bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular characterization of 5′‐deoxy‐5′‐methylthioadenosine phosphorylase‐deficient mutant clones of murine lymphoma cell line R1.1
Authors: Kadariya Yuwaraj , Nishioka Junji , Nakamura Akiko , Kato‐Nakazawa Keiko , Nobori Tsutomu
Published: August 2005

5′‐Deoxy‐5′‐methylthioadenosine phosphorylase (MTAP), the polyamine and purine metabolic enzyme, is ubiquitously expressed in normal human tissues and cells, but is deficient in a variety of human and murine malignant cell lines. MTAP‐deficient mutants were previously selected from murine lymphoma cell line R1.1 that contains abundant MTAP activity, to analyze the metabolic consequences of MTAP deficiency. Two mutants, one with partial deficiency (F clone) and the other with complete deficiency (H5 clone), were found to have the MTAP protein by immunoblotting. However, the molecular mechanism of enzyme deficiency in these mutants has not been established. In this study, we cloned the mouse cDNA and analyzed MTAP mRNA in the two mutants by reverse transcription‐polymerase chain reaction (RT‐PCR) followed by direct sequencing. Both mutants have a single nucleotide substitution at the third base of codon 223, which results in a change of cysteine to tyrosine (C223Y). The MTAP mRNA level determined by RT‐PCR was significantly lower in the mutant with complete deficiency than in wild‐type (WT) R1.1 cells, whereas the mRNA level in the mutant with partial deficiency was comparable to that in WT R1.1 cells. C223Y mutation may cause conformational alteration of the methylthioribose‐binding site and decrease the substrate binding. Thus, C223Y may account for the partial deficiency in the F clone, but the complete deficiency with a remarkably low MTAP mRNA level in the H5 clone may be a result of transcriptional abnormality in addition to C223Y mutation.
Roles of pericellular proteolysis by membrane type‐1 matrix metalloproteinase in cancer invasion and angiogenesis
Authors: Seiki Motoharu , Yana Ikuo
Published: August 2005

Behavior of cancer cells is profoundly affected by their microenvironment, which is often controlled by pericellular proteolysis or the processing of protein components, including extracellular matrices, growth factors, cytokines, receptors, cell adhesion molecules, and so on. Matrix metalloproteinases (MMPs) are a family of zinc‐dependent proteases responsible for the proteolytic events in the extracellular milieu. Among the multiple MMPs expressed in a wide range of tumors, membrane type‐1 MMP (MT1‐MMP), which is expressed especially in tumor cells with significant invasive properties, is thought to be particularly important for pericellular proteolysis. Recent studies have elucidated in part how MT1‐MMP is regulated biologically for the promotion of invasion by tumors or for angiogenesis by endothelial cells. Understanding of the proteolysis by, and the regulation of MT1‐MMP, which probably promotes cell invasion, could provide a therapeutic hint as to how to block or delay the progression of cancer.
Gene expression in colorectal cancer and in vitro chemosensitivity to 5‐fluorouracil: A study of 88 surgical specimens
Authors: Yoshinare Kentaro , Kubota Tetsuro , Watanabe Masahiko , Wada Norihito , Nishibori Hideki , Hasegawa Hirotoshi , Kitajima Masaki , Takechi Teiji , Fukushima Masakazu
Published: August 2005

To predict the sensitivity of colorectal cancer to 5‐fluorouracil (5‐FU), we compared the gene expression of surgically obtained colorectal cancer specimens with chemosensitivity to 5‐FU as detected by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H tetrazolium bromide (MTT) assay. Eighty‐eight patients with advanced and/or metastatic colorectal cancer provided written informed consent and entered the trial from September 2000 to October 2001. Fresh surgical specimens were used for the MTT assay, and sensitivity to 5‐FU was evaluated at a cutoff concentration of 50 μg/ml and 48‐h incubation time. Frozen samples were stored at −80°C until mRNA analysis of thymidylate synthetase (TS), dihydropyri‐midine dehydrogenase (DPD), thymidine phosphorylase (TP), es‐nucleoside transporter (NT), and E2F1 by real‐time RT‐PCR. The correlations between the variables were analyzed, and the predictive value of these mRNAs was assessed statistically using a receiver operating characteristic (ROC) curve. NT and DPD, TP and DPD, and TP and NT mRNA expression levels correlated significantly, while TS and E2F1 showed no correlations. High NT expression was associated with low sensitivity to 5‐FU (P<0.013), as were high DPD and E2F1 expression (P<0.022 for both). High TP mRNA expression correlated with low sensitivity to 5‐FU (P<0.034), although high TS mRNA expression did not. ROC curves indicated that DPD and NT mRNAs were possible predictors of sensitivity to 5‐FU, with cutoff values of 0.6 and 0.4, respectively. The sensitivity of colorectal cancer to 5‐FU may be regulated by DPD, the rate‐limiting enzyme of catabolism, and NT, an important transmembrane transporter of nucleosides.
Time course of carcinoembryonic antigen after resection of lung cancer: A predictor of recurrence
Authors: Yoshimasu Tatsuya , Kokawa Yozo , Oura Shoji , Hirai Issei , Sasaki Rie , Tanino Hirokazu , Sakurai Teruhisa , Okamura Yoshitaka
Published: August 2005

We investigated whether the early postoperative time course of carcinoembryonic antigen (CEA) level after resection of lung cancer could be used to predict patients' prognosis. Fifty‐three lung cancer patients were included in this study. Postoperative serum CEA levels were calculated by means of non‐linear least‐squares fitting to the equation C(t)=(C0‐Cp)exp(‐kt)+Cp, where C(t) is postoperative CEA level, t is days after surgery, C0 is CEA level at postoperative time 0, Cp is CEA level at plateau, and k is the rate constant of elimination. Postoperative CEA production (Pp) was calculated as Cp multiplied by k. Cp and Pp represent the numbers of residual tumor cells after surgery. More residual tumor cells yield higher values of Cp and Pp, and result in earlier recurrence. [Results] Kinetic parameters could be obtained for 30 patients whose preoperative CEA levels were sufficiently elevated. Cutoff levels as predictors for recurrence were 1.1 ng/ml for Cp and 0.9 ng/ml/day for Pp. The accuracy of prediction of recurrence using these cutoff levels was 79% with Cp and 89% with Pp. A very poor prognosis was observed for patients with Pp over 0.9 ng/ ml/day. [Conclusion] Analysis of the time course of changes in CEA levels after resection of lung cancer appears to be useful for predicting patient prognosis. Cp and Pp are very precise predictors of recurrence.
Svi3: A provirus common integration site in c‐myc in SL/Kh pre‐B lymphomas
Authors: Jin Guang , Tsuruyama Tatsuaki , Yamada Yoshihiro , Hiai Hiroshi
Published: August 2005

Spontaneous pre‐B lymphomas in SL/Kh mice occur by somatic acquisition of a provirus genome of endogenous murine leukemia virus (MuLV). Inverse PCR amplification and sequence analyses of a provirus and its host flanking fragment revealed a proviral insertion into c‐myc in 3 out of 60 SL/Kh pre‐B lymphomas, named Svi3 lymphomas (SL/Kh virus integration site‐3). Southern blot analysis revealed that two lymphomas had clonal integration in c‐myc exon 1 and the other, in the promoter region. In 2 out of 3 Svi3 lymphomas, a fusion transcript of provirus 3’long terminal repeat and c‐myc and a normal full‐length c‐myc transcript were obtained, but in one Svi3 lymphoma, only the normal transcript was obtained. All three Svi3 lymphomas had increased c‐myc expression, producing normal 67‐kDa c‐Myc protein. Svi3 lymphomas had more mature phenotypes in the steps of early B‐cell differentiation than Svi 1 lymphomas, in which c‐myc expression was indirectly up‐regulated by provirus integration into Stat5a.
Cyclosporine A effectively inhibits graft‐versus‐host disease during development of Epstein‐Barr virus‐infected human B cell lymphoma in SCID mouse
Authors: Gan Runliang , Yin Zhihua , Liu Tengfei , Wang Lijiang , Tang Yunlian , Song Ying
Published: August 2005

We previously constructed human peripheral blood lymphocyte (hu‐PBL)/severe combined immunodeficiency mouse (SCID) chimeras and induced human B‐cell lymphomas associated with Epstein‐Barr virus (EBV) in SCID mice. However, a number of SCID mice died of graft‐versus‐host disease (GVHD) during the early experimental course. The aim of this study was to test the efficacy of cyclosporine A (CSA) for prevention of GVHD and to define how CSA inhibits the occurrence of GVHD and the production of soluble interleukin (IL) 2 receptor (sIL‐2R) in hu‐PBL/SCID mice. No mouse died in the active EBV infection group with CSA administration, while 17 mice in three groups without CSA administration died of GVHD. Mortalities in these three groups were 55.56% (5/9), 30.43% (7/23), and 27.78% (5/18), and the medium life span was 17 days. Over the first 33 days after hu‐PBL transplantation, serum level of human sIL‐2R in hu‐PBL/SCID chimeras was stable in the active EBV infection plus CSA group, while sIL‐2R concentration gradually increased in the sera of mice with active EBV infection without CSA administration and peaked at 22 days. Thirty‐two mice developed tumors among the 43 surviving SCID mice. There was no significant difference of tumor incidence between the active EBV infection groups with CSA and without CSA administration (P>0.05). From their morphological and immunohistochemical features, as well as detection of human Alu‐sequence and EBV in tumor cells, these EBV‐induced tumors were identified as human B‐cell lymphomas. Thus, CSA can strikingly inhibit GVHD in hu‐PBL/SCID chimeras, and should therefore be effective to establish a stable SCID mouse model of human lymphoma associated with EBV. Treatment with CSA had no effect on the tumor incidence in hu‐PBL/SCID chimeras after active EBV infection. Accordingly, serum level of sIL‐2R is a valuable indicator of GVHD occurrence in hu‐PBL/SCID chimeras.
Glycosylphosphatidyl inositol‐anchored protein (GPI‐80) gene expression is correlated with human thymoma stage
Authors: Sasaki Hidefumi , Ide Nobuyuki , Sendo Fujiro , Takeda Yuji , Adachi Masakazu , Fukai Ichiro , Fujii Yoshitaka
Published: August 2005

Thymoma is one of the most common solid tumors in the mediastinum. Because there is no typical cell line for human thymoma, the development and use of molecular‐based therapy for thymoma will require detailed molecular‐genetic analysis of patients' tissues. Recent reports showed that genetic aberrations in thymoma were most frequently seen in chromosome 6q regions. We investigated the use of oligonucleotide arrays to monitor in vivo expression levels of genes in chromosome 6 regions in early‐(stage I or II) and late‐ (stage IVa) stage thymoma tissues from patients. These in vivo gene expression profiles were verified by real‐time quantitative reverse transcription polymerase chain reaction (RT‐PCR) using LightCycler for 48 thymoma patients and sandwich ELISA for 33 thymoma patients. Using both methods, a candidate gene was identified which was overexpressed in stage IV thymoma. This was a known glycosylphosphatidyl inositol (GPI)‐anchored protein (GPI‐80), which is highly homologous with Vanin‐1, a mouse thymus homing protein. Serum level of GPI‐80 was confirmed to be elevated in stage IV thymoma compared with in stage I thymoma by using sandwich ELISA. The combined use of oligonucleotide microarray, real‐time RT‐PCR, and ELISA analyses provides a powerful new approach to elucidate the in vivo molecular events surrounding the development and progression of thymoma.
Mirimostim (macrophage colony‐stimulating factor; M‐CSF) improves chemotherapy‐induced impaired natural killer cell activity, Th1/Th2 balance, and granulocyte function
Authors: Hidaka Takao , Akada Shinobu , Teranishi Akiko , Morikawa Hajime , Sato Shinji , Yoshida Yuji , Yajima Akira , Yaegashi Nobuo , Okamura Kunihiro , Saito Shigeru
Published: August 2005

The purpose of this study was to clarify the effects of mirimostim (macrophage colony‐stimulating factor; M‐CSF) on immunological functions after chemotherapy. The percentage of natural killer (NK) cells in peripheral blood mononuclear cells (PBMCs), NK cell activity, T‐helper cell 1/T‐helper cell 2 (Th1/Th2) ratio, and superoxide anion production by granulocytes (granulocyte function) were measured as immunological parameters before and after chemotherapy in 44 patients with primary ovarian cancer who received at least three consecutive courses of postoperative chemotherapy. Patients were observed during the first course of chemotherapy, and 39 patients who presented grade III or IV neutropenia were entered into this study and randomly allocated to an M‐CSF‐administered group (group 1; 19 patients) and a non‐M‐CSF‐administered group (group 2; 20 patients) for the second course. For the third course, a crossover trial was conducted. In the observation period, chemotherapy significantly impaired the immunological parameters. In particular, those parameters were significantly decreased at day 14 compared to the level before chemotherapy. The values of the parameters of group 1 were significantly higher than those of group 2. In the course of chemotherapy during which M‐CSF was administered, 19 of the 39 patients presented grade IV neutropenia, and received granulocyte colony‐stimulating factor (G‐CSF) between days 7 and 14. We compared the changes of those immunological parameters in the M‐CSF alone group and the M‐CSF+G‐CSF group, and found that the concomitant use of G‐CSF did not further improve the parameters. These results indicate that chemotherapy markedly impaired the immunological functions, and that the administration of M‐CSF significantly improved the impaired immunological functions.
lnterferon‐β gene therapy for cancer: Basic research to clinical application
Authors: Yoshida Jun , Mizuno Masaaki , Wakabayashi Toshihiko
Published: August 2005

Interferon‐β gene therapy for cancer is the first such protocol developed in Japan. Here we describe the development process of our interferon‐β gene therapy from basic research to clinical application. Interestingly, the biological and biochemical characteristics of interferon‐β gene therapy through transfer of the interferon‐(gene into tumor cells by means of cationic liposomes differed from those of conventional interferon‐β protein therapy. Interferon‐β gene transfer could induce apoptosis in interferon‐β protein‐resistant tumor cells, such as glioma, melanoma, and renal cell carcinoma. Induction of apoptosis was related to the level of intracellular mRNA of interferon‐β, prolongation of the phos‐phorylation time of molecules in the interferon‐p signal transduc‐tion pathway, such as JAK1, Trk2, and STAT1, and activation of DNase γ. In our preclinical study we developed lyophilized cat‐ionic liposomes containing interferon‐β gene (gene drug) for clinical use and confirmed their safety. Thereafter, we performed a pilot clinical trial in patients with malignant glioma and confirmed the safety and effectiveness of this interferon‐β gene therapy. In this review we also comment on the status of gene therapy regulation in Japan. Interferon‐β gene therapy is expected to become widely available for clinical use in cancer patients, and this new strategy might be extended to molecular targeting therapy, or used in combination with cell therapy or other therapies.
mRNA expression level of estrogen‐inducible gene, α1‐antichymotrypsin, is a predictor of early tumor recurrence in patients with invasive breast cancers
Authors: Yamamura Jun , Miyoshi Yasuo , Tamaki Yasuhiro , Taguchi Tetsuya , Iwao Kyoko , Monden Morito , Kato Kikuya , Noguchi Shinzaburo
Published: August 2005

We previously identified 18 genes that correlated with ER positivity by adapter‐tagged competitive‐PCR analysis of 2412 genes in human breast cancer tissues. The aim of the present study was to determine the prognostic significance of these genes. mRNA expression levels of 12 of the above 18 genes were quantified in breast cancer tissues by real‐time PCR assay, and their association with patients' prognosis (n=110) was studied according to hormone receptor (HR) status. In addition, the genes found to influence prognosis were further investigated to examine whether their mRNA expression could be induced by estrogen in MCF‐7 cells in vitro. Of the 12 genes, mRNA expression levels of two [α1‐antichymotrypsin (ACT) and stanniocalcin 2 (STC2)] were significantly (P=0.002 and P=0.007, respectively) associated with good prognosis in HR‐positive (ER and/or PR positive) breast cancer patients treated with adjuvant hormone therapy. Multivariate analysis showed that ACT mRNA level, but not STC2 mRNA level, in HR‐positive patients, was a significant prognostic factor (P=0.042), which was independent of tumor size and lymph node metastases. On the other hand, mRNA expressions of ACT and STC2 were not significantly associated with prognosis in HR‐negative patients. Estradiol treatment resulted in a significant increase in the mRNA levels of both ACT and STC2 in MCF‐7 cells. The mRNA level of ACT, which is an estrogen‐inducible gene, is a significant predictor of good prognosis in HR‐positive, but not HR‐negative, patients with breast cancers. Since HR‐positive patients were treated with adjuvant hormone therapy, we suggest that ACT mRNA level could potentially be used as a predictor of response to hormone therapy, rather than a prognostic factor (predictor of metastatic potential).
Novel models for human scirrhous gastric carcinoma in vivo
Authors: Takemura Satoru , Yashiro Masakazu , Sunami Takeshi , Tendo Masashige , Hirakawa Kosei
Published: August 2005

Human scirrhous gastric carcinoma, a diffusely infiltrating type of poorly differentiated gastric carcinoma also known as linitis plastica type carcinoma, is characterized by cancer cell infiltration and proliferation accompanied with extensive stromal fibrosis. We established two new gastric cancer cell lines, designated OUCM‐8 and OCUM‐11, which developed the characteristic biology of scirrhous gastric carcinoma upon orthotopic implantation in mice. Involvement of lymph nodes and liver metastasis was also found in both orthotopic models. Histologically, these orthotopic models showed proliferation with extensive fibrosis, resembling human scirrhous gastric cancer. Both cell lines were derived from ascites of patients with scirrhous gastric cancer. The growth of OCUM‐8 and OCUM‐11 cells following the addition of KGF, FGF, and EGF was increased significantly relative to untreated cells. An increase in the number of attached and spreading cells occurred following the addition of TGF‐β1 in both cell lines. OCUM‐11 cells showed microsatellite instability. Although subcutaneous scirrhous gastric cancer cells show medullary growth, most in vivo studies of scirrhous gastric cancer have used xenografted tumors implanted subcutaneously. Only in a few cases was it confirmed that these scirrhous gastric cancer cell lines retained the original histologic characteristics. Our orthotopic models should contribute to the elucidation of disease progression in situ and to the development of therapy for scirrhous gastric cancer.
Immunity against mouse thymus‐leukemia antigen (TL) protects against development of lymphomas induced by a chemical carcinogen, N‐butyl‐N‐nitrosourea
Authors: Tsujimura Kunio , Obata Yuichi , Matsudaira Yasue , Ozeki Satoshi , Taguchi Osamu , Nishida Keiko , Okanami Yuko , Akatsuka Yoshiki , Kuzushima Kiyotaka , Takahashi Toshitada
Published: August 2005

Mouse thymus‐leukemia antigens (TL) are aberrantly expressed on T lymphomas in C57BL/6 (B6) and C3H/He (C3H) mice, while they are not expressed on normal T lymphocytes in these strains. When N‐butyl‐N‐nitrosourea (NBU), a chemical carcinogen, was administered orally to B6 and C3H strains, lymphoma development was slower than in T3b‐TL gene‐transduced counterpart strains expressing TL ubiquitously as self‐antigens, suggesting that anti‐TL immunity may play a protective role. In addition, the development of lymphomas was slightly slower in C3H than in B6, which seems to be in accordance with the results of skin graft experiments indicating that both cellular and humoral immunities against TL were stronger in C3H than B6 mice. The interesting finding that B lymphomas derived from a T3b‐TL transgenic strain (C3H background) expressing a very high level of TL were rejected in C3H, but not in H‐2Kb transgenic mice (C3H background), raises the possibility that TL‐specific effector T cell populations are eliminated and/or anergized to a certain extent by interacting with H‐2Kb molecules.
Unique characteristics of rectal carcinoma cell lines derived from invasive carcinomas in ulcerative colitis patients
Authors: Yamashita Kazuya , Yasuda Shuichi , Kuba Tatsuru , Otani Yoshimasa , Fujiwara Mutsunori , Okayasu Isao
Published: August 2005

To identify the characteristics of ulcerative colitis (UC)‐associated carcinomas, 8 lesions, high‐grade dysplasias and invasive carcinomas, were implanted into severely combined immunodeficient (SCID) mice and/or cultured in vitro. Intramucosal neoplasias consisting of high‐grade dysplasia showed extremely slow proliferation after implantation (2/3 cases) and in vitro culture failed (4 cases). However, invasive carcinomas demonstrated rapid growth both after SCID mouse implantation and in vitro (4/4 cases). From two cases of invasive carcinomas, 6 cell lines were established, and these are the first to be described in the literature. In addition to variation in immunohistochemically determined phenotypic expression regarding α‐fetoprotein, chromogranin A and estrogen receptors, the established cell lines showed varying differentiation (moderately or poorly differentiated adenocarcinoma, adenosquamous carcinoma and poorly differentiated adenocarcinoma with multinuclear giant cells and bone formation). The results are in contrast with findings for sporadic colorectal carcinomas. Although the prevalence of DNA alterations is not frequent, loss of heterozygosity (17p and 18q) and deletion of exons 8 to 11 in DPC‐4 were revealed in all of 6 cell lines, suggesting relatively high genetic instability. We found loss or translocation of many chromosomes (#3, 5, 6, 8, 10, 11, 13, 16, 17, 18 and 19) other than chromosomes 1, 5, 8, 11, 13, 17 and 18, which are frequently involved in sporadic colorectal carcinoma cell lines. Thus, the established cell lines may be good models of tumorigenesis and progression in the chronic inflammation‐carcinoma sequence.
Expression of RCAS1 in human gastric carcinoma: A potential mechanism of immune escape
Authors: Nakamura Yuichi , Yamazaki Koichi , Oizumi Satoshi , Nakashima Manabu , Watanabe Takeshi , Dosaka‐Akita Hirotoshi , Nishimura Masaharu
Published: August 2005

RCAS1 (receptor‐binding cancer antigen expressed on SiSo cells) inhibits the in vitro growth of receptor‐expressing cells and induces apoptosis, which may contribute to the ability of tumor cells to evade host immune surveillance. In this study, we investigated RCAS1 expression in gastric cancer and precancerous lesions by immunohistochemical means. We then analyzed the relationship between RCAS1 expression and clinicopathological variables, and examined whether RCAS1 expression is associated with infiltration of tumor‐infiltrating lymphocytes (TILs) and apoptosis of TILs. Of 54 gastric cancers analyzed, RCAS1 expression was positive in 52 (96%) of them. The expression pattern of RCAS1 in gastric cancer cells could be classified as granular staining either enriched in the glandular side of the cytoplasm with polarity (P pattern) or scattered diffusely in the cytoplasm and on the cell membranes (D pattern). Nineteen of 39 intestinal‐type carcinomas (49%) showed the P pattern, and all of 13 diffuse type carcinomas (100%) showed the D pattern. In contrast, all RCAS1‐positive specimens of gastric adenoma and metaplastic mucosa were of the P pattern. The D pattern of gastric cancers was more frequently recognized in carcinomas with large size (P<0.01), in those with regional lymph node metastasis (P<0.05) and in those that had invaded beyond the submucosa (P<0.01), compared with the P pattern. On the same sections, significantly less TILs were identified in RCAS1‐positive areas than RCAS1‐negative areas. Furthermore, the rate of apoptosis of TILs was significantly higher in RCAS1‐positive areas than in RCAS1‐negative areas. The expression and distribution of RCAS1 may be involved in malignant transformation, tumor progression, histological type and tumor escape from host immune surveillance in gastric cancer.
Antitumor effect of MCC‐465, pegylated liposomal doxorubicin tagged with newly developed monoclonal antibody GAH, in colorectal cancer xenografts
Authors: Hamaguchi Tetsuya , Matsumura Yasuhiro , Nakanishi Yukihiro , Muro Kei , Yamada Yasuhide , Shimada Yasuhiro , Shirao Kuniaki , Niki Hisae , Hosokawa Saiko , Tagawa Toshiaki , Kakizoe Tadao
Published: August 2005

MCC‐465 is an immunoliposome‐encapsulated doxorubicin. The liposome is tagged with polyethylene glycol and the F(ab)2 of a monoclonal antibody named GAH, a human antibody obtained by the hybridoma technique. The epitope recognized by GAH is not well characterized, but human gastric, colorectal, and mammary cancer cells were GAH‐positive, while the normal counterparts were GAH‐negative. Pegylated liposome doxorubicin (PLD) and MCC‐465 did not show significant antitumor activity against GAH‐negative Caco‐2 xenografts. On the other hand, MCC‐465 exhibited significantly superior antitumor effects against GAH‐positive WiDr‐Tc and SW837 xenografts, compared with PLD. Immunohis‐tochemistry with GAH revealed that 94% (100 of 106) of surgical specimens of colorectal cancer were GAH‐positive. These results warrant a phase I clinical trial of MCC‐465 for patients with metastatic colorectal cancer.
Direct inhibition of EGF receptor activation in vascular endothelial cells by gefitinib (‘Iressa’, ZD1839)
Authors: Hirata Akira , Uehara Hisanori , Izumi Keisuke , Naito Seiji , Kuwano Michihiko , Ono Mayumi
Published: August 2005

The development of gefitinib (‘Iressa’, ZD1839) by targeting the EGFR tyrosine kinase is a recent therapeutic highlight. We have reported that gefitinib is antiangiogenic in vitro, as well as in vivo. In this study, we asked if the anti‐angiogenic action of gefitinib is due to a direct effect on activation of vascular endothelial cells by EGF. EGF, as well as VEGF, caused pronounced angiogene‐sis in an avascular area of the mouse cornea, and i.p. administration of gefitinib almost completely blocked the response to EGF, but not to VEGF. Immunohistochemical analysis demonstrated phosphorylation of EGFR by EGF in the neovasculature, and gefitinib markedly reduced this effect. Gefitinib also inhibited downstream activation of ERK 1/2 via EGFR in cultured microvascular endothelial (HMVE) cells. These findings suggest that the anti‐angiogenic effect of gefitinib in the vascular endothelial cells of neo‐vasculature is partly attributable to direct inhibition of EGFR activation, and that endothelial cells in malignant tumors play a critical role in the cancer therapeutic efficacy of gefitinib.
Transforming growth factor‐β signaling is differentially inhibited by Smad2D450E and Smad3D407E
Authors: Kondo Miki , Suzuki Hiroyuki , Takehara Kazuhiko , Miyazono Kohei , Kato Mitsuyasu
Published: August 2005

A missense mutant of Smad2, Smad2D450E, that was not phosphorylated by transforming growth factor β (TGF‐β) signaling, was identified in colorectal cancer. Previously, we constructed a mutant Smad3, Smad3D407E, which has an Asp to Glu mutation in the corresponding position of Smad2D450. Smad3D407E was not phosphorylated by the constitutively active form of the TGF‐β type I receptor, and inhibited the phosphorylation of co‐expressed wild‐type Smad2 and Smad3. In the present study, we examined the inhibitory effects of Smad2D450E on TGF‐β signaling and found that there are considerable differences between Smad2D450E and Smad3D407E. Smad2D450E suppressed the phosphorylation of Smad2, but did not affect the phosphorylation of Smad3, while Smad3D407E blocked the phosphorylation of both Smad2 and Smad3. Consistent with these results, Smad2D450E reduced hetero‐oligomer formation of Smad2 with Smad4, but not of Smad3 with Smad4, while Smad3D407E reduced hetero‐oligomer formation of both Smad2 and Smad3 with Smad4. However, Smad2D450E reduced the binding of Smad3 to a target DNA element as well as Smad2‐binding, and Smad2D450E had inhibitory effects on the transcriptional activity of several targets, as Smad3D407E did. These results suggested that Smad2D450E might block the Smad3‐mediated TGF‐β signaling in a hitherto unknown manner after the phosphorylation and hetero‐oligomer formation, such as in the process of nuclear translocation or transcriptional regulation, which we could not identify previously by using Smad3D407E. (Cancer Sci 2004; 95: 12–17)
Relation of cigarette smoking and alcohol use to colorectal adenomas by subsite: The self‐defense forces health study
Authors: Toyomura Kengo , Yamaguchi Keizo , Kawamoto Hideki , Tabata Shinji , Shimizu Eiichi , Mineshita Masamichi , Ogawa Shinsaku , Lee Kyong‐Yeon , Kono Suminori
Published: August 2005

While smoking has consistently been shown to be related to increased risk of colorectal adenomas, few studies have addressed the association between smoking and site‐specific colorectal adenomas. The reported association between alcohol use and colorectal adenomas has been inconsistent. We evaluated risks of adenomas at the proximal colon, distal colon, and rectum in relation to cigarette smoking and alcohol use, and their interaction. Subjects were 754 cases with histologically proven colorectal adenomas and 1547 controls with normal colonoscopy among male officials of the Self‐Defense Forces (SDF) undergoing total colonoscopy at two SDF hospitals. Statistical adjustment was made for hospital, rank, body mass index, physical activity, and either smoking or alcohol drinking. Cigarette smoking was significantly associated with an increased risk of adenomas, regardless of the location of the adenomas, but the increased risk associated with smoking was more pronounced for rectal adenomas. Alcohol use was associated with moderately increased risks of distal colon and rectal adenomas, but not of proximal colon adenomas. Cigarette smoking, but not alcohol drinking, was associated with greater increases in the risk of large adenomas and of multiple adenomas across the colorectum. There was no measurable interaction of cigarette smoking and alcohol drinking on colorectal adenomas. The findings corroborate an increased risk of colorectal adenomas associated with smoking and a weak association between alcohol use and colorectal adenomas. Further studies are needed to confirm whether smoking is more strongly related to rectal adenomas, large adenomas, or multiple adenomas. (Cancer Sci 2004; 95: 72–76)
Mechanism and biological significance of CD44 cleavage
Authors: Nagano Osamu , Saya Hideyuki
Published: August 2005

There are multiple steps in the metastasis of cancer cells. Tumor cells must first detach from the tumor mass and invade the surrounding extracellular matrix (ECM). In this step, cell surface adhesion molecules play an important role in the interaction between the cells and their microenvironments. CD44 is an adhesion molecule that interacts with hyaluronic acid (HA) and is implicated in a wide variety of physiological and pathological processes. Recently, proteolytic cleavages of CD44 have been emerging as key regulatory events for the CD44 dependent cell‐matrix interaction and signaling pathway. CD44 undergoes sequential proteolytic cleavages in the ectodomain and intramem‐branous domain, resulting in the release of a CD44 intracellular domain (ICD) fragment. The ectodomain cleavage of CD44 is triggered by multiple stimulations and contributes to the regulation of cell attachment to and migration on HA matrix. The ectodomain cleavage subsequently induces the intramembranous cleavage, which is mediated by presenilin (PS)‐dependent y‐secre‐tase. The intramembranous cleavage generates CD44ICD, which acts as a signal transduction molecule; it is translocated to the nucleus and activates transcription. An understanding of the underlying mechanism of these cleavages of CD44 could provide novel therapeutic targets for cancer cell invasion and metastasis.
hOGGl Ser326Cys polymorphism, interaction with environmental exposures, and gastric cancer risk in Japanese populations
Authors: Tsukino Hiromasa , Hanaoka Tomoyuki , Otani Tetsuya , Iwasaki Motoki , Kobayashi Minatsu , Hara Megumi , Natsukawa Syusuke , Shaura Kozo , Koizumi Yoichi , Kasuga Yoshio , Tsugane Shoichiro
Published: August 2005

Oxidative DNA damage caused by reactive oxygen species (ROS) generated by Helicobacter pylori (H. pylori) infection or smoking may be a cause of gastric cancer development. 8‐Hydroxydeoxyguanine (8‐OHdG) formation is one of the most common types of oxidative DNA damage, while human oxoguanine glycosylase 1 (hOGGl) is responsible for repairing 8‐OHdG lesions. Among several hOGGl gene polymorphisms, the SerCys polymorphism at position 326 is related to biological function. To investigate the association between Ser326Cys hOGGl polymorphism and gastric cancer in relation to the potential risk factors of gastric cancer and antioxidant dietary or nutrient intakes, we conducted a case‐control study with 142 histologically‐confirmed gastric cancer cases and 271 age, sex‐matched healthy controls in Japanese populations. Overall, neither the hOGG1 Ser/Cys nor the Cys/Cys genotype was associated with risk of gastric cancer, compared with the Ser/Ser genotype. A significant interaction was observed between hOGGl Ser/Cys or Cys/Cys genotype and atrophic gastritis (P for interaction =0.03). No significant interaction was found between hOGGl genotype and antioxidant dietary or nutrient intakes. The results of the present study suggest that patients with atrophic gastritis in conjunction with the hOGGl Cys allele might have a higher susceptibility to gastric cancer.
Inhibition of RL male 1 tumor growth in BALB/c mice by introduction of the RLakt gene coding for antigen recognized by cytotoxic T‐lymphocytes and the GM‐CSF gene by in vivo electroporation
Authors: Tanaka Motoyuki , Yamada Masaharu , Ono Toshiro , Noguchi Yuji , Uenaka Akiko , Ota Seisuke , Hata Hidenori , Harada Mine , Tanimoto Mitsune , Nakayama Eiichi
Published: August 2005

A DNA vaccine for inducing a tumor immune response was investigated using a well‐characterized murine model tumor antigen. We demonstrated that in vivo electroporation augmented the induction of IFNγ enzyme‐linked immunospot (ELISPOT) and cytotoxic T lymphocyte (CTL) generation against pRL1a peptide in BALB/c spleen cells upon immunization with RLakt plasmid. Immunization without in vivo electroporation resulted in only a marginal induction of IFNmG ELISPOT and CTL generation. Furthermore, co‐injection of GM‐CSF and RLakt plasmids significantly enhanced the induction of IFNmG ELISPOT and CTL generation compared to the injection of RLakt plasmid alone. Inhibition of RL male 1 tumor growth was observed by injecting BALB/c mice with GM‐CSF and RLakt plasmids using in vivo electroporation, although no effect was observed against an established tumor using the same treatment. No growth inhibition was observed without in vivo electroporation. Immunization with either RLakt plasmid alone, or GM‐CSF and pCIneo control plasmids using in vivo electroporation did not inhibit RL male 1 tumor growth.
Potent antitumor effect elicited by superantigen‐linked tumor cells transduced with heat shock protein 70 gene
Authors: Huang Changxin , Yu Hai , Wang Qingqing , Ma Wenxue , Xia Dajing , Yi Pingyong , Zhang Lihuang , Cao Xuetao
Published: August 2005

Heat shock proteins (HSP) induce antitumor‐specific immunity via a unique mechanism, but HSP alone fails to produce a satisfactory antitumor efficacy. We considered that the potent immune‐activation of superantigen (SAg) might assist HSP to elicit a strong tumor‐antigen‐specific immunity. We initially prepared B16 melanoma cells linked to SAg SEA via a fusion protein with a trans‐membrane sequence (TM), and demonstrated that SEA thus anchored on the tumor cell surface could elicit strong antitumor immunity. We then prepared cells transduced with an inducible heat shock protein 70 (HSP70) gene, and bearing SEA‐TM fusion protein on the cell surface, and used these cells as a dual‐modified vaccine. In this study, either in a therapeutic setting or in a pre‐immune model, the SEA‐anchored vaccine or the HSP70 gene‐modified vaccine induced marked tumor suppression, prolonged survival, augmented lymphocyte proliferation and higher NK and CTL activity in C57BL/6 mice compared with their controls (P<0.01), though they were less effective than the dual‐modified vaccine. Among these vaccines, the dual‐modified vaccine showed the best therapeutic efficacy in B16 melanoma‐bearing mice and gave the greatest protection against wild‐type B16 melanoma challenge. The results indicated that the dual‐modified vaccine could induce a potent tumor‐antigen‐specific immune response in addition to an increase of non‐specific immunity. This study offers a novel approach to bridging specific and non‐specific immunity for cancer therapy.
DE‐310, a novel macromolecular carrier system for the camptothecin analog DX‐8951f: Potent antitumor activities in various murine tumor models
Authors: Kumazawa Eiji , Ochi Yusuke
Published: August 2005

DE‐310 is a novel macromolecular conjugate composed of DX‐8951f, a camptothecin analog, and a carboxymethyldextran polyalcohol carrier, which are covalently linked via a peptidyl spacer. In a murine Meth A (fibrosarcoma) solid tumor model, once daily×5 treatments (qd×5) with DX‐8951f at the maximum tolerated dose (MTD) were required to shrink the tumor, and DX‐8951f (qd×5) at 1/4 MTD was required to inhibit tumor growth. A single treatment (qd×1) with DE‐310 at the MTD or 1/4 MTD shrank the tumor, with no body weight loss occurring at 1/4 MTD. Even at 1/16 MTD, DE‐310 inhibited tumor growth. In a long‐term assay, Meth A solid tumors disappeared in mice treated with DE‐310 (qd×1) at the MTD and 1/2 MTD, and all 6 mice remained tumor‐free on the 60th day after administration. Repeated injection (4 times) on schedules of every 3 days, 7 days or 14 days demonstrated that multiple treatment with DE‐310 produced greater tumor growth delay than a single treatment with DE‐310. Against 5 human tumor (colon and lung cancer) xenografts in mice, DE‐310 (qd×1) was as effective as DX‐8951f administered once every 4 days, 4 times. The life‐prolonging activity of DE‐310 was assessed in lung (3LL, Lewis lung carcinoma) and liver (M5076, histiocytoma) metastasis models. Against 3LL, DE‐310 (qdx1) at the MTD to 1/3 MTD significantly prolonged survival, with an increase in life span (ILS) of 4.8‐ to 1.6‐fold, respectively, over that in untreated control mice. Also, DE‐310 (qd×1) significantly prolonged survival in the liver metastasis model of M5076. These results demonstrate that DE‐310 is a promising agent for the treatment of cancer.
p53‐independent anti‐tumor effects of the nitrogen‐containing bisphosphonate zoledronic acid
Authors: Kuroda Junya , Kimura Shinya , Segawa Hidekazu , Sato Kiyoshi , Matsumoto Seiji , Nogawa Masaki , Yuasa Takeshi , Kobayashi Yutaka , Yoshikawa Toshikazu , Ottmann Oliver G. , Maekawa Taira
Published: August 2005

Zoledronic acid (ZOL), a nitrogen‐containing bisphosphonate, exerts anti‐tumor effects by inhibiting the prenylation of small GT‐Pases. We have also reported that ZOL shows an anti‐leukemic effect by inducing apoptosis throughout the S phase to the G2/M boundary. Here, we studied the effects of ZOL on various cell cycle regulators, including p53, cyclin‐dependent kinases (CDKs), CDK inhibitors and cyclins, using BV173 leukemia and HCT116 colorectal carcinoma cell lines, harboring wild‐type (wt‐) p53. ZOL induced the accumulation of neither p53 nor p21WAF1/CIP1 during the execution of apoptosis in BV173 cells. Therefore, we investigated the dependence of ZOL‐induced apoptosis on intact p53 by using wt‐p53 HCT116 and a p53‐degraded HCT116 subline, and observed no significant difference. p57KIP2 was upregulated by ZOL in BV173 cells, but not in HCT116 cells. Flow cytometric analyses showed that ZOL also impaired the cell cycle‐dependent expression patterns of cyclins A, B and D3 in BV173. In conclusion, the p53‐independent anti‐tumor activities of ZOL suggest that it may be an attractive agent for treating cancers, including those with chemoresistance resulting from the loss of p53 function. ZOL also affected the coordinate expression patterns of several cell cycle regulators during the execution of anti‐tumor activity.
Presence of human mycoplasma DNA in gastric tissue samples from Korean chronic gastritis patients
Authors: Kwon Hyuk‐Joon , Kang Jeong‐Ok , Cho Sun‐Hee , Kang Hee‐Bum , Kang Kyung‐Ah , Kim Jeong‐Ki , Kang Yoon‐Suk , Song Byung‐Cheol , Kang Hyun‐Wook , Shim Mi‐Ja , Kim Hee‐Sun , Kim Young‐Bae , Hahm Ki‐Baeg , Kim Bum‐Joon , Kook Myeong‐Cherl , Chung Myung‐Hee , Hyun Jin‐Won
Published: August 2005

We aimed to determine whether mycoplasmas are present in Korean chronic gastritis, and to understand their roles in gastric cancer tumorigenesis, because mycoplasmas resemble Helicobacter pylori in terms of ammonia production and induction of inflammatory cytokines in immune and non‐immune cells. The presence and identity of mycoplasmas were assessed by semi‐nested PCR and sequencing, and the results were compared with pathologic data. Fifty‐six samples collected from Korean chronic gastritis patients were used for this study. Twenty‐three (41.1%) were positive for mycoplasmas. Eighteen sequenced samples contained a single human mycoplasma or two mycoplasmas, which were identified as Mycoplasma faucium (13/18), M. fermentans (3/18), M. orale (1/18), M. salivarium (2/18), and M. spermatophilum (1/18). Mycoplasma‐infected chronic gastritis samples showed significantly more severe neutrophil infiltration than non‐infected samples (P=0.0135). Mycoplasma profiles in the oral cavity (M. salivarium is major) and stomach were different, and the presence of significant proinflammatory responses in mycoplasmapositive patients suggests that the mycoplasmas are not simply contaminants. Further studies are required to understand whether mycoplasmas play a role in gastric tumorigenesis.
Suppression of VEGFR‐3 signaling inhibits lymph node metastasis in gastric cancer
Authors: Shimizu Kenji , Kubo Hajime , Yamaguchi Koji , Kawashima Kazuhiko , Ueda Yoshihide , Matsuo Koichi , Awane Masaaki , Shimahara Yasuyuki , Takabayashi Arimichi , Yamaoka Yoshio , Satoh Seiji
Published: August 2005

In gastric cancer, lymph node metastasis is one of the major prognostic factors and forms the basis for surgical removal of local lymph nodes. Recently, several studies have demonstrated that overexpression of lymphangiogenic growth factor VEGF‐C or VEGF‐D induces tumor lymphangiogenesis and promotes lymphatic metastasis in mouse tumor models. We examined whether these processes could be inhibited in naturally metastatic tumors by blocking of their cognate receptor VEGFR‐3 signaling pathway. Using a mouse orthotopic gastric cancer model which has a high frequency of lymph node metastasis, we estimated lymphatic vessels in gastric cancers by immunostaining for VEGFR‐3 and other specific lymphatic markers, LYVE‐1 and prox‐1. Then we systemically administered anti‐VEGFR‐3 blocking antibodies. This treatment resulted in the inhibition of regional lymph node metastasis and reduction of lymphatic vessel density in the primary tumors. In addition, increased density of LYVE‐1‐positive lymphatic vessels of primary tumors was closely correlated with lymph node metastasis in human samples of gastric cancer. Antilymphangiogenesis by inhibiting VEGFR‐3 signaling could provide a potential strategy for the prevention of lymph node metastasis in gastric cancer.
Nitric oxide generated by iNOS reduces deformability of Lewis lung carcinoma cells
Authors: Igawa Satoshi , Hayashi Izumi , Tanaka Naohiko , Hiruma Hiromi , Majima Masataka , Kawakami Tadashi , Hirose Minoru , Masuda Noriyuki , Kobayashi Hirosuke
Published: August 2005

Previous studies have indicated that NO plays a crucial role in the metastasis of tumor cells and that tumor cells produce nitric oxide (NO) via inducible nitric oxide synthase (iNOS). Since the deformability of tumor cells is an important factor governing their metastatic potential, in this study we investigated the regulation of tumor cell deformability by NO. Lewis lung tumor cells (3LL cells) were also incubated with a cytokine mixture (IL‐1β, IFNγ, and TNFα). The nitrite/nitrate content of the supernatant was then measured by the Griess method, and iNOS expression was evaluated by RT‐PCR in vitro. Nitrite/nitrate was produced in response to administration of the cytokine mixture, and iNOS mRNA was expressed in the cytokine‐treated cells. The deformability of the 3LL cells was evaluated by measuring the peak pressure generated during their passage through a microfilter at a constant flow rate. Both the cytokine mixture and NO donor (NOC 18) significantly increased the filtration pressure, and the staining of the cells with rhodamine‐phalloidin revealed assembly of F‐actin in the cell membrane. In conclusion, NO plays a role in the decreased deformability of tumor cells, suggesting that NO is one of the factors that regulates metastasis.
Carbohydrate‐mediated cell adhesion in cancer metastasis and angiogenesis
Authors: Kannagi Reiji , Izawa Mineko , Koike Tetsufumi , Miyazaki Keiko , Kimura Naoko
Published: August 2005

Malignant transformation is associated with abnormal glycosylation, resulting in the synthesis and expression of altered carbohydrate determinants including sialyl Lewisa and sialyl Lewisx. The sialyl Lewisa and sialyl Lewisx determinants appear in the sera of patients with cancer, and are extensively utilized for serum diagnosis of cancers in Japan. Sialyl Lewisa and sialyl Lewisx are involved in selectin‐mediated adhesion of cancer cells to vascular endothelium, and these determinants are thought to be closely associated with hematogenous metastasis of cancers. Recent progress in this area includes the following: 1. Substantial increases in solid clinical statistics that further confirm the contribution of these determinants in the progression of a wide variety of cancers; 2. Elucidation of the ligand specificity of the three family members of selectins and evaluation of the roles of these molecules in cancer cell adhesion; and 3. Advances in the study of the mechanism that leads to the enhanced expression of the sialyl Lewisa/x determinants in malignant cells. These recent results have confirmed that these determinants are not merely markers for cancers, but are functionally implicated in the malignant behavior of cancer cells. The results also suggested that the increase of these determinants in malignant cells is an inevitable consequence of the malignant transformation of cells. Considerable new knowledge has also been accumulated regarding the therapeutic implications for suppression of hematogenous metastasis targeting this cell adhesion system.