Biomedical subjects

M Namba

Publications and source records attributed to M Namba.

At least 127 records · Page 7Linked to original sources

PubMed1996

Two-dimensional electrophoretic studies on down-regulated intracellular transferrin in human fibroblasts immortalized by treatment with either 4-nitroquinoline 1-oxide or 60Co gamma rays.

The immortalization of human cells is an important early step of carcinogenesis. To investigate the mechanisms of immortalization, we established three immortalized human fibroblast cell lines by treatment with either 4-nitroquinoline 1-oxide (4NQO) or 60Co gama rays. Using two-dimensional gel electrophoresis (2-D PAGE), we identified proteins which are down-regulated in these immortalization cell lines. Their isoelectric points ranged between 5.5 and 6.3, and their molecular masses were approximately 80 kDa. The proteins were also decreased in another human fibroblast cell line immortalized with simian virus 40 (SV40) and two human cervical cell lines. The proteins were present in a cytoskeletal fraction, reacted with anti-transferrin antibody and some of them were phosphorylated. The proteins were identified as transferrin. Although the 2-D PAGE patterns of our proteins and serum transferrin of different origin were distinct from each other, their isoelectric points and molecular weights were similar. We implicate some functions of intracellular transferrin, other than an iron transporter, in immortalization of human cells and discuss a new aspect of transferrin metabolism in the cells.

4-Nitroquinoline-1-oxide↗

PubMed1996

Islet-infiltrating t lymphocytes in insulin-dependent diabetic patients express CD80 (B7-1) and CD86 (B7-2).

Insulin-dependent diabetes mellitus (IDDM) results mainly from T cell mediated pancreatic beta cell destruction. To fully activate antigen specific T cells, current evidence suggests that two signals are required. One signal is delivered via the antigen specific T cell receptor (TCR) when engaged by major histocompatibility complex presented antigen (MHC:Ag), the other via the T cell's CD28 when engaged by CD80/86. Recent studies have demonstrated that transgenic mice expressing CD80 on their pancreatic beta cells are susceptible to autoimmune beta cell destruction. To further explore whether CD80/86 expression plays a role in IDDM pathogenesis, we analysed pancreatic biopsy specimens from 16 recent-onset IDDM patients (13 men and 3 women; age 29.7 +/- 8.8 years) for CD80/86 expression. While no biopsy revealed any islet cell specific CD80 or CD86 expression, biopsies from six of the nine patients with insulitis revealed both CD80 and CD86 expression on the islet infiltrating cells. Triple immunofluorescent staining for CD80/86, CD3, and glucagon revealed that the CD80/86-positive cells were also CD3-positive. Of the CD3-positive cells, 19.4% expressed CD80 and 21.7% expressed CD86. CD80 and CD86-positive cells were similarly distributed throughout the inflamed islets. These data suggest that CD28 engagement with CD80/86 may play a pathogenic role in the beta cell destruction underlying IDDM.

Adult↗

PubMed1996

Telomere elongation observed in immortalized human fibroblasts by treatment with 60Co gamma rays or 4-nitroquinoline 1-oxide.

Telomeres are the tandemly repeated (TTAGGG)n sequences that make up the structural and functional ends of all chromosomes in mammals. Many lines of evidence indicate that telomeres stabilize chromosomes, prevent aberrant recombination, and direct chromosome attachment to the nuclear membrane. Since DNA polymerase requires a labile primer to initiate unidirectional 5'-3' DNA synthesis, some bases at the 3' end of each template strand are not copied unless special mechanisms bypass this end-replication problem. To overcome this problem, most eukaryotic cells use telomerase, an enzyme that elongates telomeres. However, this enzyme has not been detected in normal human cells, and these cells lose telomeres with cell division. Cellular senescence might be the result of this loss. Thus, activation of telomerase seems to be critical for the immortalization of human cell lines. In addition, substantial evidence indicates that immortalization in itself is a rate-limiting step for the malignant transformation of human cells. We have treated normal human fibroblasts (AD387, KMS-6, and OUMS-24 lines) intermittently with either 60Co gamma rays or 4-nitroquinoline 1-oxide (4NQO) during serial subcultivations, and have obtained three immortalized cell lines, SUSM-1, KMST-6, and OUMS-24F. In KMS-6 and OUMS-24, the mean terminal restriction fragment length significantly decreased as the population-doubling level increased. The rate of telomere loss was 40 and 50 bp/population doubling in the KMS-6 and OUMS-24 cell lines, respectively. Once these normal cell lines were immortalized, their telomeres became elongated. Similar data were obtained for AD387 cells and their immortalized SUSM-1 cells. These results suggest that telomeres play a critical role in cellular senescence and in the immortalization processes of human cells.

4-Nitroquinoline-1-oxide↗

PubMed1996

Cytokine-induced apoptotic cell death in a mouse pancreatic beta-cell line: inhibition by Bcl-2.

Cytokines are thought to contribute to the induction of pancreatic beta-cell destruction in insulin-dependent diabetes mellitus. The molecular mechanisms that underlie beta-cell death were investigated by studying cytokine-induced cell death in beta-cell lines. A combination of three cytokines (interleukin-1 beta, tumour necrosis factor-alpha, and interferon-gamma) induced apoptotic cell death in the mouse pancreatic beta-cell line beta TC1, as judged from the appearance of cells with hypodiploid nuclei and oligonucleosomal DNA fragmentation. The same treatment also induced apoptosis in the mouse pancreatic alpha-cell line alpha TC1 and the NOD/Lt mouse beta-cell line NIT-1, although to a lesser extent than in beta TC1 cells. The abundance of endogenous Bcl-2 in beta TC1 cells was lower than that in the other two cell lines. Overexpression of human Bcl-2 in beta TC1 cells partially protected them from cytokine-induced cell death. These results suggest that apoptosis may be responsible, at least in part, for cytokine-induced beta-cell destruction and that Bcl-2 prevents apoptosis in pancreatic islet cells.

Animals↗

PubMed1996

Effects of okadaic acid on cell growth, anchorage-independent growth, and co-cultures of normal (KMS-6), immortalized (KMST-6), and neoplastically transformed (KMST-6T and KMST-6/RAS) human fibroblasts.

The effects of okadaic acid (OA) on normal human (KMS-6), its immortalized (KMST-6) and neoplastically transformed (KMST-6T and KMST-6/RAS) cells were investigated as a model of two stage carcinogenesis. The presence of OA inhibited cell growth of the normal and immortalized cells but not that of the neoplastic KMST-6T cells. In contrast, cell growth of the other neoplastic KMST-6/RAS cells transformed with the Ha-ras oncogene was inhibited by OA. OA enhanced colony formation of KMST-6T cells in soft agar, but it suppressed that of KMST-6/RAS cells. Co-cultures of KMST-6T cells with normal KMS-6 cells showed an increase in focus formation of KMST-6T cells in the presence of OA, whereas focus formation of KMST-6/RAS cells decreased. These results indicate that OA has growth-promoting effects on certain types of transformed human cells.

Carcinogens↗

PubMed1996

Establishment and characterization of a human colon cancer cell line, OUMS-23, from a patient with familial adenomatous polyposis.

A human colon carcinoma cell line designated OUMS-23 has been established from metastatic pericardial fluid of a male familial adenomatous polyposis patient with colon cancer. Since 1984, the epithelial cells have been maintained in culture. Ultrastructural studies revealed the presence of numerous microvilli on the cell surface and desmosomes between the adjacent cells. The cells secreted carcinoembryonic antigen into the culture medium (15 ng/10(6) cells-1 24 h-1). The cells expressed heat-stable placental-type-like alkaline phosphatase, whereas the normal counterparts expressed tissue-unspecific alkaline phosphatase. Karyotypic analysis showed that the cell line was of human origin and that the chromosome number was broadly distributed between 53 and 118. Southern blot analysis of the APC gene revealed no abnormalities in OUMS-24 cells, while Northern blot analysis demonstrated that the expression of the gene was about one-half that of the normal human fibroblasts. No mutations at the "hot spots" of codons 12 and 61 of H-, K- and N-ras proto-oncogenes were detected in the cells. The cells could grow in soft agar at a cloning efficiency of 6.5%, and upon transplantation into nude mice the cells formed tumors, which were diagnosed as differentiated adenocarcinoma.

Adenomatous Polyposis Coli↗

PubMed1996

Disordered expression of glycolytic and gluconeogenic liver enzymes of juvenile visceral steatosis mice with systemic carnitine deficiency.

A quantitative study of the effect of carnitine deficiency on expression of glycolytic and gluconeogenic enzymes was performed using juvenile visceral steatosis mice which are systemically deficient in carnitine. The amounts of glucokinase and L-type pyruvate kinase mRNA were reduced in homozygotes, compared to heterozygotes and normal controls at 2 and 8 weeks. Liver-type phosphofructokinase, however, did not differ significantly. The abundance of fructose 1,6-bisphosphatase mRNA was unchanged at 2 and 8 weeks. The level of phosphoenolpyruvate carboxykinase mRNA was increased slightly at 2 weeks, but not at 8 weeks. A part of these changes could not be explained by the plasma glucose or insulin level. Carnitine administration restored the mRNA of these enzymes to normal levels. These results suggest that carnitine deficiency affects the expression of these liver enzymes.

Age Factors↗

PubMed1996

Dominant TCR alpha-chain clonotypes and interferon-gamma are expressed in the pancreas of patients with recent-onset insulin-dependent diabetes mellitus.

In order to clarify the nature of T lymphocytes infiltrating the pancreatic islets of patients with insulin-dependent diabetes mellitus (IDDM), we analysed T cell receptor (TCR) gene transcripts expressed in pancreatic biopsy specimens of patients with recent-onset IDDM. We also investigated the expression of cytokines (interferon-gamma: IFN-gamma; tumour necrosis factor-alpha: TNF-alpha; interleukin-4: IL-4; interleukin-6: IL-6) in the same specimens. The TCR V beta repertoire was not restricted either in the pancreas or the peripheral lymphocytes of IDDM patients. In contrast, the TCR V alpha repertoire was restricted in the pancreas, but not in the peripheral blood lymphocytes, of IDDM patients. The sequence analysis of the complementarity-determining region 3 (CDR3) of the TCR alpha revealed the presence of dominant clonality in alpha chains of T cells in the patients. IFN-gamma mRNA was highly expressed in the pancreas of IDDM patients, while IL-4 mRNA was deficient. A lower level of expression of IL-6 mRNA was detected in the IDDM pancreas than in the control tissue. These results indicate that T cells bearing a distinct TCR alpha chain are selectively retained and activated within the pancreas of recent-onset IDDM.

Adolescent↗

PubMed1996

IFN-gamma in combination with IL-3 accelerates platelet recovery in mice with 5-fluorouracil-induced marrow aplasia.

The effects of interferon-gamma (IFN-gamma) on platelet recovery were examined in mice with marrow aplasia induced by i.p. injection of 250 mg/kg of 5-fluorouracil (5-FU). The cytokine was administrated by microosmotic pump, with an ability to deliver a consistent intact dose of cytokine for 7 consecutive days. Administration of 250 IU/kg/day of IFN-gamma in combination with 10(3) U/kg/day of IL-3, which alone had no effect on platelet counts, diminished the nadir for platelet count and shortened the duration of thrombocytopenia. The effect was comparable to that of higher doses of IL-3 (10(5) U/kg/day). The administration of 250 IU/kg/day of IFN-gamma in combination with 10(3) U/kg/day of IL-3 also induced megakaryocyte proliferation in bone marrow cell cultures. Single administration of either 250 IU/kg/day of IFN-gamma or 10(3) U/kg/day of IL-3 had no significant effects. The effect of this combination was also comparable to that of a higher dose of IL-3 (10(5) U/kg/day). We suggest that IFN-gamma accelerates megakaryocyte development, which leads to platelet production in chemotherapy-induced marrow aplasia. The administration of IFN-gamma in combination with IL-3 might be useful for the management of marrow aplasia.

Anemia, Aplastic↗

PubMed1996

Selective expression of mutated p53 in human cells immortalized with either 4-nitroquinoline 1-oxide or 60Co gamma rays.

Many studies on in vitro transformation of human cells indicate that the cells must be immortalized before they can be neoplastically transformed, indicating that immortalization is a critical step in multistep neoplastic transformation of human cells. We immortalized three human cell lines by repeated treatment with either 60Co gamma rays or a chemical carcinogen, 4-nitroquinoline 1-oxide, and found that all three immortalized cell lines have mutations in the tumor suppressor gene, p53. Direct sequencing of the reverse-transcribed mRNA and immunoprecipitation of p53 protein revealed that mutant p53 is selectively expressed in all the immortalized cell lines, whereas the genomic fragments of the immortalized cells contain wild-type and mutated p53 alleles. Although the mutated p53 is selectively expressed in the immortalized cells, expression of the wild-type p53 was induced by treatment of the cells with a hypomethylating reagent, 5-azacytidine, indicating that the wild-type p53 allele might be inactivated by hypermethylation of DNA. Actually, the entire genomic locus including the promoter region of p53 is hypermethylated in all the immortalized cell lines. Expression and phosphorylation of Rb was normal in these three cell lines. Thus, inactivation of both wild type p53 alleles and selective expression of mutated p53 seem to be key factors in the immortalization of human fibroblasts.

4-Nitroquinoline-1-oxide↗

PubMed1996

Immortalization of human cells and its mechanisms.

One of the contributions of in vitro neoplastic transformation studies on human cells is the confirmation that cells become malignant by a multistep process. In this review, we use the term "neoplastic transformation" to indicate that the cells become capable of forming a histologically malignant tumor, that is, one that invades and/or erodes normal tissue and is serially transplantable in a susceptible animal host. Phenotypically, the cells first become immortalized and then neoplastic. If normal human cells do not overcome aging by becoming immortalized, they cannot be neoplastically transformed. However, normal human cells are stringently destined to cellular aging and very rarely become immortalized by oncogenic DNA viruses (SV40, papillomavirus types 16 and 18, adenovirus types 5 and 12), chemicals, X-rays, or spontaneously. Once human cells get immortalized, they relatively easily become neoplastic. Evidence shows that immortalization is a critical step in the neoplastic transformation process of human cells. Therefore, investigation of the mechanisms of immortalization is indispensable in understanding the multistep carcinogenesis of human cells. Although the precise mechanisms of immortalization are unknown at present, data indicate that the p53 cascade appears to be important for the immortalization of human cells. The loss of functions of normal p53 may induce genomic instability, resulting in mutations of some other unknown genes that are more directly involved in the immortalization of human cells. These unknown genes, which are presumably located at chromosomes 1, 4, 6, 7, 9, 11, 13, 17, 18, and X are now under extensive investigation.

Cell Transformation, Neoplastic↗

PubMed1996

Immortalization of cells from brains derived from a strain (MSM/MSfB6C3F1) of wild mouse.

Escape from cellular aging is the rate-limiting step of multistep carcinogenesis. While normal human cells invariably undergo cellular aging and almost never spontaneously immortalize, cells derived from rodents such as mice are relatively easily immortalized. In this experiment, we studied the immortalization patterns of cells obtained from brain tissues of an inbred strain (MSM/MSfB6C3F1) derived from wild mice. We established 12 cell strains derived from 12 mouse brains in order to investigate whether these cells show cellular aging in the same fashion as human cells or whether these cells are immortalized as easily as rodent cells reported previously. As a result, all cell strains were immortalized up to about 200 days in culture. One strain immortalized very early, in the first 50 days, four strains immortalized in the last 200 days, and the other seven strains became immortal between 150 and 200 days in culture. All immortalized cell strains showed varying amounts of chromosome abnormalities, numerically and structurally, but no specific changes related to immortalization were detected. Before immortalization, three types of cells, glial-like, polygonal flat-thin, and fibroblast-like cells, were observed in culture, but after immortalization most of the cultures became fibroblastic. From these results, we concluded that fibroblast-like cells derived from brains of these mice immortalized in like fashion to fibroblasts of other inbred mice.

Animals↗

PubMed1996

Cloning of cDNA with possible transcription factor activity at the G1-S phase transition in human fibroblast cell lines.

Normal human fibroblasts have a finite proliferative capacity in vitro. Thus, immortalization of human cells is associated with cellular aging. We have established an immortalization-sensitive cell line from fibroblasts of Wilms' tumor patients which have a partial deletion of chromosome 1 1p. This cell line was easily immortalized by introducing SV4OT. By differential hybridization using both SV4OT-introduced crisis cells and young cells, we cloned a gene that was highly expressed in 1 1p-cells at the time of the crisis and named this gene C-1. Nucleotide sequence analysis of C-1 revealed that it contains a helix-loop-helix domain, indicating that it may be a transcription factor. Expression of the C-1 gene was transiently induced early in the G0-to-S phase transition in two normal human (OUMS-24 and HSF-412) and a non-tumorigenic immortal human (OUMS-24F) fibroblast cell lines, while the other immortal SUSM-1 cells highly expressed the C-1 gene in the middle G1 phase. These results suggest that the C-1 gene product may function as a transcription factor related to the cell cycle.

Amino Acid Sequence↗

PubMed1996

Spheroid cultures of human hepatoblastoma cells (HuH-6 line) and their application for cytotoxicity assay of alcohols.

Spheroid cultures of human hepatoblastoma cells (HuH-6 line) were established by rotating 3 x 10(6) cells/3 ml culture medium in 25-ml Erlenmeyer flasks on a gyratory shaker. The size of the spheroids rapidly increased until 4 days of culture, and thereafter their size gradually increased until 8 days of culture. A considerable amount of lactate dehydrogenase (LDH) was detected in the culture medium at 24h after seeding because of cell damage by subculturing, but thereafter the amount released was small, indicating that the spheroids were in healthy condition. Albumin production, one of the differentiated functions of hepatocytes, was higher in spheroid cultures than in monolayer cultures. Using this spheroid culture model, the cytotoxic effects of alcohols on HuH-6 cells were studied by measuring the activity of LDH released in the medium from damaged cells. The results indicate that the increasing order of toxicity of the alcohols was as follows: methanol < ethanol < propanol.

Albumins↗

PubMed1996

Comparison of various methods of assaying the cytotoxic effects of ethanol on human hepatoblastoma cells (HUH-6 line).

The sensitivity of five kinds of cytotoxicity assays using ethanol on human hepatoblastoma cells (HUH-6 line), which were cultured as monolayers or spheroids, was compared. Ethanol was chosen as a test because it acts on cell membranes directly without being metabolized and exerts its cytotoxicity. The assay methods used were as follows: 3- (4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH), colony formation, cell growth and DNA assays. The sensitivity of the assays was: LDH < DNA < cell growth < MTT < colony formation. LDH assay had the advantage that the same culture could be used for multiple assays, but when a small number of cells were assayed, no significant increase in the release of LDH was detected in the assay cultures compared with the control cultures. Although the DNA and cell growth assays were more sensitive than the LDH assay, the extent of cell damage may be underestimated because the damaged cells and DNA present in the cultures are included in the assay samples. On the other hand, both MTT and colony formation assays showed a high sensitivity. The MTT assay was done within 24 h after ethanol was added to the cultures and was applicable to both monolayer and spheroid cultures, while the colony formation assay required 1-2 weeks and it was applicable only to monolayer cultures. Taken together, the MTT assay was the most suitable method to evaluate the cytotoxic effects of ethanol on HUH-6 cells cultured as either monolayers or spheroids.

Cell Survival↗

PubMed1996

High prevalence of antibodies to glutamic acid decarboxylase in comparison to islet cell antibodies in patients with long-standing insulin-dependent diabetes mellitus.

To elucidate the clinical significance of antibodies to glutamic acid decarboxylase (GAD Ab) compared to islet cell antibodies (ICA) in recent-onset and long-standing insulin-dependent diabetes mellitus (IDDM). We examined GAD Ab and ICA in 29 recent-onset and 85 long-standing patients with IDDM. GAD Ab was detected by a radioimmunoassay kit using purified pig brain GAD as an antigen. The prevalence of GAD Ab in the recent-onset diabetic patients was 55.2%, slightly lower than that of ICA (65.5%). In contrast, the prevalence of GAD Ab in long-standing diabetic patients was 42.4%, which was significantly higher than that of ICA (23.5%) (p < 0.01). GAD Ab were consistently detected in approximately 40% of patients with long-standing disease, while ICA decreased according to duration of disease. The GAD Ab titer in ICA-positive patients (mean +/- SD, 1588.2 +/- 6755.1; range, 6-38574) was significantly higher than that in ICA-negative patients (mean +/- SD, 13.4 +/- 17.9; and range, 5-72 units) (p < 0.001). These findings suggest that GAD Ab are more useful than ICA to know participation of immune disorders in long-standing patients with IDDM.

Adult↗

PubMed1996

Expression and distribution of glucose-6-phosphatase catalytic subunit messenger RNA and its changes in the diabetic state.

Glucose-6-phosphatase (G6Pase) plays a major role in gluconeogenic pathway. To analyze its regulation, we have cloned a full-length cDNA for G6Pase catalytic subunit from the rat liver. In the cloned cDNA, a 492 base insertion, respective to the previously reported sequence, was found in the 3'-noncoding region. In both ends of this insertion, 5'- and 3' 2' splice site motifs were identified. However, spliced mRNA was hardly observed in the rat RNA. Abundant expression was observed in the liver and the kidney by Northern analysis. Expression was also observed in the spleen, adrenal gland and small intestines. Reverse-transcription (RT) polymerase chain reaction (PCR) analysis revealed that G6Pase mRNA was also expressed in a variety of tissues including pancreatic islets. Its expression was increased in the ketotic diabetic mice livers and was corrected by insulin treatment. However, no appreciable changes were observed in kidneys. Broad tissue distribution in expression and the tissue specificity in regulation were thus considered to be the important features of G6Pase gene.

Amino Acid Sequence↗

PubMed1996

Hepatocyte nuclear factor-4 alpha gene mutations in Japanese non-insulin dependent diabetes mellitus (NIDDM) patients.

A mutation in the hepatocyte nuclear factor-4 alpha (HNF-4 alpha) gene has been recently reported to cause maturity-onset diabetes of the young (MODY) (Yamagata, Furuta, et al., 1996). The mutation can also be a good candidate for the responsible gene of non-insulin dependent diabetes mellitus (NIDDM). The existence of the mutated allele of Q268X (C to T substitution within the exon 7 of HNF-4 alpha gene) was searched in 514 alleles of Japanese NIDDM patients by polymerase chain reaction-restriction fragment length polymorphism analysis. No mutation was found in these patients. The result showed that the Q268X mutation of HNF-4 alpha gene was not frequent among general NIDDM patients and that it cannot serve as the major diabetogenic gene in the Japanese ethnic group.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗