxidation. The coadministraion of 25 mg/kg ATX‐S10 followed by ultrasonic exposure at 2 MHz stopped the growth of implanted colon 26 tumors at an intensity at which ultrasound alone showed only a slight antitumor effect.
Enhancer‐promoter Activity of Human Papillomavirus Type 16 Long Control Regions Isolated from Cell Lines SiHa and CaSki and Cervical Cancer Biopsies
Authors: Kozuka Takuyo , Aoki Yukimasa , Nakagawa Keiichi , Ohtomo Kuni , Yoshikawa Hiroyuki , Matsumoto Koji , Yoshiike Kunito , Kanda Tadahito
Published: August 2005

Expression of human papillomavirus 16 (HPV‐16) oncogenes is markedly higher in cervical cancer cells than in precancerous cells, and the elevated expression is believed to be required for the malignant phenotypes. We compared cancer cell lines CaSki (with 200 to 400 copies of HPV‐16 DNA per cell) and SiHa (with one to two copies of HPV‐16 DNA per cell) for the E7 expression in cells and the enhancer‐promoter activity of the isolated viral long control region (LCR). Although these parameters per cell were 10‐fold higher in CaSki than in SiHa, the levels of the E7 mRNA and protein per HPV DNA copy were 10‐ to 20‐fold higher in SiHa than in CaSki. Characterization of the isolated LCRs showed that, whereas the LCR from CaSki resembled the prototype in structure and activity, the LCR from SiHa, with a deletion of 38 base pairs, enhanced transcription from P97 as assayed by using a plasmid capable of expressing luciferase. The upregulation appeared to be due to removal of one of the silencer YY1‐binding sites. Furthermore, we isolated and characterized LCRs from 51 cervical cancer patients’ biopsies. Among them, one with a deletion including YY1‐binding sites and the other with a substitution in a YY1‐motif were found to enhance the transcription. These findings suggest that mutation affecting YY1‐motifs in the LCR is one of the mechanisms enhancing the viral oncogene expression in the course of progression of cancer cells.
Microsatellite Instability and k‐ras, p53 Mutations in Thyroid Lymphoma
Authors: Takakuwa Tetsuya , Hongyo Tadashi , Syaifudin Mukh , Kanno Hiroyuki , Matsuzuka Fumio , Narabayashi Isamu , Nomura Taisei , Aozasa Katsuyuki
Published: August 2005

Patho‐epidemiological studies showed that thyroid lymphoma (TL) arises in inflammatory lesions of chronic lymphocytic thyroiditis (CLTH). Replication error (RER) is found in inflammatory lesions and associated cancer, suggesting that chronic inflammation could be a risk factor for neoplastic development through causing RER. To clarify whether RER is involved in the pathogenesis of TL, we examined the microsatellite instability (MSI) in 9 cases with CLTH and 19 with TL, including 10 diffuse large B‐cell lymphoma (DLBL), 4 follicle center cell lymphoma, 3 marginal zone B‐cell lymphoma of extranodal (MALT) type, and 2 lymphoplasmacytic type. Sixteen distinct microsatellite repeats were analyzed. Mutations of p53 and k‐ras genes were also examined. When alterations at 2 or more microsatellite loci were judged as positive, only 5 DLBL cases exhibited MSI. The frequency of MSI in DLBL was significantly higher than that in other types of TL and CLTH (P < 0.05). Four of 19 cases (21.1%) showed point mutation of the k‐ras gene. The k‐ras mutations occurred in the cases with DLBL with RER, and four of five cases with RER had a k‐ras mutation, indicating a close association between RER and k‐ras mutation. p53 mutations were not found in the CLTH. Two of 19 TL cases showed mutations of p53 gene. There was no significant association between RER and p53 mutation. These findings indicate that genomic instability contributes to the progression of TL from low grade to high grade, but not to the development of low grade lymphoma in CLTH lesions.
PTEN/MMAC1 Mutation and Frequent Loss of Heterozygosity Identified in Chromosome 10q in a Subset of Hepatocellular Carcinomas
Authors: Fujiwara Yoshiyuki , Hoon Dave S. B. , Yamada Terumasa , Umeshita Koji , Gotoh Mitsukazu , Sakon Masato , Nishisho Isamu , Monden Morito
Published: August 2005

Frequent allelic losses on chromosome 10q have been reported in several types of cancers, suggesting the presence of a putative tumor suppressor gene(s) on the chromosomal arm. We examined loss of heterozygosity (LOH) on chromosome 10q in 37 hepatocellular carcinomas (HCC) using eleven dinucleotide microsatellite markers, spanning the entire chromosome arm of 10q. Twelve (32%) out of 37 informative cases showed allelic losses of at least one locus on 10q and eight tumors showed a partial deletion of 10q. Analysis of deletion mapping of these eight cases identified two commonly deleted regions within the distal part of 10q (10q24‐q26), a 20‐cM interval flanked by D10S597 and D10S216 and a 24‐cM interval flanked by D10S216 and D10S590. Moreover, we detected a somatic missense mutation (Met→Val) of a candidate tumor suppressor gene PTEN/MMAC1, located at 10q23.3, in one HCC with LOH of 10q. Our findings indicated the presence of putative tumor suppressor gene(s) in the distal region of 10q that might be involved in the development and progression of HCC. Inactivation of PTEN/MMAC1 gene located outside the commonly deleted region of 10q might also play an important role in a subset of HCCs.
Mapping of Target Regions of Allelic Loss in Primary Breast Cancers to 1‐cM Intervals on Genomic Contigs at 6q21 and 6q25.3
Authors: Utada Yoshihito , Haga Shunsuke , Kajiwara Tetsuro , Kasumi Fujio , Sakamoto Goi , Nakamura Yusuke , Emi Mitsuru
Published: August 2005

Allelic losses on the long arm of human chromosome 6 are frequently observed in cancers of the ovary, prostate, and breast. To identify the locations of putative tumor suppressor genes on 6q, we examined 192 primary breast cancers for patterns of allelic loss at 16 polymorphic microsatellite loci distributed along this chromosome arm. Allelic losses at one or more loci were observed in 105 (55%) of the tumors examined. Detailed deletion mapping with appropriate yeast artificial chromosome (YAC) contigs identified two distinct commonly deleted regions; one was confined to a 1‐cM interval at 6q21 flanked by D6S1040 and D6S262 and the other to a 1‐cM interval at 6q25.3 flanked by D6S305 and D6S411. Allelic losses at 6q21 were more frequent in invasive solid tubular and scirrhous carcinomas than in tumors of less aggressive histologic types (P=0.0006). Allelic loss at 6q25.3 was associated with loss of progesterone receptor (P=0.0256). Our results suggest the presence of two tumor suppressor genes for breast cancer on 6q that are likely to be associated with tumor progression and/or loss of hormonal dependency.
Helicobacter pylori and the t(11;18)(q21;q21) Translocation in Gastric Low‐grade B‐Cell Lymphoma of Mucosa‐associated Lymphoid Tissue Type
Authors: Nakamura Tsuneya , Nakamura Shigeo , Yonezumi Masakatsu , Suzuki Takashi , Matsuura Akira , Yatabe Yasushi , Yokoi Takio , Ohashi Kazuhiko , Seto Masao
Published: August 2005

The reported regression of mucosa‐associated lymphoid tissue (MALT) type gastric low‐grade Bcell lymphoma following treatment for Helicobacter pylori (H. pylori) infection has not yet been comprehensively analyzed, especially in relation to the recently identified c‐IAP2‐MALT1/MLT gene alteration resulting from the t(11;18)(q21;q21) chromosomal translocation found in MALT lymphoma. The relationship between MALT lymphomas and H. pylori was investigated in 30 patients who received an antibacterial treatment. Patients were followed up by means of endoscopy and biopsy. Molecular genetic analyses focused on the presence or absence of the immunoglobulin heavy chain (IgH) gene and/or MALT1/MLT gene alteration resulting from t(11;18)(q21;q21) translocation. H. pylori was positive in 26 of the 30 patients. The overall success rate of cure of H. pylori infection was 96% (25/26). Thirteen patients (52%) showed complete remission (CR) of lymphoma, nine (36%) partial remission (PR), and three (12%) registered no change (NC). Statistical analysis revealed significant differences between CR and PR/NC patients in age (< 60 or 60), in lymphoma location (single or multiple sites) and in the presence or absence of gene rearrangement before eradication (P< 0.05). Endoscopy showed a cobblestone appearance only in PR cases and polypoid features predominantly in NC cases. Two NC patients with polypoid gross appearance showed rearrangements involving either c‐IAP2 or MALT1 gene in Southern blot analysis, while none of seven other resected patients with non‐polypoid superficial gross appearance showed rearrangement. Gastric MALT lymphoma could be pragmatically subdivided into three groups, CR (MALT‐A), PR (MALT‐B), and NC (MALT‐C) on the basis of the reaction to eradication of H.pylori. We speculate that MALT‐A may represent an incipient neoplasm or dysplasia, MALT‐B a neoplasm activated by antigenic stimulation of H. pylori, and MALT‐C a lymphoma independent of H. pylori. Polypoid lesions in MALT‐C were associated with c‐IAP2‐MALT1/MLT gene alteration resulting from t(11;18)(q21;q21). This classification is thought to be clinically significant for deciding the most appropriate mode of treatment of MALT‐type lymphoproliferative disorders.
Expression of Glutathione S‐Transfer α, P1–1 and T1–1 in the Human Gastrointestinal Tract
Authors: de Bruin Wieke C. C. , Wagenmans Muriel J. M. , Peters Wilbert H. M.
Published: August 2005

Glutathione S‐transferases (GSTs) form a family of enzymes, which play an important role in the prevention of cancer by detoxifying numerous potentially carcinogenic compounds. GSTs catalyze the conjugation of glutathione to such harmful molecules, and enable their secretion. Human GSTs can be divided into five main classes. The θ class of isoenzymes was only recently identified and limited (immunohistochemical) data on these enzymes are available. In the present study, paraffinembedded sections of different gastrointestinal tissues were analyzed immunohistochemically for GSTα, GSTP1–1 and GSTT1–1 expression using specific antibodies. GSTα, GSTP1–1 and GSTT1–1 were highly expressed in all gastrointestinal tissues examined, with a unique cellular distribution. GSTT1–1 is the first GST isoenzyme demonstrated in duodenal Paneth cells and glands of Brunner. The common expression of GSTα, GSTT1–1 and GSTP1–1 in many cell types along the human gastrointestinal tract suggests an important role in the protection against carcinogens and other xenobiotics.
Increased Expression after X‐Irradiation of MUC1 in Cultured Human Colon Carcinoma HT‐29 Cells
Authors: Kang Yun , Hirano Kazuya , Suzuki Norio , Enomoto Atsushi , Morita Akinori , Irimura Tatsuro , Sakai Kazuo
Published: August 2005

The effect of X‐irradiation on production of MUC1 was studied with human colon carcinoma HT‐29 cells. As evaluated by immunocytochemical staining, the percentages of MUC1‐positive cells in cells at 4 days after 6 Gy irradiation and in unirradiated control cells were 52±3.5% (n=6) and 26±2.8% (n=6), respectively. Flow‐cytometric analysis of living cells showed that MUC1 began to rise from day 1, reaching a plateau by day 4 after 6 Gy irradiation. Western blot analysis with monoclonal antibody MY.1E12 against glycosylated MUC1 (mature form) showed dose‐dependent increases of two bands (500 and 390 kDa) corresponding to two polymorphic MUC1 alleles. Premature forms of MUC1 (350 and 240 kDa) were detectable with monoclonal antibody HMFG‐2 only in irradiated cells, suggesting that new core protein synthesis had been induced. The transcriptional activity of the MUC1 gene was analyzed in terms of transient expression of MUC1‐CAT reporter plasmids containing 5′‐flanking sequences of the MUC1 gene fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. The results of CAT assay indicate that enhanced expression of MUC1 in irradiated HT‐29 cells was due to upregulation of MUC1 transcription, and required the upstream promoter.
Significance of Thymidine Phosphorylase/Platelet‐derived Endothelial Cell Growth Factor in Carcinoma of the Papilla of Vater
Authors: Zhao Bin , Kimura Wataru , Futakawa Noriaki , Abe Hideki , Kitayama Joji , Nagawa Hirokazu , Makuuchi Masatoshi
Published: August 2005

The expression of thymidine phosphorylase (TP) in carcinoma of the papilla of Vater was studied to clarify its significance in tumor progression and in determining prognosis. Fifty‐nine cases of surgically resected carcinoma of the papilla of Vater were studied. Immunohistochemical staining was performed to evaluate the expression of TP, microvessel count and p53 overexpression. TP expression was demonstrated in tumor cells in 62.7% (37/59) of the cases. A higher frequency of regional lymph node metastasis was found in TP‐positive tumors than in TP‐negative tumors (P=0.006). TP‐positive tumors were more advanced than TP‐negative tumors with regard to clinical stage (P=0.035). TP‐positive tumors had significantly higher microvessel density (27.6±10.1) than TP‐negative tumors (20.4±10.0, P=0.01). Moreover, TP expression was significantly correlated with a poor prognosis (P=0.02). These suggest that in carcinoma of the papilla of Vater, TP production by tumor cells is correlated with tumor progression through its regulatory effect on neovascularization.
Expression of SART3 Tumor‐rejection Antigen in Gastric Cancers
Authors: Niiya Fumihiko , Nishizaka Shinya , Matsunaga Kazuko , Koufuji Kikuo , Mori Masaki , Katai Hitoshi , Yamana Hideaki , Itoh Kyogo
Published: August 2005

We previously reported SART3 as a tumor‐rejection antigen recognized by histocompatibility leukocyte antigen (HLA)‐A24‐restricted cytotoxic T lymphocytes (CTLs). In this study, we investigated the expression of the SART3 antigen in gastric cancers, as a candidate for use in specific immunotherapy. The SART3 antigen was detected in 9 of 10 (90%) gastric cancer cell lines, 35 of 52 (67.3%) gastric cancer tissues, and 0 of 20 non‐tumorous gastric tissues. SART3‐derived peptides corresponding to positions 109–118 and 315–323 induced HLA‐A24‐restricted and tumorspecific CTLs from peripheral blood mononuclear cells (PBMCs) of gastric cancer patients. These peptide‐induced CTLs recognized HLA‐A24+ SART3+ gastric cancer cells, but not HLA‐A24+ SART3− or HLA‐A24− SART3+ gastric cancer cells. Therefore, the SART3 peptides could be useful in specific immunotherapy of gastric cancer patients.
Effects of Reproduction on Spontaneous Development of Endometrial Adenocarcinomas and Mammary Tumors in Donryu Rats
Authors: Nagaoka Takaharu , Takegawa Kiyoshi , Takeuchi Masaki , Maekawa Akihiko
Published: August 2005

Effects of reproduction on spontaneous development of uterine endometrial adenocarcinomas and mammary tumors in Donryu rats were investigated. While the incidence of endometrial adenocarcinomas in Donryu rats was not influenced by a single reproductive experience (SRE), it showed a tendency to decrease in animals having three reproductive experiences (TRE), compared to the nulliparous case (NRE). In addition, both SRE and TRE animals showed delayed occurrence and decreased incidences and mean numbers of mammary tumors, along with reduced incidences of proliferative lesions in the pituitary gland and mucinous epithelium in the vagina. The appearancetime and incidences of persistent estrus in TRE rats were delayed and low, respectively, compared to the SRE and NRE values. The hormonal environment was altered in both groups, the prolactin level in TRE especially being decreased. These results suggest that suppression of the occurrence of endometrial adenocarcinomas and mammary tumors in rats experiencing reproduction is associated with change in the hormonal milieu.
Chinese Medicinal Herb, Acanthopanax gracilistylus, Extract Induces Cell Cycle Arrest of Human Tumor Cells in vitro
Authors: Shan Bao En , Zeki Kazuya , Sugiura Tsutomu , Yoshida Yasuhiro , Yamashita Uki
Published: August 2005

We investigated the effect of a Chinese medicinal herb, Acanthopanax gracilistylus (AG), extract (E) on the growth of human tumor cell lines in vitro. AGE markedly inhibited the proliferation of several tumor cell lines such as MT‐2, Raji, HL‐60, TMK‐1 and HSC‐2. The activity was associated with a protein of 60 kDa, which was purified by gel‐filtration chromatography. Cell viability analyses indicated that the treatment with AGE inhibits cell proliferation, but does not induce cell death. The mechanism of AGE‐induced inhibition of tumor cell growth involves arrest of the cell cycle at the G0/G1 Stage without a direct cytotoxic effect. The cell cycle arrest induced by AGE was accompanied by a decrease of phosphorylated retinoblastoma (Rb) protein. Furthermore, cyclin‐dependent kinases 2 and 4 (Cdk2 and Cdk4), which are involved in the phosphorylation of Rb, were also decreased. These results suggest that AGE inhibits tumor cell growth by affecting phosphorylated Rb proteins and Cdks.
Negative p53/Positive p21 Immunostaining Is a Predictor of Favorable Response to Chemotherapy in Patients with Locally Advanced Bladder Cancer
Authors: Koga Fumitaka , Kitahara Satoshi , Arai Kyoko , Honda Mikihiko , Sumi Shuhei , Yoshida Ken‐ichiro
Published: August 2005

The relationship between clinical response to DNA‐damaging drugs and p53 and p21 status in patients with locally advanced transitional cell carcinoma (TCC) of the bladder was assessed. The response to intraarterial chemotherapy (IAC) comprising 100 mg/m2 of cisplatin (CDDP) and 40 mg/m2 of pirarubicin (THP) and the prognosis were assessed in 23 patients (the mean follow‐up period was 19 months). The p53 gene status of tumors was analyzed at exons 5–8 using polymerase chain reaction‐single strand conformation polymorphism analysis in 19 patients, and paraffinembedded tumor sections were immunostained for p53 and p21 in 23 patients. The overall objective response rate (incidence of good responders) was 70%. The negative p53 group (n=17) showed a significantly higher objective response rate than the positive p53 group (n=6) (82% vs. 33%; P=0.045). The p53 gene status or p21 staining status was not significantly associated with responsiveness. When the p53 and p21 immunostaining results were combined, good responders were more accurately predicted than by p53 staining status alone; the negative p53/positive p21 group (n=12) showed an objective response rate of 92%, which was significantly higher than that of the positive p53 and/or negative p21 group (45%, n=11) (P=0.027). Cause‐specific survival of the negative p53 group was significantly superior to that of the positive p53 group (P=0.015). Negative p53/positive p21 immunostaining is a possible predictor of favorable chemotherapeutic response in patients with TCC of the bladder.