bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Inhibition of O6‐Alkylguanine‐DNA Alkyltransferase in Rats on Carcinogenesis by Methylnitrosourea and Ethylnitrosourea
Authors: Lijinsky William , Pegg Anthony E. , Anver Miriam R. , Moschel Robert C.
Published: August 2005

Many alkylating agents are potent carcinogens and there is considerable evidence that the formation of O6‐alkylguanine in DNA can lead to mutations and the initiation of neoplastic growth. The repair of O6‐methyl‐ or O6‐ethylguanine in DNA is known to be brought about by the action of a protein termed O6‐alkylguanine‐DNA alkyltransferase. In order to investigate the role of this activity in the carcinogenic action of methylnitrosourea and ethylnitrosourea, O6‐benzylguanine, a potent inhibitor of the alkyltransferase, was used. Groups of 20 female F344 rats were treated with the nitrosourea (0.2 mmol) by gavage in 10 weekly doses and a parallel group was also treated with 4 mg of O6‐benzylguanine, 2 h prior to each dose of the nitrosourea. This dose of O6‐benzylguanine was sufficient to reduce the alkyltransferase activity to zero in the liver for at least 8 h but activity had returned to about 60% of normal within 24 h. Animals were maintained until they became moribund, when they were killed, or until death related to tumors. The median week of death in the animals receiving methylnitrosourea was reduced from 60 wk to 52 wk by co‐treatment with O6‐benzylguanine. There was a smaller reduction from 55 to 50 wk in the rats receiving ethylnitrosourea. The treatment with O6‐benzylguanine caused no significant change in the incidence of the principal tumors induced by the alkylnitrosoureas and there were no liver tumors produced by the combined treatments. These results show that the level of inactivation of alkyltransferase produced by this dose of O6‐benzylguanine was not sufficient to greatly alter the potent carcinogenic effect of these doses of alkylnitrosoureas in this system.
Macrophage Colony‐stimulating Factor Induces the Growth and Differentiation of Normal Pregnancy Human Cytotrophoblast Cells and Hydatidiform Moles but Does Not Induce the Growth and Differentiation of Choriocarcinoma Cells
Authors: Saito Shigeru , Ibaraki Tamotu , Enomoto Masahiro , Ichijo Motohiko , Motoyoshi Kazuo
Published: August 2005

In the present study, we examined whether or not macrophage colony‐stimulating factor (M‐CSF; CSF‐1) is involved in the growth and differentiation of human chorionic, hydatidiform mole and choriocarcinoma cells. M‐CSF promotes the growth of early gestation chorionic cells, hydatidiform mole cells, and a human term placenta cell line (tPA30‐1). However, the growth of choriocarcinoma cells, BeWo, Jar, Jeg‐3, and NUC‐1, was not influenced at all by M‐CSF. M‐CSF promoted the secretion of human chorionic gonadotropin (hCG) and human placental lactogen (hPL), which are secreted from differentiated trophoblast, from early gestation chorionic cells and from hydatidiform mole cells. However, the secretion of hCG and hPL from choriocarcinoma cells was not affected by M‐CSF. When M‐CSF localization was examined by immunohistochemical staining, M‐CSF was detected in chorionic and hydatidiform mole cells, but was absent in choriocarcinoma cells. These results suggest that the growth and differentiation of normal chorionic and hydatidiform mole cells are M‐CSF‐dependent, while the growth and differentiation of choriocarcinoma cells are not.
BE‐23372M, a Novel and Specific Inhibitor for Epidermal Growth Factor Receptor Kinase
Authors: Tanaka Seiichi , Okabe Takayoshi , Chieda Shinya , Endo Kaori , Kanoh Tomoko , Okura Akira , Yoshida Eisaku
Published: August 2005

The fungal metabolite BE‐23372M is a structurally novel protein kinase inhibitor. Its IC50 for epidermal growth factor (EGF) receptor kinase was 0.03 μM. IC50 values of BE‐23372M for other protein tyrosine kinases, erbB‐2, p43v‐abl, insulin receptor kinase, and p60c‐src were 0.42, 1.0, 3.3, and 4.5 μM, respectively, and the IC50 for protein kinase C, a serine/threonine kinase, was 4.1 μM. Cdc2 kinase, casein kinases I and II and cAMP‐dependent protein kinase were not inhibited by 20 μM BE23372M. A kinetic study showed that BE‐23372M was competitive with respect to the substrate peptide and to ATP. Autophosphorylation of solubilized EGF receptor kinase was clearly inhibited by 0.1 μM BE‐23372M. Autophosphorylation of EGP receptor in A431 cells was also inhibited. These results show that BE‐23372M is a potent and specific EGF receptor kinase inhibitor. It should be a valuable tool for EGF receptor kinase research.
bcl‐2 Regulation in Normal Resting Lymphocytes and Lymphoblasts
Authors: Kendo Eisaku , Yoshino Tadashi , Nomura Shintaro , Nakamura Shuji , Takahashi Kiyoshi , Teramoto Norihiro , Hayashi Kazuhiko , Akagi Tadaatsu
Published: August 2005

Expression of the bcl‐2 protein and bcl‐2 mRNA at the individual cell level was semiquantitatively examined in normal quiescent peripheral blood lymphocytes and pokeweed mitogen‐ or concanavalin A and interleukin‐2‐induced lymphoblasts in vitro by microscopic fluorometry using immunofluorescence and fluorescein‐labeled in situ hybridization. Approximately 90% of normal quiescent T and B lymphocytes expressed bcl‐2 protein at a level which was compatible with that of bcl‐2 mRNA. On the contrary, most mitogen‐induced lymphoblasts showed a posttranscriptional suppression of bcl‐2 protein expression. However, bcl‐2 protein was not downregulated by the posttranscriptional suppression in all lymphocytes activated in vitro, but approximately 15% of the lymphoblasts still expressed bcl‐2 protein at a higher level than nontransformed quiescent small lymphocytes; thus bcl‐2 protein expression in lymphoblasts showed a distinct bimodal pattern. Furthermore, it was supposed that lymphoblasts with no detectable bcl‐2 protein might fall into apoptosis but the remainder, expressing high levels of bcl‐2 protein, could escape apoptosis. Thus, the bcl‐2 gene may play an important role as a regulator of apoptosis in the human immune system.
Selective Increases in Isoform PP1α of Type‐1 Protein Phosphatase in Ascites Hepatoma Cells
Authors: Takizawa Norio , Mizuno Yusuke , Saadat Mostafa , Kikuchi Kunimi
Published: August 2005

The amounts of four isoforms of the catalytic subunit of type‐1 protein phosphatases, PP1α, PP1γ1, PP1γ2, and PP1δ, have been determined in extracts of rat ascites hepatomas, AH131A, AH13, AH13NMOR, AH143A, and Yoshida sarcoma, and compared to those of rat liver by Western blot analysis. The amount of PP1α was increased over three times in all five hepatomas. The amount of PP1γ1 was increased over two times in AH13, AH13NMOR, and AH143A. The amount of PP1δ was selectively increased about 4 times in AH131A and AH143A. The PP1γ2 protein was undetectable in both liver and hepatomas. There was good parallelism between the general increase in only PP1α protein in the hepatomas and the previous data demonstrating the general increase in PP1α mRNA in numerous ascites hepatomas. These results suggest that PP1α plays important roles in the expression of malignant phenotype, that its amount is under strict control at the transcription level, and that PP1γ1 and PP1δ play different roles in the expression of some phenotype(s) of the ascites hepatomas.
Frequency of Atypical Mitosis in Intestinal Metaplasia of the Gastric Mucosa in Japanese Patients
Authors: Rubio Carlos A. , Kato Yo , Kitagawa Tomoyuki
Published: August 2005

The morphologic characteristics of the mitotic figures present in intestinal metaplasia (IM) of the gastric mucosa were investigated in 70 concecutive gastrectomy specimens from Japanese nationals. The specimens contained, in addition, early gastric cancer of intestinal type (n=44) or of diffuse type (n=22). The remaining 4 were recorded as mixed. One hundred or more consecutive mitoses/specimens were studied by high‐power microscopy in hematoxylin and eosin‐stained preparations (at × 1000). A total of 7259 mitoses were recorded (mean 103.7 mitoses/case). Of these, 1089 mitoses (i.e. 19.1%) were considered as atypical according to a previous classification. The percentage of atypical mitoses was found to be unrelated to the gender, to the increasing age of the patients, to the histologic type of the adenocarcinoma contained in the specimens, or to the anatomic site (e.g. corpus or antrum or tumor proximity). Comparative studies were done with gastrectomy specimens from Swedish nationals (a population with a 4‐times‐lower incidence of gastric carcinoma than the Japanese). The results showed a much lower frequency of mitotic figures/specimen and only occasional atypical mitosis. Since atypical mitosis has so far been reported only for neoplastic lesions in the gastrointestinal tract, it is suggested that IM with atypical mitosis may be a genuine precancerous lesion in the gastric mucosa in Japanese subjects.
Development and Characterization of Chimeric Anti‐carcinoembryonic Antigen Monoclonal Antibodies and Their Fab Fragments
Authors: Kamigaki Takashi , Ohyanagi Harumasa , Yamamoto Masahiro , Kaneda Teruo , Goto Takashi , Ohmura Takao , Yokoyama Kazumasa , Saitoh Yoichi
Published: August 2005

In an attempt to reduce the immunogenicity of two different murine anti‐carcinoembryonic antigen (CEA) monoclonal antibodies (MAbs), KM10 and A10, we produced recombinant mouse/human chimeric MAbs and the respective Fab fragments carrying the variable regions of the murine MAbs. Chimeric A10 Fab fragment was expressed in Escherichia coli, and produced in large quantities in a mini‐jar fermentation system. In competitive binding assays, chimeric MAbs and their Fab fragments showed identical specificity to human CEA epitopes, as compared to the parental MAbs or Fab fragments. Both chimeric Fab fragments exhibited strong immunohistochemical reactivity with various gastrointestinal carcinomas and no reactivity with CEA‐related antigens, such as NCA (nonspecific cross‐reacting antigen) or BGPI (biliary glycoprotein I). Furthermore, chimeric KM10 MAb elicited substantially higher antibody‐dependent cellular cytotoxicity than the murine MAb. Complement‐dependent cytotoxicity in vitro was much weaker with chimeric KM10 MAb. These results indicate that chimeric MAbs or Fab fragments could potentially replace the parental murine antibodies or their Fab fragments in therapy or diagnosis of human gastrointestinal carcinomas.
Augmented Systemic Immunity in Mice Implanted with Tumor Necrosis Factor‐α Gene‐transduced Meth‐A Cells
Authors: Fujii Shigeyuki , Liu Yingxian , Neda Hiroshi , Itoh Yoshinori , Koshita Yoshikazu , Takahashi Minoru , Watanabe Naoki , Kohgo Yutaka , Niitsu Yoshiro
Published: August 2005

Syngeneic BALB/c mice bearing methylcholanthrene‐induced fibrosarcoma (Meth‐A) cells transduced with a tumor necrosis factor (TNF) gene showed a longer life span and tumor regression as compared with mice carrying TNF‐non‐producing Meth‐A cells. To elucidate the mechanism of the reduced tumorigenicity of TNF‐producing Meth‐A, we compared systemic immune responses between mice bearing high TNF producer (C5) and unmodified Meth‐A cells (M0). The results indicated that the cytotoxic activity of lymphokine‐activated killer cells (LAK) induced from spleen cells of mice bearing C5 was higher against both M0 and C5 than that of LAK from mice bearing M0. Also, C5 was more sensitive to LAK induced from spleen cells of C5‐ and M0‐ bearing mice than M0. We also found that cytotoxic T lymphocyte from spleen cells of mice transplanted with C5 was more cytotoxic to M0 than that from mice with M0. In addition, the population of Lyt2 (CD8)‐positive T cells was higher in freshly isolated spleen cells of mice transplanted with C5 than from mice with M0. Finally, we observed a higher expression of MHC class 1 antigen on C5 than on M0. These observations suggest that the augmented host systemic immunity in mice carrying TNF gene‐modified Meth‐A cells is one of the mechanisms of the reduced tumorigenicity of C5 and that the increased systemic immunity can be ascribed to the increased immunogenicity of the tumor cells. Thus, the use of TNF gene‐modified tumor cells as vaccines appears to be promising for therapeutic and/or prophylactic application.
Detection of Ultraviolet Photoproducts in Mouse Skin Exposed to Natural Sunlight
Authors: Qin Xiusheng , Zhang Shaomin , Zarkovic Mirjana , Nakatsuru Yoko , Shimizu Seiichiro , Yamazaki Yukari , Oda Hideaki , Nikaido Osamu , Ishikawa Takatoshi
Published: August 2005

In the present study, we for the first time investigated the formation of ultraviolet (UV) photoproducts, cyclobutane pyrimidine dimers (CPDs), pyrimidine‐pyrimidone (6–4) photoproducts (64PPs) and Dewar isomers, in vivo in shaved and depilated C3H/HeN mouse skin exposed to natural sunlight (NSL) at noon for 5 min to 1 h in mid‐summer, using a highly sensitive immunohistochemical method. This method permits the quantitative analysis of UV‐photoproducts in formalin‐fixed, paraffin‐embedded sections with specific antibodies against CPDs, 64PPs and Dewar isomers. We demonstrated that the induction of CPDs in vivo in mouse skin by NSL was exposure time‐dependent, but the accumulation of 64PPs or Dewar isomers was comparatively low in the skin sections from mice exposed to NSL in vivo. The results indicate that CPDs are the main photoproducts in vivo induced by sunlight and that their formation and repair may be important in connection with carcinogenesis in sun‐exposed areas of human skin.
p53 Gene Deficiency Does Not Enhance Instability of Mouse Minisatellites in Somatic Cells of Normal Tissues
Authors: Ohashi Manabu , Hatakeyama Katsuyoshi , Aizawa Shinichi , Kominami Ryo
Published: August 2005

The effect of p53‐deficiency on somatic mutation of minisatellites in normal tissues was examined using p53‐deficient (−/−) mice. In total, 248 mice consisting of three different genotypes, +/+, +/− and −/−, were obtained and DNA from their embryos was probed with two minisatellites, Pc‐1 and Pc‐2. The somatic mutation was detected by Southern blot hybridization as the presence of a third nonparental band reflecting mosaicism in tissues. Mutation frequency of Pc‐1 for (+/+) and (+/−) was 1.3% and 1.4%, respectively, which is consistent with previous studies. On the other hand, none of the mice lacking the p53 gene (−/−) exhibited mutation. The Pc‐2 probe did not show any somatic mutation in the three groups. These results suggest that the p53 deficiency does not enhance the genomic instability of the minisatellite loci in normal somatic cells.
Acquisition of Invasive Phenotype in Gallbladder Cancer Cells via Mutual Interaction of Stromal Fibroblasts and Cancer Cells as Mediated by Hepatocyte Growth Factor
Authors: Matsumoto Kunio , Date Kazuhiko , Shimura Hideo , Nakamura Toshikazu
Published: August 2005

Growth and motility of carcinoma cells are regulated through their interactions with host stromal cells, i. e., tumor‐stromal interactions. Hepatocyte growth factor (HGF), a ligand for c‐Met tyrosine kinase, is a stromal‐derived regulator of growth, motility, and morphogenesis. HGF stimulated proliferation and motility of GB‐d1 gallbladder carcinoma cells from a patient with gallbladder cancer. HGF induced in vitro invasion of GB‐d1 cells into a collagen gel matrix, and this potent, invasive effect was not seen with epidermal growth factor, transforming growth factor‐β1, basic fibroblast growth factor, or platelet‐derived growth factor. Although GB‐d1 did not produce HGF, the cells did produce a factor which enhances HGF production in human skin fibroblasts, and this factor proved to be interleuldn‐1β (IL‐1β). When GB‐d1 cells were co‐cultured with fibroblasts such that a collagen gel matrix was layered between the GB‐d1 cells and fibroblasts, GB‐d1 cells invaded the gel, but invasion of the cells in the co‐culture system was inhibited by antibodies against HGF and partially inhibited by antibodies against IL‐1β. Thus, GB‐d1 cell‐derived IL‐1β stimulates HGF production in stromal fibroblasts and HGF up‐regulated in the fibroblasts induces invasion of GB‐d1 cells. The looped interaction of carcinoma cells and stromal fibroblasts mediated by HGF and a HGF‐inducer such as IL‐1β may be one mechanism which would explain the acquisition of malignant phenotype through tumor‐stromal interactions.
Image Quantitation of Intestinal Metaplasia in Entire Gastrectomy Specimens from Swedish and Japanese Patients
Authors: Rubio Carlos A. , Matthies Marianne , Itabashi Masayuki , Hirota Teruyuki
Published: August 2005

The aim of this work was to investigate the extension of intestinal metaplasia (IM), as well as to quantitate various components of IM (namely sialomucins, sulfomucins and Paneth cells), in entire gastrectomy specimens from Swedish and Japanese patients. The length of the gastric mucosa was assessed by morphometry. The percent of sections with IM was regarded as the extension of IM in the specimens. Histochemically labeled sialomucins, sulfomucins and Paneth cells (the 3 main findings in gastric IM) were quantified in separate sections with the aid of an image analyzer. In total, 1,321 sections corresponding to 6 gastrectomy specimens were quantified. Sialomucins and sulfomucins were more extensively distributed in the 4 specimens with carcinoma than in the 2 without carcinoma (one having a peptic ulcer and the other, hereditary gastric cancer syndrome (HGCS) without carcinoma). On the other hand, quantitative analysis in Swedish specimens indicated that the highest values for sialomucins, sulfomucins and Paneth cells were present in HGCS. When Swedish and Japanese specimens with adenocarcinoma were compared, only sulfomucins (denoting Types II and III IM) were significantly higher in those carrying an intestinal‐type carcinoma (ITC) than in those with diffuse‐type carcinoma (DTC). The results substantiate those obtained with gastric biopsies by other authors. On the other hand, the mucosal extension and the amount of sulfomucins are not comparable parameters (since that mucin was not equally distributed, but “concentrated” in certain areas in the mucosa). One possible conclusion is that the focal distribution of acidic mucins and of Paneth cells in the gastric mucosa may strongly influence their detection rate in gastric biopsies. Thus, haphazard biopsy of the gastric mucosa may fail to sample areas with sulfomucins in population studies aiming to detect individuals at risk. Such sampling errors in gastric biopsies may explain the conflicting results on this subject appearing in the literature.
Rejection of Mouse Renal Cell Carcinoma Elicited by Local Secretion of Interleukin‐2
Authors: Hara Isao , Hotta Hak , Sato Noriyuki , Eto Hiroshi , Arakawa Soichi , Kamidono Sadao
Published: August 2005

We introduced the interleukin‐2 (IL‐2) gene into mouse renal cell carcinoma (RenCa) in order to examine the mechanism of tumor rejection. IL‐2 gene‐transfected RenCa (RenCa/IL‐2Hi) exhibited marked retardation of tumor growth when implanted in a syngeneic host. Growth retardation of RenCa/IL‐2Hi was also observed in athymic nude mice even after depletion of natural killer (NK) cells by treatment with anti‐asialo GM1 antibody. Histological analysis of RenCa/IL‐2Hi tumors disclosed non‐specific inflammatory changes in syngeneic hosts. Co‐injection of Bacillus Calmette Guerin with RenCa/IL‐2Hi considerably enhanced the anti‐tumor effects. Taken together, these findings strongly suggest that in situ IL‐2 production leads to tumor rejection through non‐specific inflammatory responses without participation of T cells and NK cells. On the other hand, the syngeneic mice that had rejected RenCa/IL‐2Hi acquired immunity against parental RenCa, suggesting possible participation of memory T cells in the second rejection of the tumor.
p53 Antisense Oligonucleotide Inhibits Growth of Human Colon Tumor and Normal Cell Lines
Authors: Hirota Yasuhide , Horiuchi Tadashi , Akahane Kouichi
Published: August 2005

We examined the relationship between the expression of mutant p53 proteins and tumor cell growth using a p53 antisense oligonucleotide (5′‐CCCTGCTCCCCCCTGGCTCC‐3′). The oligonucleotide inhibited the growth of three human colon tumor cell lines (DLD‐1, SW620 and WiDr), which produce only mutant p53 proteins with different mutation sites. Treatment of DLD‐1 cells with the p53 antisense oligonucleotide caused a decrease in the level of p53 mutant protein. Synthesis of DNA in DLD‐1 and SW620 cells was inhibited more potently than that of RNA or protein after antisense treatment. Furthermore, these cells were accumulated in the S phase when DNA synthesis was inhibited. Meanwhile, the antisense oligonucleotide also inhibited the growth of three human normal cell lines (WI‐38, TIG‐1 and Intestine 407). While treatment of WI‐38 and TIG‐1 cells with the antisense oligonucleotide inhibited synthesis of DNA more potently than that of RNA or protein, these normal cells were accumulated in the G0/G1 phase. These results suggest that p53 proteins, either with or without mutation, play a pivotal role in the growth of tumor and normal cells, but that mutant and wild‐type p53 proteins may function differently in cell growth.
Transduction of the Macrophage Colony‐stimulating Factor Gene into Human Multidrug Resistant Cancer Cells: Enhanced Therapeutic Efficacy of Monoclonal Anti‐P‐glycoprotein Antibody in Nude Mice
Authors: Sone Saburo , Tsuruo Takashi , Sato Shigeo , Yano Seiji , Nishioka Yasuhiko , Shinohara Tsutomu
Published: August 2005

To develop a therapeutic modality for overcoming multidrug‐resistant (MDR) cancer with anti‐MDR1 antibody, we examined the effect of macrophage colony‐stimulating factor (M‐CSF) gene transfection into MDR AD10 cells on therapy of MDR cancer with anti‐MDR1 antihody (MRK17) in nude mice. MDR human ovarian cancer (AD10) cells were transduced with the human M‐CSF gene inserted into an expression vector to establish gene‐modified cells capable of producing low (ML‐AD10), intermediate (MM‐AD10) and high (MH‐AD10) amounts of M‐CSF. Systemic administration of MRK17 resulted in significant dose‐dependent inhibition of subcutaneous growth of ML‐AD10 tumors. In contrast, systemic administration of recombinant M‐CSF in combination with MRK17 did not augment the therapeutic efficacy of MRK17 alone, but rather promoted the growth of the parent AD10 cells. To test the efficacy of in vivo M‐CSF gene therapy combined with antibody, we mixed the parent AD10 cells with MH‐AD10 cells producing a large amount of M‐CSF, and inoculated the mixed cells subcutaneously. Treatment with MRK17 inhibited growth of the mixed cells more than that of the parent cells alone. Thus, combined therapy with anti‐MDR1 mAb and M‐CSF gene modification of MDR cancer cells may provide a new immunotherapeutic modality for overcoming MDR in humans.
Biological Activity and Intracellular Metabolism of ZD1694 in Human Leukemia Cell Lines with Different Resistance Mechanisms to Antifolate Drugs
Authors: Takemura Yuzuru , Kobayashi Hiroyuki , Miyachi Hayato , Gibson William , Kimbell Rosemary , Jackman Ann L.
Published: August 2005

The biological activity and cellular metabolism of ZD1694, a novel folate‐based thymidylate synthase (TS) inhibitor, were analyzed in a human leukemia cell line, MOLT‐3, and its antifolate‐resistant sublines with different mechanisms of resistance to methotrexate (MTX), trimetrexate (TMQ) and N10‐propargyl‐5,8‐dideazafolic acid (CB3717). MOLT‐3/CB371740, which was selected for CB3717 resistance, demonstrated impaired membrane drug transport via reduced folate carrier (RFC) and lower accumulation of [3H]ZD1694‐polyglutamates in the cells with a shift in the polyglutamate distribution profile to shorter chain length polyglutamates, indicating an alteration in polyglutamation capacity in this subline. Impaired RFC and reduced rate of polyglutamation could explain the cross‐resistance (12‐fold) of this subline to ZD1694. On the other hand, there was little or no cross‐resistance to this drug in a subline (MOLT‐3/TMQ800) reportedly resistant to TMQ through impaired membrane transport for TMQ and an increase in dihydrofolate reductase (DHFR) activity. Total amount of ZD1694 polyglutamated to a level higher than diglutamate was approximately 1.7‐fold higher in the TMQ‐resistant cells than that in the parent cells, but a low degree of increase in TS activity in the cells counteracted the supposed increase in sensitivity to ZD1694. MOLT‐3/TMQ800‐MTX10000 cells, which were established by sequential exposure of the TMQ‐resistant cells to MTX and were previously shown to amplify mutated DHFR with low affinity for MTX, showed a decreased accumulation of polyglutamated ZD1694 as compared with the parent line and this was consistent with cross‐resistance to ZD1694 in this subline. Overproduction of variant DHFR scarcely influenced the sensitivity to this drug. These results indicate that ZD1694 could overcome antifolate resistance through a mechanism such as amplified DHFR activity, and the biological activity of this drug against the cells paralleled the amount of polyglutamated drug inside the cells. Determination of polyglutamation capacity in tumor cells may allow prediction of sensitivity to this drug.
Determinants of Myelosuppression in the Treatment of Non‐small Cell Lung Cancer with Cisplatin‐containing Chemotherapy
Authors: Matsui Kaoru , Masuda Noriyuki , Uchida Yasuo , Fukuoka Masahiro , Negoro Shunichi , Yana Takashi , Kusunoki Yoko , Kudoh Shinzoh , Kawase Ichiro , Kawahara Masaaki , Ogawara Mitsumasa , Kodama Nagahisa , Kubota Kaoru , Furuse Kiyoyuki
Published: August 2005

Data on 16 potential risk factors for myelosuppression were assessed in 134 patients who received either vindesine and cisplatin (VP) or mitomycin C, vindesine and cisplatin (MVP) for inoperable stage III or IV non‐small cell lung cancer in a randomized trial. Determinant factors for myelosuppression were evaluated by using univariate analysis and the logistic regression model. Recursive partitioning and amalgamation (RPA) was also used to define patient subgroups frequently suffering from severe bone marrow toxicity. Overall, 33 (25%) of 134 patients experienced at least one episode of grade 4 leukopenia. In univariate analysis, age, body surface area, serum creatinine, and pretreatment hemoglobin concentration were associated with severe leukopenia. A multivariate analysis using the logistic regression method showed that only raised creatinine level was an independent predictor for grade 4 leukopenia (P=0.049). The RPA model generated three distinct subgroups based on age, body surface area and regimen. The three subgroups were distinguished by the frequency of severe (grade 4) leukopenia (50%, 25%, and 2.4%, respectively) (P<0.001). Grade 4 leukopenia occurred more frequently in patients in class 3 (age ≥65 years and treatment with MVP). The RPA model was useful in identifying the risk factors for myelosuppression induced by cisplatin‐based chemotherapy, and in defining patient subgroups with elevated risk of toxicity.
Increased MCL–1 Expression Is Associated with Poor Prognosis in Ovarian Carcinomas
Authors: Shigemasa Kazushi , Katoh Osamu , Shiroyama Yuko , Mihara Shoji , Mukai Keiji , Nagai Nobutaka , Ohama Koso
Published: August 2005

To investigate the potential role of the BCL–2 gene family (BAX, BCL–2, MCL–1, and BCL‐XL) in ovarian cancer development and progression, mRNA expression levels of these genes were measured using semi‐quantitative PCR in epithelial ovarian tumor tissues and normal ovaries. The immunohistochemical expression of MCL–1 in ovarian tumors was also examined. The expression levels of BAX and MCL–1 mRNA were significantly higher in ovarian cancers and in adenomas than in normal ovaries (P<0.05). In contrast, the BCL–2 mRNA expression level in ovarian cancers was significantly lower than in ovarian adenomas and in normal ovaries (P<0.05). Expression of BCL‐XL mRNA was no different between normal ovaries and ovarian tumors. Log‐rank testing showed that low BAX mRNA expression and high MCL–1 mRNA expression significantly correlate with poor survival for patients with stage III ovarian carcinomas (BAX, P=0.05; MCL–1, P=0.02). Immunohistochemical analysis showed that diffuse‐positive expression of MCL–1 protein in mucinous carcinomas was significantly higher than in mucinous low malignant potential (LMP) tumors (P=0.03). In ovarian cancer cases, diffuse‐positive expression of MCL–1 protein significantly correlates with advanced clinical stage, high histologic grade, and poor survival (stage, P<0.01; grade, P=0.01; survival, P=0.01). These results suggest that increased MCL–1 expression may play an important role in replacing the functions of increased BAX and decreased BCL–2 in ovarian carcinoma cells, thereby promoting cell survival, and resulting in a poor prognosis for patients with ovarian cancer.
Evaluation of a Gemcitabine‐Doxorubicin‐Paclitaxel Combination Schedule through Flow Cytometry Assessment of Apoptosis Extent Induced in Human Breast Cancer Cell Lines
Authors: Serrano Maria J. , Sánchez‐Rovira Pedro , Algarra Ignacio , Jaén Ana , Lozano Ana , Gaforio José J.
Published: August 2005

Combination chemotherapy with gemcitabine (Gem), doxorubicin (Dox), and paclitaxel (Pac) (GAT) has been considered attractive as first‐line treatment in metastatic breast cancer. We compared the potential of various schedules of GAT to induce apoptosis on MDA‐MB–231, MCF7, and T47D human breast cancer cell lines. The extent of apoptotic induction was analyzed by flow cytometry with 7–aminoactinomycin D (7AAD) staining. Differences between various schedules in terms of apoptotic induction were statistically significant (P<0.05). The most effective apoptotic induction regimen was achieved by the sequence: Dox for 16 h followed by Pac+Gem. Schedules employing a 16–h interval between drug administrations induced higher levels of apoptosis in human breast cancer cell lines compared with schedules using a 4–h interval. The therapeutic efficacy of the experimental results shown in this paper has been clinically corroborated in a phase II trial in metastatic breast cancer patients.
Inhibition of Nucleotide Excision Repair by Fludarabine in Normal Lymphocytes in vitro, Measured by the Alkaline Single Cell Gel Electrophoresis (Comet) Assay
Authors: Yamauchi Takahiro , Kawai Yasukazu , Ueda Takanori
Published: August 2005

Alkylating agents or platinum analogues initiate several excision repair mechanisms, which involve incision of the DNA strand, excision of the damaged nucleotide, gap filling by DNA resynthesis, and rejoining by ligation. The previous study described that nucleotide excision repair permitted incorporation of fludarabine nucleoside (F‐ara‐A) into the repair patch, thereby inhibiting the DNA resynthesis. In the present study, to clarify the repair kinetics in view of the inhibition by F‐ara‐A, normal lymphocytes were stimulated to undergo nucleotide excision repair by ultraviolet C (UV) irradiation in the presence or absence of F‐ara‐A. The repair kinetics were determined as DNA single strand breaks resulting from the incision and the rejoining using the alkaline single cell gel electrophoresis (comet) assay. DNA resynthesis was evaluated in terms of the uptake of tritiated thymidine into DNA. The lymphocytes initiated the incision step maximally at 1 h, and completed the rejoining process within 4 h after UV exposure. UV also initiated thymidine uptake, which increased time‐dependently and reached a plateau at 4 h. A 2–h pre‐incubation with F‐ara‐A inhibited the repair in a concentration‐dependent manner, with the maximal inhibition by 5 μM. This inhibitory effect was demonstrated by the reduction of the thymidine uptake and by the inhibition of the rejoining. A DNA polymerase inhibitor, aphidicolin, and a ribonucleotide reductase inhibitor, hydroxyurea, were not so inhibitory to the repair process as F‐ara‐A at equimolar concentrations. The present findings suggest that inhibition of nucleotide excision repair may represent a novel therapeutic strategy against cancer, especially in the context of resistant cells with an increased repair capacity.
Dexamethasone‐resistant Human Pre‐B Leukemia 697 Cell Line Evolving Elevation of Intracellular Glutathione Level: An Additional Resistance Mechanism
Authors: Inoue Hitoshi , Takemura Haruyuki , Kawai Yasukazu , Yoshida Akira , Ueda Takanori , Miyashita Toshiyuki
Published: August 2005

Glucocorticoids remain among the most important drugs in the treatment of acute lymphoblastic leukemia (ALL). Although the mechanisms of glucocorticoid resistance have been studied in some T‐cell leukemic cell lines, less work has been done with B‐cell lines. We established a dexamethasone (DEX)‐resistant human pre‐B lineage leukemia cell line (697/DEX) and investigated the mechanism of resistance. 697/DEX was over 430–fold more resistant to DEX compared with the parental cells (697/Neo). Overexpression of Bcl–2 protein was not observed in 697/DEX, different from the mechanism of resistance in Bcl–2–virus‐infected cells (697/Bcl–2). Although the expression of p‐glycoprotein (Pgp) in 697/DEX was positive, its functional activity was not detected. The numbers of glucocorticoid receptors (GR) in 697/DEX and 697/Bcl–2 were significantly lower than those in 697/Neo. In addition, 697/DEX and 697/Bcl–2 had higher levels of glutathione (GSH) than 697/Neo. In the presence of l‐buthionine‐(S, R)‐sulfoximine (BSO), an inhibitor of GSH synthesis, both 697/DEX and 697/Bcl–2 recovered their sensitivity to DEX. Interestingly, cell death by the depletion of GSH did not involve caspase–3/7 activation in 697/Bcl–2 and 697/DEX, different from 697/Neo, suggesting a death mechanism through caspase‐independent programmed cell death or necrosis. In conclusion, DEX‐resistance in 697/DEX was related not only to a GR decrease, but also to an increase in intracellular GSH level in the DEX‐resistant B‐cell leukemia cell line. Circumvention of DEX‐resistance with BSO may offer an approach to overcoming resistance to chemotherapy in B‐cell lineage ALL.
Genetic Polymorphisms of the UDP‐Glucuronosyltransferase 1A7 Gene and Irinotecan Toxicity in Japanese Cancer Patients
Authors: Ando Maki , Ando Yuichi , Sekido Yoshitaka , Ando Masahiko , Shimokata Kaoru , Hasegawa Yoshinori
Published: August 2005

Irinotecan often causes unpredictably severe, occasionally fatal, toxicity involving leukopenia or diarrhea. It is converted by carboxyesterase to an active metabolite, SN–38, which is further conjugated and detoxified to SN–38–glucuronide by UDP‐glucuronosyltransferase (UGT). We genotyped the UGT1A7 gene by direct sequencing analysis and polymerase chain reaction‐restriction fragment length polymorphism in 118 cancer patients and 108 healthy subjects. All the patients had received irinotecan‐containing chemotherapy and were evaluated to see whether the variant UGT1A7 genotype would increase the likelihood of severe toxicity of irinotecan consisting of grade 4 leukopenia and/or grade 3 or more diarrhea. Among the 26 patients with severe toxicity, the allele frequencies were 61.5% for UGT1A7*1, 15.4% for UGT1A7*2, and 23.1% for UGT1A7*3. On the other hand, the frequencies were 63.6% for UGT1A7*1, 15.8% for UGT1A7*2, and 20.7% for UGT1A7*3 among the 92 patients without severe toxicity. None of the 118 patients had UGT1A7*4. Neither univariate analysis (odds ratio, 1.13; 95% confidential interval, 0.46–2.75) nor multivariate logistic regression analysis (odds ratio, 0.74; 95% confidential interval, 0.26–2.07) found any significant association between carrying at least one of the variant alleles and the occurrence of severe toxicity. The distribution of UGT1A7 genotypes in 108 healthy subjects was not significantly different from that in the patients (P=0.99 and 0.86 for those with and without severe toxicity, respectively), but significantly less than that in Caucasians reported previously (P<0.001). The results suggested that determination of UGT1A7 genotypes would not be useful for predicting severe toxicity of irinotecan.
Stability of a Recombinant Adenoviral Vector: Optimization of Conditions for Storage, Transport and Delivery
Authors: Ugai Hideyo , Watanabe Sanae , Suzuki Erika , Tsutsui‐Nakata Hatsumi , Yokoyama Kazunari K. , Murata Takehide
Published: August 2005

Recombinant viral vectors have been developed for use as therapeutic agents and for the introduction of exogenous genes into living cells. However, little is known about the viability and stability of such recombinant viruses during storage, transport and delivery under various conditions. We describe here an analysis of the stability of an adenoviral vector in crude solutions of cell lysates during freezing and thawing and during storage at various temperatures in the presence and in the absence of glycerol. For example, the titer of adenoviruses in crude lysates of infected cells was reduced only ten‐fold or three‐fold after two hundred rounds of freezing and thawing or after incubation at 28°C for 14 days, respectively. Our observations indicate that recombinant adenoviral vector was more stable than expected both during freezing and thawing and during storage at low temperatures. Our results confirm the importance of appropriate conditions for the delivery and transport of recombinant adenoviral vectors.
Close Association between Fas Ligand (FasL; CD95L)‐positive Tumor‐associated Macrophages and Apoptotic Cancer Cells along Invasive Margin of Colorectal Carcinoma: A Proposal on Tumor‐Host Interactions
Authors: Sugita Junichi , Ohtani Haruo , Mizoi Takayuki , Saito Kazuya , Shiiba Kenichi , Sasaki Iwao , Matsuno Seiki , Yagita Hideo , Miyazawa Masaaki , Nagura Hiroshi
Published: August 2005

Anti‐tumor immune responses are considered to be one of the key host reactions in human colorectal cancer, with T cells as important effector cells. For the induction of tumor‐specific immunity, processing of cancer cells and pruning of T cells by antigen‐presenting cells are important. The present study was designed to clarify the relationship between Fas ligand (FasL; CD95L) expression and apoptotic cancer cells. Immunohistochemistry using frozen sections taken from 58 patients with colorectal cancer revealed that stromal cells composed mainly of tumor‐associated macrophages expressed FasL, leaving cancer cells negative for FasL. These macrophages were abundantly distributed along the invasive margin. In situ hybridization revealed that these macrophages as well as cancer cells expressed FasL mRNA, whereas macrophages in the normal colon mucosa rarely expressed FasL. Apoptotic cancer cells recognized by monoclonal antibody M30 CytoDEATH were localized not only in cancer cell nests, but also in the stroma along the invasive margin showing a dissociated pattern, which was particularly evident in the areas of FasL+ macrophages. Furthermore, these macrophages, phenotypically similar to dendritic cells, occasionally contained M30+ apoptotic cancer cells in the cytoplasm. Clinicopathologic analyses in 123 cases revealed 1) a positive correlation between the degree of dissociated M30+ apoptotic cancer cells and the number of macrophages along the invasive margin and 2) an inverse association between the degree of dissociated M30+ apoptotic cancer cells and the occurrence of hematogenous metastasis after surgical resection of the primary tumor. In conclusion, the present study shows the impor‐ tance of FasL+ activated macrophages as one of the host defense mechanisms against cancer cell spread in human colorectal cancer.
Temperature‐sensitive Ovarian Carcinoma Cell Line (OvBH‐1)
Authors: Bar Julia K. , Haroziska Antonina , Kartarius Sabine , Montenarh Mathias , Wyrodek Ewa , Parkitna Jan M. Rodriguez , Kochman Marian , Ozyhar Andrzej
Published: August 2005

OvBH‐1 cells from a patient with ovarian clear cell carcinoma were established and their biochemical status was analyzed. Cells grown at 37°C exhibited normal cell cycle distribution, whereas the cells shifted to 31°C were arrested in the G2/M phase of the cell cycle. Immunochemical analysis using anti‐p53 antibodies (DO‐1, PAb240, PAb421, and PAbl620) revealed that only the DO‐1 antibody reacted with p53 with a high and similar percentage at both temperatures. PAb240 reacted with a low percentage of cells at 37°C and no reaction was observed at 31°C. PAb421 antibody stained a significantly lower percentage of cells at 37°C than at 31°C. Cells were not stained with PAbl620 antibody and were negative for antibodies against p21WAF1 and MDM2 proteins independently of the temperature. Sequencing of all coding exons of the p53 gene demonstrated only a neutral genetic polymorphism, i.e. a G‐to‐A substitution (GAG to GAA) at nucleotide position 13 432. Thus, the observed temperature sensitivity of OvBH‐1 cells cannot be ascribed to a p53 primary structure mutation. Based upon immunochemical analyses, we consider, however, that p53 in nuclei of OvBH‐1 cells is in a highly unstable conformation. Furthermore, the N‐terminal portion of the p53 protein at Ser20 has not been modified, and Lys373 and/or Ser378 of the C‐terminus is acetylated and/or phosphorylated. The nuclear location signal of p53 is preserved. Induction of MDM2 protein is uncoupled from the cell regulatory machinery and the induction of p21WAF1 by p53 is unpaired in OvBH‐1 cells.
Expression of Ly‐6D on the Surface of Normal and Neoplastic Mammary Epithelial Cells of the Mouse
Authors: Tsukada Yasukatsu , Sasaki Tetsuo , Hanyu Kazuhide , Enami Jumpei
Published: August 2005

Rat were immunized with mouse mammary epithelial cells and monoclonal antibodies (MAbs) were obtained to identify antigens stably expressed on the surface of both normal and neoplastic mammary epithelial cells of the mouse. Examination of the reactivities of the MAbs by immunofluorescence staining of tissue sections showed that one of the antibodies, MAb 2A2, reacted with luminal epithelium of the mammary gland and spontaneous mammary carcinomas of SHN mice. Further examination of the tissue lysates by western blot analysis revealed that MAb 2A2 reacts with a 17‐kDa antigen expressed in normal mammary epithelial cells and mammary carcinoma cells. The antigen recognized by MAb 2A2 was absent in the lysates of liver, lung, salivary gland, kidney, small intestine, ovary and uterus. After immunoaffinity purification of the MAb 2A2‐ recognized antigen and determination of its N‐terminal amino acid sequence, we identified the antigen as Ly‐6D, also known as ThB, which belongs to a family of glycosyl‐phosphatidylinositol‐anchored cell surface proteins. Northern blot analysis further demonstrated that Ly‐6D mRNA is expressed in the mammary gland. Based on these observations we concluded that Ly‐6D is stably expressed on the surface of both normal and neoplastic mammary epithelial cells of the mouse. Ly‐6D will serve as a useful epithelial cell surface marker for the study of mammary gland development, as well as for breast cancer research.
Allelic Losses in Mouse Skin Tumors Induced by γ‐Irradiation of p53 Heterozygotes
Authors: Miyazawa Tomonori , Sato Hiroki , Hatakeyama Katsuyoshi , Kitagawa Tomoyuki , Kominami Ryo
Published: August 2005

Skin tumors were induced by γ‐irradiation in F1 mice between C3H/He or BALB/c and MSM carrying a p53‐deficient allele. The incidence was 39.1% (34/87) in p53 (KO/+) mice of the C3H/MSM genetic background and 14.3% (19/133) in those of the BALB/MSM background. Interestingly, most of the tumors (82%) lost the wild‐type p53 allele and no skin tumor was found in p53 (+/+) F1 mice. This suggests a requirement of p53 loss for the skin cancer development. Genome scan localized a chromosomal locus showing frequent allelic losses near D12Mit2, which may harbor a tumor suppressor gene. In addition, 23 loci distributed on 13 chromosomes exhibited allelic losses at frequencies of more than 20%. The genome‐wide occurrence of allelic losses suggests that genomic instability of the skin tumors may be implicated in radiation‐induced carcinogenesis. The present study is the first to report a mouse model system useful for the analysis of radiation induction of skin cancer in man.
Expression of Cyclin‐dependent Kinase Inhibitor p27/Kipl and AP‐1 Coactivator p38/Jabl Correlates with Differentiation of Embryonal Rhabdomyosarcoma
Authors: Tsuchida Rika , Miyauchi Jun , Shen Lisong , Takagi Masatoshi , Tsunematsu Yukiko , Saeki Morihiro , Honna Toshiro , Yamada Seiko , Teraoka Hirobumi , Kato Jun‐ya , Mizutani Shuki
Published: August 2005

Cyclin‐dependent kinase (CDK) inhibitor p27/Kip1 (p27) is a diagnostic and prognostic marker of various malignancies. Low expression of p27 reflects poor differentiation and poor prognosis, and an inverse correlation between the expression of p27 and degree of tumor malignancy has been reported. Because p27 mutation is extremely rare in human tumors, it is important to study the expression of p27 and its inactivator, p38/Jab1 (JAB1). Here we analyzed the expression of p27 and JAB1 by immunohistochemistry in embryonal rhabdomyosarcoma (E‐RMS). We first confirmed the expression of p27 and JAB1 in normal human tonsillar epithelium, and observed a coordinated expression pattern depending on cell differentiation. Subsequently, specimens of eight poorlyand three well‐differentiated E‐RMS were examined for the expression of p27 and JAB1. The analyses revealed that four out of eight poorly‐differentiated E‐RMS were negative for p27, with positivity for nuclear JAB (NJAB) (‐ /± for p27/NJAB) in three and negativity for any JAB‐1 expression (‐ / ‐) in one. The remaining four poorly‐differentiated E‐RMS expressed p27 in the nuclei, together with predominant NJAB (±/±). In three well‐differentiated E‐RMS, only one expressed nuclear p27 and all of these three expressed no NJAB (±/ ‐ for p27/NJAB), but expressed predominant cytoplasmic JAB1 (CJAB). These findings suggest that JAB1 may play an important role in determining the differentiation stage of rhabdomyosarcoma cells by modulating the activity of CDK inhibitor p27.
p185HER‐2/neu and p21CIP1/WAF1 Expression in Primary Tumors and Lymph Node Metastases in Non‐small Cell Lung Cancer
Authors: Akita Kenji , Inagaki Hiroshi , Sato Shigeki , Niimi Takashi , Maeda Hiroyoshi , Ninomiya Shigemitsu , Achiwa Hiroyuki , Kawaguchi Haruhiko , Yamakawa Yosuke , Fujii Yoshitaka , Shimizu Shigeki , Eimoto Tadaaki , Ueda Ryuzo
Published: August 2005

p185HER‐2/neu, a tyrosine kinase receptor, is one of the target molecules for cancer therapy, and its expression may reduce the sensitivity of tumor cells to anti‐cancer drugs. p21CIP1/WAF1 is a cyclindependent kinase inhibitor, and its expression may also be involved in chemoresistance. Non‐small cell lung cancer (NSCLC) is a potentially systemic disease, and systemic therapies play an important role in its treatment. However, there have been no studies comparing the expression of these molecules between primary and metastatic tumors. We investigated the expression of p185HER‐2/neu and p21CIP1/WAF1 in 57 paired samples of primary NSCLC tumors and corresponding lymph node metastases by immunohistochemistry. Expression of each of p185HER‐2/neu and p21CIP1/WAF1 was highly correlated between primary tumors and lymph node metastases, and similar correlations were also obtained when adenocarcinoma and squamous cell carcinoma cases were analyzed individually. However we failed to detect any correlation between p185HER‐2/neu and p21CIP1/WAF1 expression. Our results suggested that expression of both p185HER‐2/neu and p21CIP1/WAF1 is concordant between primary and metastatic tumors.