bluuebunny/crossref_metadata_2025 huggingface dataset:
p53 Status in Multiple Human Urothelial Cancers: Assessment for Clonality by the Yeast p53 Functional Assay in Combination with p53 Immunohistochemistry
Authors: Yamamoto Shinji , Tada Mitsuhiro , Lee Chyi Chia R. , Masuda Chikayoshi , Wanibuchi Hideki , Yoshimura Rikio , Wada Seiji , Yamamoto Keisuke , Kishimoto Taketoshi , Fukushima Shoji
Published: August 2005

Multifocal synchronous or metachronous tumor development is a common observation in human urothelial cancer cases. However, the underlying mechanism has remained obscure. We have employed a new tool to investigate the p53 gene status, the yeast p53 functional assay, in combination with immunohistochemistry in a total of 50 tumor samples from 32 cases with urothelial cancers, including 8 with multiple synchronous tumor development and 2 demonstrating metachronous tumors. p53 mutations were found in 13 cases (9 with missense mutations, 3 with deletion, 1 with splicing mutation) by the yeast p53 functional assay. p53 protein overexpression was seen in all 9 cases with missense mutations, but in only one of the 4 cases with nonsense mutations. Two tumors without p53 mutation also showed positive p53 immunoreactivity. Overall, p53 abnormalities including mutations and/or protein overexpression were found in 15 (47%) cases. p53 abnormalities were significantly more frequent in non‐papillary and in high grade tumors. Loss of the wild type allele in addition to a p53 mutation was suggested in 8 of the 15 (53%) cases. All 4 cases with mutations in multiple synchronous tumors had identical p53 mutations in the separate urothelial cancers, strongly suggestive of monoclonality. The one case with multiple metachronous tumors, in contrast, was characterized by variation in the p53 status, indicative of different clonal origins. In conclusion, combined assessment for p53 status as used here (yeast p53 functional assay plus immunohistochemistry) may provide insights into the molecular mechanisms of urothelial carcinogenesis.
Mutations in Zinc‐binding Domains of p53 as a Prognostic Marker of Esophageal‐cancer Patients
Authors: Kihara Chikashi , Seki Toyokazu , Furukawa Yoichi , Yamana Hideaki , Kimura Yasutoshi , van Schaardenburgh Pepijn , Hirata Koichi , Nakamura Yusuke
Published: August 2005

Some investigators have suggested that mutations of the p53 gene may be molecular markers for poor prognosis of cancer patients, although others have reported conflicting results. We examined esophageal cancers from 138 patients to investigate whether mutational status of p53 could be correlated either with prognosis or with response to chemotherapy or radiation. We detected p53 mutations in the tumors of 78 (56.5%) patients. Kaplan‐Meier analysis showed that these 78 patients tended to have shorter survival times and greater resistance to either form of therapy than patients whose tumors carried two wild‐type p53 alleles. The difference became more evident when we focused on mutations in zinc‐binding domains of p53 (L2 and L3); the prognosis was significantly poorer among the 29 patients with tumors in this category than among patients whose tumors had no p53 mutations, or p53 mutations outside L2 or L3 (P=0.0060). Moreover, those tumors as a group were more resistant to chemotherapy or radiation than the others (P=0.0105). Our results underscore the importance of the zinc‐binding domains of p53 with respect to clinical prognosis for patients with esophageal carcinomas.
Germ‐line Mutation Analysis in Patients with von Hippel‐Lindau Disease in Japan: An Extended Study of 77 Families
Authors: Yoshida Minoru , Ashida Shingo , Kondo Keiichi , Kobayashi Kazuki , Kanno Hiroshi , Shinohara Nobuo , Shitara Nobuyuki , Kishida Takeshi , Kawakami Satoshi , Baba Masaya , Yamamoto Isao , Hosaka Masahiko , Shuin Taro , Yao Masahiro
Published: August 2005

We have previously reported on the analysis of germ‐line mutations in Japanese von Hippel‐Lindau disease (VHL) patients and found mutations in 26 families. We have now extended these studies to include an additional 41 VHL families. Germ‐line mutation of the VHL, gene was screened by DNA‐SSCP, direct sequencing, and Southern blot analysis. To summarize all of the data we have studied in this and our previous report, germ‐line mutations have been detected in 55 of 77 (73%) (type 1: 41 / 62 (66%) and type 2: 14/15 (93%)) families. We found similarities in the nature of germ‐line mutations including mutational incidence, location, and DNA substitution patterns between Japanese and Western VHL. These similarities may reflect the predominance of endogenous mutational processes. We also found several interesting characteristics in Japanese VHL. Twenty of 41 (49%) intragenic mutations were unique and not reported in the Western VHL. Four mutations (Arg113Stop, Gln132Stop, Leu158Val, and Cys162Tyr) previously characterized as type 1 mutations were identified in the type 2 (with pheochromocytoma) Japanese families. Genotype‐phenotype correlation study suggested non‐missense mutations predicted to result in the loss of VHL function were associated with the occurrence of renal cell carcinoma, as in sporadic tumors. Our data add to the diversity of VHL germ‐line mutations and provide a better understanding of VHL disease in terms of both clinical management and molecular pathogenesis.
Molecular Identification of a Human Carcinoma‐associated Glycoprotein Antigen Recognized by Mouse Monoclonal Antibody FU‐MK‐1
Authors: Tomita Yoshihiro , Arakawa Fumiko , Yamamoto Takafumi , Kuwahara Motohisa , Watanabe Ryoji , Iwasaki Hiroshi , Kikuchi Masahiro , Kuroki Masahide
Published: August 2005

Mouse monoclonal antibody FU‐MK‐1, raised against a human gastric adenocarcinoma, recognizes an antigen (termed MK‐1 antigen) present on the majority of carcinomas. The present study aimed to identify the MK‐1 molecule and to establish its relationship to other carcinoma antigens. Immunoprecipitation studies of human tumor cell lines revealed that FU‐MK‐1 recognizes a monomeric membrane glycoprotein with two forms, 40 kDa (major form) and 42 kDa (minor form), and with a molecular mass of 35 kDa following treatment with the N‐glycosylation inhibitor tunicamycin. The partial amino acid sequence of a main fragment of the MK‐1 molecule obtained by spontaneous cleavage under hypotonic conditions was examined, and the 17 contiguous NH2‐terminal amino acids were found to be identical with residues 81–97 of the 314‐residue GA733–2 protein [Szala et al.; Proc. Natl. Acad. Sci. USA, 87, 3542–3546 (1990)]. Hence, the GA733–2 cDNA was cloned and the specificity of FU‐MK‐1 was confirmed using four recombinant forms of the GA733–2 antigen expressed in COS‐1 cells. Immunoprecipitation with FU‐MK‐1 of the cell lysate transfected with the full‐length GA733–2 cDNA revealed two bands corresponding to those obtained from the tumor cell lines. FU‐MK‐1 also precipitated three other recombinant proteins consisting of amino acids 1–265, 1–201, and 1–139 of the GA733–2 protein, respectively. Furthermore, immunoblotting analysis indicated that FU‐MK‐1 binds to a small fragment (6 kDa) generated from a tumor cell line under hypotonic conditions, suggesting that the FU‐MK‐1 epitope exists on the distal 6‐kDa peptide of the extracellular domain of the GA733–2 molecule. We thus conclude that the MK‐1 antigen is the GA‐733–2 antigen, which is currently being used as a target in clinical trials with monoclonal antibodies.
Tumor‐selective Blood Flow Decrease Induced by an Angiotensin Converting Enzyme Inhibitor, Temocapril Hydrochloride
Authors: Hori Katsuyoshi , Saito Sachiko , Takahashi Hiroto , Sato Haruhiko , Maeda Hiroshi , Sato Yasufumi
Published: August 2005

To enhance chemotherapeutic efficacy against cancer, it is important to deliver anticancer drugs preferentially to cancer cells and to retain the drugs there for a prolonged time. The in vivo prolongation of the exposure time of anticancer drugs in tumors can be accomplished by decreasing tumor tissue blood flow (tBF) after anticancer drug administration. The present study demonstrated that temocapril hydrochloride, an angiotensin converting enzyme inhibitor, decreases tumor tBF markedly in LY80 tumor, a subline of Yoshida sarcoma in the rat, without affecting the blood flow in liver, kidney, bone marrow, and brain. In tumor areas with flow of above 20 ml/min/ 100 g, the tBF decreased by approximately 50% due to temocapril. In tumor areas with tBF of about 20 ml/min/100 g, it became less than 3 ml/min/100 g with temocapril and did not recover during the 2 h experiment. These findings were obtained not only in large tumors, but also in microfoci growing within a transparent chamber. Furthermore, even when temocapril was administered under the condition of increased tumor tBF by administering angiotensin II, tumor tBF decreased immediately. Using this technique, it should be possible to trap anticancer drugs selectively in tumor tissue for an extended period of time.
Selective Inhibition of Hepatoma Cells Using Diphtheria Toxin A under the Control of the Promoter/Enhancer Region of the Human α‐Fetoprotein Gene
Authors: Kunitomi Michito , Takayama Eiji , Suzuki Satoshi , Yasuda Tatsuji , Tsutsui Ken , Nagaike Kazuhiro , Hiroi Sadayuki , Tadakuma Takushi
Published: August 2005

We constructed a plasmid containing human α‐fetoprotein (AFP) promoter/enhancer to direct the cell type‐specific expression of diphtheria toxin fragment A (DTA), designated as pAF‐DTA, to AFP‐producing hepatocellular carcinoma cells. The transfection was carried out with cationic liposomes (DMRIE‐C) and the expression of the DTA gene was confirmed by a northern blot analysis. When pAF‐DTA was transfected, the growth of AFP‐positive HuH‐7 cells was inhibited, whereas growth inhibition was not observed in AFP‐negative MKN45 cells. In this experiment, the secretion of AFP was similarly suppressed, but the secretion of carcinoembryonic antigen from MKN45 was not altered. pAF‐DTA could also exert its growth inhibitory effect on PLC, a cell line with a low level of AFP. However, no inhibitory effect of pAF‐DTA was observed on the proliferation of primary hepatocyte cells. Furthermore, transfection experiments in which HuH‐7 and splenic stromal cells were co‐cultured revealed the growth inhibition by pAF‐DTA to be selective in HuH‐7 cells. Finally, the growth of HuH‐7 transplanted on BALB/c nu/nu mice was inhibited by the direct injection of pAF‐DTA/liposome complex into a tumor mass. These results suggest that use of pAF‐DTA may be potentially useful as a novel approach for the selective treatment of tumor cells producing AFP even at low levels, without affecting other types of cells.
Dose‐ and Sex‐related Carcinogenesis by N‐Bis(2‐hydroxypropyl)nitrosamine in Wistar Rats
Authors: Moreira Eduardo L. T. , de Camargo João L. V. , Rodrigues Maria A. M. , Barbisan Luis F. , de Favero Salvadori Daisy M.
Published: August 2005

An initiation‐promotion medium‐term bioassay for detection of chemical carcinogens, developed in the male F344 rat, uses 0.1% N‐bis(2‐hydroxypropyl)nitrosamine (DHPN) among five genotoxic chemicals for the initiation of carcinogenesis in multiple organs. To establish this bioassay in the Wistar strain, the effects of two dose levels of DHPN were evaluated on the main DHPN rat target organs: lung, thyroid gland, kidneys and liver. Four groups of male and female animals were studied: Control—untreated group; Multi‐organ initiated group (also referred to as DMBDD, based on the initials of the five initiators)—treated sequentially with N‐diethylnitrosamine (DEN, i.p.), N‐methyl‐N‐nitrosourea (MNU, i.p.), N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine (BBN, drinking water), N, ďN′‐dimethylhydrazine (DMH, s.c.) and DHPN (drinking water) for 4 weeks; a third group treated with 0.1% DHPN in drinking water for 2 weeks and the last group treated with 0.2% DHPN in drinking water for 4 weeks. The animals were sacrificed after 30 weeks. DHPN at 0.2% induced preneoplasia in the liver and kidneys of rats of both sexes, the number and area of the putative preneoplastic liver glutathione S‐transferase‐positive hepatocyte foci being significantly increased in these animals. It also induced benign and malignant tumors in female and in male rats. However, there was no relationship between the increased incidence of preneoplastic lesions and tumor development in the 0.2% DHPN‐exposed groups of both sexes. DHPN at 0.1% induced only a few preneoplastic lesions in the liver and kidney and no tumors in both male and female rats. A clear dose and sex‐related carcinogenic activity of DHPN was registered, although Wistar rats of both sexes showed a relative resistance to the carcinogenic activity of this compound.
Analysis of Novel Metastasis‐associated Gene TI‐227
Authors: Ishiguro Tatsuaki , Nagawa Hirokazu , Naito Mikihiko , Tsuruo Takashi
Published: August 2005

TI‐227, is a cancer metastasis‐associated gene isolated from the B16‐F10 mouse melanoma subline that preferentially metastasizes to the lung following intravenous injection. TI‐227, is more highly expressed in the B16‐F10 and colon26‐n117 sublines, which preferentially metastasize to the lung following intravenous injection, than in the B16‐BL6 and colon26‐nl22 sublines, which preferentially metastasize to the lung following subcutaneous injection. Since TI‐227, could not be detected in normal tissues, there is a possibility that it is expressed specifically in tumors that preferentially metastasize to the lung following intravenous inoculation. Although the 5’ end of TI‐227 was analyzed, its total sequence contained no obvious open reading frame. However comparison with the isolated human counterpart, TI‐227H, gene, revealed a highly homologous region near the 3’ end, which was expected to contain a very short open reading frame.
Mutation of the SRC Gene in Endometrial Carcinoma
Authors: Sugimura Masaki , Kobayashi Kanji , Sagae Satoru , Nishioka Yoshihiro , Ishioka Shin‐ichi , Terasawa Katsuhiko , Tokino Takashi , Kudo Ryuichi
Published: August 2005

Recently, an activating mutation of the SRC Gene has been implicated in about one‐tenth of advanced colon cancers. The SRC 531 mutation results in truncation of SRC directly C‐terminal to the regulatory Tyr 530 and appears to activate the Tyr 530. To investigate whether mutation of SRC plays an important role in the development and progression of gynecological tumors, we performed mutational analysis of the entire coding region of SRC in 70 ovarian carcinomas, 68 endometrial carcinomas and 3 endometrial stromal sarcomas by means of polymerase chain reaction‐single strand conformation polymorphism (PCR‐SSCP) followed by nucleotide sequencing and restriction fragment length polymorphism (RFLP) analysis. We found one truncated mutation at codon 531 (Gln to Stop) in an endometrial carcinoma. However, we found no mutation of this gene in ovarian carcinoma or endometrial stromal sarcoma. Our results suggest that mutation of SRC may be implicated in a small proportion of endometrial carcinomas.
Screening of BRCA1 Mutation Using Immunohistochemical Staining with C‐Terminal and N‐Terminal Antibodies in Familial Ovarian Cancers
Authors: Kashima Katsunori , Oite Takashi , Aoki Yoichi , Takakuwa Koichi , Aida Hiroshi , Nagata Hiroshi , Sekine Masayuki , Wu Hong Jun , Hirai Yasuo , Wada Yuichi , Yamamoto Kaichiro , Hasegawa Kazuo , Sonoda Takahiko , Maruo Takeshi , Nagata Ichiro , Ohno Masayuki , Suzuki Mitsuaki , Kobayashi Iwao , Kuzuya Kazuo , Takahashi Takeshi , Torii Yuichi , Tanaka Kenichi
Published: August 2005

We examined the subcellular localization of BRCA1 proteins using immunohistochemical staining with C‐terminal (GLK‐2 antibody) and N‐terminal (Ab‐2 antibody) monoclonal antibodies in 44 familial ovarian cancers. Among these, 24 cases were associated with 13 independent germ‐line mutations of BRCA1, and loss of heterozygosity (LOH) at one or more BRCA1 microsatellite markers was found in all 21 informative tumors tested. With GLK‐2 antibody, cytoplasmic staining was observed in 15 of 16 tumors (93.8%) with mutation in exon 11, and BRCA1 staining was absent in 8 of 8 tumors (100%) with mutation in exons other than exon 11. When immunohistochemical staining was performed with Ab‐2 antibody, both nuclear and cytoplasmic staining were observed in 14 of 16 tumors (87.5%) with mutation in exon 11. Interestingly, nuclear staining was observed in 3 of 3 tumors (100%) with mutation downstream of exon 11, even though no staining was detected in 5 of 5 tumors (100%) with mutation upstream of exon 11. On the other hand, in familial ovarian cancers without BRCA1 mutations, nuclear staining or both nuclear and cytoplasmic staining was observed in 18 of 20 specimens (90%) and 20 of 20 specimens (100%) with GLK‐2 antibody and with Ab‐2 antibody, respectively. These results suggest that an immunohistochemical assay in combination with employing the C‐terminal and the N‐terminal antibodies appears to have potential as a reliable and useful technique for the screening of BRCA1 mutations, at least to predict the status of mutation, upstream or downstream of exon 11.
A Novel Class of Antitumor Prodrug, 1‐(2′‐Oxopropyl)‐5‐fluorouracil (OFU001), That Releases 5‐Fluorouracil upon Hypoxic Irradiation
Authors: Shibamoto Yuta , Zhou Ling , Hatta Hiroshi , Mori Mayuko , Nishimoto Sei‐ichi
Published: August 2005

We have been developing prodrugs of anticancer agents such as 5‐fluorouracil (5‐FU) that are activated by irradiation under hypoxic conditions via one‐electron reduction. Among them, OFU001 [1‐(2′‐oxopropyl)‐5‐fluorouracil] is a prototype radiation‐activated prodrug. In this study, we investigated the radiation chemical reactivity and the biological effects of OFU001. This prodrug is presumed to release 5‐FU through incorporation of hydrated electrons into the antibonding σ* orbital of the C(1′)‐N(1) bond. Hydrated electrons are active species derived from radiolysis of water, but are readily deactivated by O2 into superoxide anion radicals () under conditions of aerobic irradiation. Therefore, 5‐FU release occurs highly specifically upon irradiation under hypoxic conditions. OFU001 dissolved in phosphate buffer released 5‐FU with a G‐value (mol number of molecules that are decomposed or produced by 1 J of absorbed radiation energy) of 1.9×10−7 mol/ J following hypoxic irradiation, while the G‐value for 5‐FU release was 1.0×10−8 mol/J following aerobic irradiation. However, the G‐values for decomposition of OFU001 were almost the same, i.e., 3.4×10−7 mol/J following hypoxic irradiation and 2.5×10−7 mol/J following aerobic irradiation. When hypoxically irradiated (7.5–30 Gy) OFU001 was added to murine SCCVII cells for 1–24 h, a significant cell‐killing effect was observed. The degree of this cytotoxicity was consistent with that of authentic 5‐FU at the corresponding concentrations. On the other hand, cytotoxicity was minimal when the cells were treated with aerobically irradiated or unirradiated OFU001. This compound had no radiosensitizing effect against SCCVII cells under either aerobic or hypoxic conditions when the drug was removed immediately after irradiation. Since hypoxia is generally most marked in tumors and irradiation is applied at the tumor site, this concept of prodrug design appears to be potentially useful for selective tumor treatment with minimal adverse effects of anticancer agents.
Absence of Germline CHK2 Mutations in Familial Gastric Cancer
Authors: Kimura Kenji , Shinmura Kazuya , Yoshimura Kimio , Shimizu Kimihiro , Katai Hitoshi , Beppu Yasuo , Moriya Hideshige , Yokota Jun
Published: August 2005

Recently, the CHK2 gene was identified as being a candidate gene responsible for Li‐Fraumeni syndrome (LFS). Gastric cancer is often clustered in families with LFS, so it is possible that germline CHK2 mutation is also present in familial gastric cancer (FGC). We therefore defined the genomic structure of the CHK2 gene, designed intronic primers, and searched for germline CHK2 mutations in 25 FGC cases by polymerase chain reaction‐single strand conformational polymorphism analysis of the entire coding region. In all of the 25 cases, at least two siblings had histories of gastric cancer. There were no FGC cases that showed germline CHK2 mutations. Thus, it was indicated that germline CHK2 mutations do not contribute to the familial clustering of gastric cancer.
Chemopreventive Effect of Curcumin on N‐Nitrosomethylbenzylamine‐induced Esophageal Carcinogenesis in Rats
Authors: Ushida Jun , Sugie Shigeyuki , Kawabata Kunihiro , Pham Quan Vhin , Tanaka Takuji , Fujii Keiko , Takeuchi Hideyuki , Ito Yatsuji , Mori Hideki
Published: August 2005

Modifying effects of curcumin (derived from the rhizome of Curcuma longa L.) during the initiation or post‐initiation phase of N‐nitrosomethylbenzylamine (NMBA)‐induced esophageal carcinogenesis were investigated in male F344 rats. Five‐week‐old rats were divided into 5 groups, and groups 1, 2 and 3 were given intraperitoneal injections of NMBA (0.5 mg/kg body weight/injection 15 times) for 5 weeks from 7 weeks old to induce esophageal neoplasms. Groups 2 and 3 were fed the diet containing 500 ppm curcumin during the initiation and post‐initiation phases, respectively. Group 4 was given the diet containing curcumin throughout the experiment, and group 5 was kept on the basal diet alone and served as an untreated control. Incidence and multiplicity of esophageal neoplasms of group 1 (NMBA alone) were 66.7% and 0.83 ±0.70, respectively. Those of groups 2 and 3 were significantly less than those of group 1 (39.3%, 0.46±0.64, P < 0.05; 33.3%, 0.36±0.56, P < 0.05, respectively). Furthermore, the incidence and multiplicity of esophageal preneoplastic lesions (moderate or severe epithelial dysplasia) of group 2 (57.1%, 0.61±0.57; 40%, 0.29±0.46) or 3 (56.7%, 0.67±0.66; 23.3%, 0.23±0.43) were less than those of group 1 (100%, 1.67±0.70; 70.8%, 0.92±0.72) (P < 0.05). In this experiment, feeding of curcumin significantly decreased the expression of cell proliferation biomarkers (5‐bromo‐2′‐deoxyuridine labeling index) in the non‐lesional esophageal epithelium (P < 0.01). These findings indicate that curcumin inhibits NMBA‐induced esophageal carcinogenesis when given during the post initiation as well as initiation phase. This inhibition may be related to suppression of the increased cell proliferation induced by NMBA in the esophageal epithelium.
Co‐expression of Thymidine Phosphorylase and Heme Oxygenase‐1 in Macrophages in Human Malignant Vertical Growth Melanomas
Authors: Torisu‐Itakura Hitoe , Furue Masutaka , Kuwano Michihiko , Ono Mayumi
Published: August 2005

Expression of thymidine phosphorylase (TP) is often associated with tumor angiogenesis and/or prognosis in patients. Further, infiltration of macrophages is closely correlated with the depth of tumor and angiogenesis in melanomas. In this study, we examined the expression of TP and an activated macrophage‐specific enzyme, heme oxygenase‐1 (HO‐1), involved in malignancy in 22 cases with melanomas. TP was strongly expressed not only in CD68‐positive macrophages in and around tumors, but also in S100 protein‐positive melanoma cells, fibroblasts and keratinocytes. By contrast, HO‐1 was specifically expressed in macrophages, but only slightly in melanoma cells and other cell types in the stroma of melanomas. We thus observed apparent co‐expression of TP and HO‐1 in macrophages infiltrating in the late stage of malignant melanomas. There appeared increasing numbers of TP‐positive cells in Clark level IV and V melanoma compared with Clark level I (in situ) melanoma, and there was also a close correlation between numbers of TP‐positive cells and HO‐1‐positive cells. Both TP‐ and HO‐1‐positive macrophages could be observed in the stroma in and around tumors in vertical growth melanomas.
Regulation of Granulocyte Colony‐stimulating Factor and Parathyroid Hormonerelated Protein Production in Lung Carcinoma Cell Line OKa‐C‐1
Authors: Uemura Yoshiki , Nakata Hideshi , Kobayashi Makoto , Harada Ryuji , Asahi Yasutomo , Taguchi Hirokuni
Published: August 2005

Previously we have established a clonal squamous cell carcinoma cell line OKa‐C‐1 derived from lung cancer of a patient with marked leukocytosis and hypercalcemia. OKa‐C‐1 cells simultaneously produce granulocyte colony‐stimulating factor (G‐CSF) and parathyroid hormone‐related protein (PTHrP) at the single cell level and cause paraneoplastic syndromes in nude mice bearing the tumor. It is known that the production of G‐CSF and PTHrP is individually regulated by inflammatory cytokines in various malignant cells. To investigate the common factors in the regulation of G‐CSF and PTHrP production in OKa‐C‐1 cells, we examined the effects of some inflammatory agents [lipopolysaccharide (LPS), phorbol‐12‐myristate‐13‐acetate (PMA), tumor necrosis factor‐α (TNF‐α), interleukin‐1 (IL‐1) β and IL‐6] on G‐CSF and PTHrP production, by means of enzyme‐linked immunosorbent assay (ELISA), immunoradiometric assay (IRMA) and quantitative reverse transcription‐polymerase chain reaction (RT‐PCR). TNF‐α or IL‐1β induced both G‐CSF and PTHrP production in the conditioned medium. TNF‐α synergized with IL‐1β to significantly increase G‐CSF production. In addition, TNF‐α and IL‐1β strongly induced G‐CSF mRNA with peaks at 2 and 6 h respectively. Although PTHrP production was also strongly induced by TNF‐α PTHrP mRNA expression was more strongly induced by PMA than by TNF‐α. Thus, TNF‐α and IL‐1β could be common factors that individually and synergistically regulate G‐CSF and PTHrP production in OKa‐C‐1 cells. Moreover, G‐CSF and PTHrP production could be not only transcriptionally, but also posttranscriptionally regulated by other factors.
ICAM‐1 (Intercellular Adhesion Molecule‐1) Gene Transfection Inhibits Lymph Node Metastasis by Human Gastric Cancer Cells
Authors: Sunami Takeshi , Yashiro Masakazu , Chung Kosei Hirakawa‐Y.‐S.
Published: August 2005

Lymph node metastasis is one of the prognostic factors in gastric cancer. We have previously reported that decreased intercellular adhesion molecule‐1 (ICAM‐1) expression on cancer cells is associated with lymph node metastasis using a gastric cancer cell. In this study, we transfected ICAM‐1 gene into a gastric cancer cell line, 2MLN, and analyzed the effect on lymph node metastasis in vitro and in vivo. A significantly greater amount of peripheral blood mononuclear cells (PBMC) adhered to ICAM‐1 transfected 2MLN cells, 2MLN/ICAM cells, than to 2MLN/Vector cells. The lysis of 2MLN/ICAM cells by PBMC was significantly increased compared with that of 2MLN/Vector cells. The tumor growth rate of 2MLN/ICAM cells was significantly decreased in vivo. Lymph node metastases caused by 2MLN/ICAM cells were recognized as being fewer in number and smaller, while many lymph node metastases were caused by 2MLN cells. Histologic findings showed that leukocytes were heavily infiltrated in both the 2MLN/ICAM tumors and metastatic lesions, while only a few leukocytes were observed in the lesions associated with 2MLN cells. The above findings indicate that ICAM‐1 gene transduction could prove to be an effective gene therapy for lymph node metastasis of gastric cancer.
p53 Mutations in Prostatic Intraepithelial Neoplasia and Concurrent Carcinoma: Analysis of Laser Capture Microdissected Specimens from Non‐transition and Transition Zones
Authors: Takayama Hitoshi , Shin Masaru , Nonomura Norio , Okuyama Akihiko , Aozasa Katsuyuki
Published: August 2005

Prostatic intraepithelial neoplasia (PIN) is characterized by intraluminal proliferation of epithelial cells and is divided into high‐grade (HGPIN) and low‐grade (LGPIN) lesions. HGPIN is regarded as the most likely precursor of prostatic cancer (PCA). Microdissected DNA selectively extracted from paraffin‐embedded sections of 27 cases with PCA were analyzed for p53 mutation in exons 5–8 by single‐strand conformation polymorphism of polymerase chain reaction‐amplified DNA fragments (PCR‐SSCP) followed by direct sequencing. These patients received total prostatectomy (27 cases). After a review of histologic sections, DNA was extracted from 193 locations; 111 lesions from 27 cases with HGPIN (75 lesions from non‐transition zone and 36 from transition zone), 55 lesions from 27 cases with PCA (30 lesions from non‐transition zone and 25 from transition zone), and 27 from 27 benign glands. Analysis revealed 27 mutations of the p53 gene in 24 lesions from 12 cases. Benign glands adjoining PIN and/or PCA had no mutations. All mutations were point mutations: 17 missense, 7 silent, and 2 nonsense. Mutations were detected in 6 cases (22.2%) or 13 of 111 lesions (11.7%) with HGPIN and 8 cases (29.6%) or 11 of 55 lesions (20.0%) with PCA. In a given case, HGPIN and PCA lesions had different p53 mutations from each other, suggesting multiclonal development of prostatic precancerous lesions. The frequency of p53 mutation of PCA was significantly higher in the non‐transition zone (33.3%) than in the transition zone (4%), and higher in the stage T3 cases (30.3%) than in the stage T2 cases (4.5%, 1 of 22 lesions) (both P < 0.05). Frequency of p53 mutation of PIN in the non‐transition zone (14.7%) was higher than that in the transition zone (5.6%), although the difference was not significant. The frequency rate of p53 mutation in HGPIN close to PCA (≤2 mm) was significantly higher (24%) than that in HGPIN lesions > 2 mm from PCA (3%). All these findings indicate that the p53 gene mutations are involved in prostatic carcinogenesis and explain why the non‐transition zone is the predominant site of PCA.
Alterations of the Transforming Growth Factor‐(β Signaling Pathway in Hepatocellular Carcinomas Induced Endogenously and Exogenously in Rats
Authors: Sasaki Yasutaka , Tsujiuchi Toshifumi , Murata Nao , Tsutsumi Masahiro , Konishi Yoichi
Published: August 2005

To elucidate involvement of the transforming growth factor‐β (TGF‐β) signaling pathway in endogenous and exogenous liver carcinogenesis, we investigated mutations of TGF‐β receptor type II (TGF‐βRII), Smad2 and Smad4 genes, and expression of TGF‐βRII in hepatocellular carcinomas (HCCs) induced by a choline‐deficient L‐amino acid‐defined (CDAA) diet and by N‐nitrosodiethyl‐amine (DEN). Male Fischer 344 rats received a CDAA diet continuously and HCCs were sampled after 75 weeks. Administration of DEN was followed by partial hepatectomy (PH), with colchicine to induce cell cycle disturbance and a selection pressure regimen, HCCs being obtained after 42 weeks. Total RNAs were extracted from individual HCCs and mutations in TGF‐PRH, Smad2 and Smad4 were investigated by reverse transcription (RT)‐polymerase chain reaction (PCR)‐restric‐tion‐single‐strand conformation polymorphism (SSCP) analysis followed by sequencing analysis. Mutations of Smad2 were detected in 2 out of 12 HCCs (16.7%) induced by the CDAA diet, a GGT‐to‐GGC transition (Gly to Gly) at codon 30 and a TCT‐to‐GCT (Ser to Ala) transversion at codon 118, without any TGF‐βRII or Smad4 alterations. No mutations of TGF‐βRII, Smad2 and Smad4 were encountered in eleven HCCs induced by the exogenous carcinogen. Semi‐quantitative RT‐PCR revealed reduced expression of TGF‐βRII in 2 HCCs (16.7%) without Smad2 mutations out of 12 HCCs induced by the CDAA diet and none of 11 induced by DEN. These results suggest that the TGF‐p signaling pathway may be disturbed in endogenous liver carcinogenesis in rats.
Anti‐angiogenic Treatment for Peritoneal Dissemination of Pancreas Adenocarcinoma: A Study Using TNP‐470
Authors: Kato Hiroaki , Ishikura Hiroshi , Kawarada You , Furuya Mitsuko , Kondo Satoshi , Kato Hiroyuki , Yoshiki Takashi
Published: August 2005

We established peritoneal dissemination‐prone subcultures (PCI‐43p3) using nude mice by a repetitive in vivo selection of intraperitoneally inoculated PCI‐43, a pancreas adenocarcinoma cell line. The subcultures showed upregulated expression of matrix metalloproteinase (MMP)‐9, but not MMP‐2 in culture supernatants. They also produced increased amounts of vascular endothelial growth factor (VEGF), which was not associated with alterations in isoforms of VEGF mRNA. PCI‐43p3 cells attached to cultured mesothelial cell monolayers more readily than did the parent PCI‐43 cells. The angiogenesis inhibiting agent, TNP‐470, at 30 mg/kg was administered to the model mice, resulting in a prominent suppression of the establishment of peritoneal nodules. The suppression was dependent on the duration of TNP‐470 treatment. TNP‐470 treatment significantly suppressed proliferation of tumor cells in disseminated nodules, assessed in terms of immunostaining for proliferating cell nuclear antigen (PCNA). TNP‐470 did not affect the in vitro attachment between PCI‐43p3 and mesothelial cells. The combined data show that anti‐angiogenic treatment profoundly suppresses the in vivo process of peritoneal dissemination.
Prevention of Hepatic and Peritoneal Metastases by the Angiogenesis Inhibitor FR‐118487 after Removal of Growing Tumor in Mice
Authors: Tanaka Tatsuo , Konno Hiroyuki , Baba Shozo , Kanai Toshikazu , Matsumoto Keigo , Matsuda Iwao , Ohba Kohji , Ohta Manabu , Kamiya Kinji , Nakamura Satoshi
Published: August 2005

We established a mouse “primary tumor resection model” in which a transplanted tumor was resected after an orthotopic transplantation of colorectal cancer tissue to estimate the therapeutic effect of an angiogenesis inhibitor on metastasis. The angiogenesis inhibitor FR‐118487 is a member of the fumagUlin family. Here, 1 mg/kg/day of FR‐118487 was subcutaneously administered to nude mice for 1 week, 2 weeks, or 4 weeks through an osmotic pump. Liver metastasis developed in 7 of 9 control mice, 2 of 6 mice that underwent the tumor resection 2 weeks after transplantation (early resection), and in all 7 of the mice that underwent the tumor resection 4 weeks after transplantation (late resection). In the short treatment trial, the FR‐118487 administration immediately after the early resection completely inhibited both hepatic and peritoneal metastases, whereas its administration after the late resection had no effect on liver metastasis. In the prolonged treatment trial, inhibitory effects of prolonged treatment with FR‐118487 on both hepatic and peritoneal metastases after the late resection were clearly demonstrated. The mice of the resection‐alone group all died within 106 days after tumor inoculation, due to metastases of colon carcinoma. In contrast, half of the mice that underwent resection and then received antiangiogenic therapy were alive at the end of the observation period (160 days after transplantation). In conclusion, the combination of surgery and subsequent antiangiogenic therapy may be useful to prevent the distant metastasis of colorectal cancer and to improve the prognosis of patients with colorectal cancer.
Strain Difference in Regulation of Pituitary Tumor Transforming Gene (PTTG) in Estrogen–induced Pituitary Tumorigenesis in Rats
Authors: Yin Hong , Fujimoto Nariaki , Maruyama Satoshi , Asano Kohsuke
Published: August 2005

Recently a novel oncogene, PTTG (pituitary tumor transforming gene) was isolated from a rat pituitary tumor cell line whose expression is apparently correlated with pituitary tumorigenesis. In the rat, estradiol (E2) is known to induce anterior pituitary hyperplasia. The effects of E2, however, vary greatly among rat strains. Therefore we examined the expression of PTTG and its regulation by E2 in F344, Wistar, Brown–Norway and Donryu rats. Four–week–old females were ovariecto–mized and a pellet containing 10 mg of E2 was given s.c. Total RNA was isolated from the pituitary gland and PTTG mRNA was measured with a competitive RT–PCR technique. The F344 strain was the most susceptible to E2 induction of pituitary tumorigenesis, followed by Wistar and Brown–Norway, while no increase in pituitary weight was noted in Donryu rats. PTTG mRNA in the gland was induced by E2 within 48–72 h in F344 and Wistar, but not in Brown–Norway or Donryu strains. These data suggest that PTTG expression may at least in part be responsible for strain differences in E2–induced pituitary tumorigenesis
Clinicopathological Characteristics of Estrogen Receptor‐(3‐positive Human Breast Cancers
Authors: Miyoshi Yasuo , Taguchi Tetsuya , Gustafsson Jan‐Åke , Noguchi Shinzaburo
Published: August 2005

Recent studies have identified the presence of estrogen receptor (ER)‐β in addition to ER‐α in human breast cancers, but the Clinicopathological characteristics of ER‐β‐positive tumors remain to be established. In this study, we have conducted an immunohistochemical analysis of ER‐α and ER‐β expression in human breast cancers. In addition, we investigated the correlation of ER‐α and ER‐β expression with progesterone receptor (PR) status, determined by enzyme immunoassay, and with various Clinicopathological factors. Of 79 tumors, 49 (62%) were positive for ER‐α and 24 (30%) were positive for ER‐β, and there was no significant association between ER‐α and ER‐β expression. ER‐α‐positive tumors were significantly more likely to be PR‐positive than were ER‐α‐negative tumors (P<0.0001), but there was no significant association between ER‐β expression and PR status. However, the PR values of ER‐α‐positive and ER‐β ‐positive tumors (65±17 fmol/mg protein, mean±SE) were marginally significantly lower than those of ER‐α‐positive and ER‐β ‐negative tumors (340±109) (P=0.08). ER‐β positivity was significantly associated with small tumor size (<2 cm) and high histological grade (P<0.05), and this association was also observed when only ER‐α‐positive tumors were considered. These results suggest that determination of ER‐β status might be clinically useful for further defining the characteristics of ER‐α‐positive tumors
Reduced Expression of Syndecan‐1 Affects Metastatic Potential and Clinical Outcome in Patients with Colorectal Cancer
Authors: Fujiya Mikihiro , Watari Jiro , Ashida Toshifumi , Honda Mitsunori , Tanabe Hiroki , Fujiki Takanori , Saitoh Yusuke , Kohgo Yutaka
Published: August 2005

Syndecan‐1 is a transmembrane heparansulfate proteoglycan which regulates cell‐to‐cell or cell‐to‐extracellular matrix interactions and may influence malignant cell behavior. We investigated the alterations of syndecan‐1 expressions in colorectal cancers and analyzed the relationship between histological and clinical characteristics. Syndecan‐1 protein expression in colorectal cancer tissues was investigated with immunohistochemical staining of resected specimens. In situ hybridization was performed using syndecan‐1 riboprobe to confirm the transcriptional signals. Syndecan‐1 mRNA expression in cancer cell lines cultured with or without methylation inhibitor was also analyzed by quantitative PCR. Out of 105 specimens tested, less than 25% of tumor cells were stained with anti‐syndecan‐1 monoclonal antibody in 36 (34.3%). In situ hybridization showed a similar staining profile to that of immunohistochemistry. Syndecan‐1 mRNA expression was increased by the methylation inhibitor 5‐aza‐2′‐deoxycytidine, suggesting that the hypermethylation is involved in the suppression of syndecan‐1 expression. Clinically, the incidence of metastasis to lymphnode or liver in patients with syndecan‐1‐negative tumors was significantly high. Among Tl colorectal cancers displaying a primary invasive phase, lymphnode metastasis, undifferentiated characters and ‘budding’ of cancer cells were more common in syndecan‐1‐negative tumors. The survival rate in patients with syndecan‐1‐negative tumors was decreased significantly in a stage‐independent manner. These results suggest that the reduction of syndecan‐1 expression in colorectal cancer cells, which is supposed to be regulated at the transcription level, is closely related to invasive character. The evaluation of syndecan‐1 expression in colorectal cancer may allow prediction of patients' survival after surgery
Hypoxia Reduces Hormone Responsiveness of Human Breast Cancer Cells
Authors: Kurebayashi Junichi , Otsuki Takemi , Moriya Takuya , Sonoo Hiroshi
Published: August 2005

Resistance to hormonal therapy frequently occurs following successful treatment in breast cancer. The mechanism responsible for this acquired resistance is still unknown. It has been suggested that a hypoxic tumor microenvironment promotes malignant progression of cancer, i.e., hypoxia may promote estrogen‐independent growth (a more malignant phenotype) of breast cancer. To clarify this hypothesis, the effects of hypoxia on the growth responses to hormonal agents and the expression levels of estrogen receptor (ER)‐α and progesterone receptor (PgR) were investigated in two human breast cancer cell lines, ML‐20 and KPL‐1. The expression level of ER‐α was significantly decreased by hypoxia (1% O2) in a tune‐dependent manner in both cell lines. Hypoxia also significantly reduced the growth‐promoting effect of estradiol (E2) and the growth‐inhibitory effects of an antiestrogen, ICI 182 780, and a progestin, medroxyprogesterone acetate, in both cell lines. In addition, hypoxia markedly suppressed the induction of PgR mRNA and protein by E2 in both cell lines. To clarify further the effect of hypoxia on ER‐α expression, the expression levels of hypoxia‐inducible factor‐la (HIF‐lα), a marker of hypoxia and ER‐α were immunohistochemically examined in 36 breast cancer specimens. ER‐α expression (both its proportion and intensity) was significantly lower in nuclear HIF‐lα‐positive tumors than in negative tumors. These findings indicate that hypoxia down‐regulates ER‐α expression as well as ER‐α function in breast cancer cells. These processes may lead to an acquired resistance to hormonal therapy in breast cancer
Cyclin D1 Antisense Oligonucleotide Inhibits Cell Growth Stimulated by Epidermal Growth Factor and Induces Apoptosis of Gastric Cancer Cells
Authors: Saikawa Yoshiro , Kubota Tetsuro , Otani Yoshihide , Kitajima Masaki , Modlin Irvin M.
Published: August 2005

The cyclin Dl protein is one of the cell cycle regulators required for cell cycle progression through Gl phase to S phase. The cyclin Dl‐cyclin‐dependent kinase (CDK) system is thought to control the cell cycle through mediating extracellular signals from mitogens, such as epidermal growth factor (EGF). In this study, we attempted to examine the therapeutic effect of cyclin Dl antisense oligonucleotides (AS/D1) on cell proliferation and apoptosis of the gastric cancer cell line MKN‐74, in the presence and absence of EGF‐stimulation. Evaluation of cell survival and DNA synthesis revealed that enhanced cell growth following EGF‐stimulation was completely inhibited by a 24 h pre‐incubation with 100 nM AS/D1. This inhibition was down to 19.3% compared with maximal DNA synthesis after stimulation with 3 nM EGF alone. Western blotting demonstrated that while EGF‐stimulation led to cyclin Dl over‐expression, AS/D1 inhibited cyclin Dl protein expression. We also demonstrated the induction of apoptosis in MKN‐74 cells by AS/D1. In conclusion, EGF‐stimulated MKN‐74 cell proliferation was inhibited by AS/D1, which could overcome EGF‐induced cyclin Dl over‐expression. AS/D1 also affected cell survival by inducing apoptosis through cell cycle arrest following cyclin Dl depletion. Thus, AS/D1 may be a candidate for use as a novel cancer therapy specifically targeted against the over‐expression of cyclin Dl enhanced by EGF in malignant cells
Antitumor Effect of the Idiotypic Cascade Induced by an Antibody Encapsulated in Poly(d, l‐lactide‐co‐glycolide) Microspheres
Authors: Ma Jie , Luo Dong , Qi Weimin , Cao Liren
Published: August 2005

A major difficulty encountered during development of antibody vaccines is their weak immunogenicity. In this study, a monoclonal antibody CS20.5 to human breast cancer antigen CA15.3 was coencapsulated in poly(d, l‐lactide‐co‐glycolide) microspheres with monophosphoryl lipid A. The antitumor effect of this formulation was investigated in a murine model. The induced Ab2 biologically mimics antigen as it competed with CA15.3 for the same idiotope on Abl. Ab3 induction was also observed. After five sequential administrations of encapsulated antibody, mice showed statistically significant tumor regression. These results indicate that this formulation may serve as a potential treatment for breast cancer.
Intracellular Levels of Two Cyclosporin Derivatives Valspodar (PSC 833) and Cyclosporin A Closely Associated with Multidrug Resistance‐modulating Activity in Sublines of Human Colorectal Adenocarcinoma HCT‐15
Authors: Uchiyama‐Kokubu Noriko , Watanabe Toru , Cohen Dalia
Published: August 2005

P‐Glycoprotein, which mediates multidrug resistance (MDR) in cancer chemotherapy, is a principal target of Cyclosporin A and [3′‐keto‐Bmt1]‐[Val2]‐cyclosporin (valspodar; PSC 833). To clarify mechanisms contributing to the different MDR‐modulating activities of valspodar and Cyclosporin A, we investigated the relation of the intracellular levels of the two Cyclosporin derivatives to their modulating effect on MDR in different P‐glycoprotein‐expressing human colorectal carcinoma HCT‐15 cells (parental HCT‐15 and adriamycin‐resistant sublines). In this study, valspodar was found to be much more potent than Cyclosporin A in both sensitizing resistant cells to MDR‐related anticancer drugs (e.g., adriamycin, vincristine and paclitaxel (taxol)) and increasing 2‐[6‐amino‐3‐imino‐3H‐xanthen‐9‐yl]benzoic acid methyl ester (rhodamine 123) retention and [G‐3H]vincristine sulfate ([3H]vincristine) accumulation in these cells. Furthermore, a good correlation was detected between P‐glycoprotein levels and the MDR‐reversing effect of valspodar. In contrast, the effects of Cyclosporin A could not be linked to P‐glycoprotein levels in the MDR cells. In addition, the intracellular accumulation of valspodar was found to be 3–6 fold higher than that of Cyclosporin A in four sublines and verapamil, an inhibitor of P‐glycoprotein‐mediated transport, enhanced the accumulation of Cyclosporin A, but not valspodar. These results suggested that valspodar accumulation is not actively regulated by the P‐glycoprotein‐mediated efflux system
High Expression of Thymidylate Synthase Leads to Resistance to 5‐Fluorouracil in Biliary Tract Carcinoma in vitro
Authors: Habara Kazuto , Ajiki Tetsuo , Kamigaki Takashi , Nakamura Takeshi , Kuroda Yoshikazu
Published: August 2005

To evaluate the effect of chemotherapy of 5‐fluorouracil (5‐FU) in human biliary tract carcinoma, we studied 5‐FU sensitivity, thymidylate synthase (TS) content, and dihydropyrimidine dehydroge‐nase (DPD) activity in 4 human biliary tract carcinoma cell lines compared to 12 various digestive carcinoma cell lines of human organs in vitro. 5‐FU sensitivity in the cell lines was analyzed by MTT assay. TS content was analyzed by the [6‐3H]FdUMP binding assay method, and DPD activity was analyzed by thin‐layer chromatography (TLC). 5‐FU lC50 values of biliary tract carcinoma cell lines were significantly higher than those of the carcinoma cell lines of the other digestive organs: 97, 45, 119, and 194 tunes the concentration of the other digestive, pancreas, colon, and gastric carcinoma cell lines, respectively. TS content of biliary tract carcinoma cell lines was also significantly greater than that of the carcinoma cell lines of the other organs. No difference in DPD activity, however, was recognized between the carcinoma cell lines of each organ. TS content in the cell lines significantly correlated with 5‐FU sensitivity, but DPD activity did not. Therefore, in the present study, TS expression was concluded to influence the high resistance to 5‐FU of biliary tract carcinoma in comparison with the carcinomas of the other digestive organs.