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Biomedical subjects

H Shiku

Publications and source records attributed to H Shiku.

At least 181 records · Page 10Linked to original sources
PubMed1995

Differences in sensitivity to induction of apoptosis among rat fibroblast cells transformed by HTLV-I tax gene or cellular nuclear oncogenes.

The tax gene of human T lymphotropic virus type I has been implicated in the genesis of adult T cell leukemia (ATL). It has been reported that expression of tax induces neoplastic transformation in the rat fibroblast cell line Rat-1, and that co-expression with the ras gene can transform rat embryo fibroblasts. Possible activation of cellular oncogenes including c-myc and c-fos by tax has been implicated in these tax functions. In this study, comparative analysis of biological properties of tax and cellular nuclear oncogenes c-myc and c-fos was performed in Rat-1 cells. While all three oncogenes could transform Rat-1 cells, significant differences in the sensitivity to induction of apoptosis were observed between cells transformed with each oncogene. Induction of apoptosis by serum starvation was observed in tax-transfected Rat-1 cells but to a lesser extent than that in those transfected with c-myc or c-fos. In contrast, exposure to a DNA-damaging agent, etoposide, resulted in enhanced apoptotic death only in c-myc-transfected Rat-1 cells. Our findings indicate that the pathways for apoptosis induction may not be identical among these three oncogenes, and that the relatively low apoptosis-inducing activity and sufficient transforming capacity of tax might be associated with transformation of T cells and the low susceptibility of the transformed T cells (ATL cells) to chemotherapeutic agents.

Animals↗
PubMed1995

Inhibitory action of nm23 proteins on induction of erythroid differentiation of human leukemia cells.

We recently identified a differentiation inhibitory factor (I-factor) in mouse myeloid leukemia M1 cells as a murine homolog of the human nm23-H2 gene product. nm23 genes encode proteins that participate in tumor metastasis regulation and in various fundamental cellular processes, although their mechanisms of action are still unknown. Although all nm23 proteins contain nucleoside diphosphate (NDP) kinase activity, it has not been established that the enzyme activity mediated the various functions of nm23 proteins. In the present experiment, we examined the effect of nm23 proteins on various differentiation induction systems of human leukemic cells including HL-60, U937, HEL/S, KU812F, K562, and HEL cells. Native human erythrocyte NDP kinase protein inhibited the induction of erythroid differentiation of HEL, KU812 and K562 cells, but not the induction of monocytic or granulocytic differentiation of HL-60, U937 and HEL/S cells. The erythroid differentiation of HEL cells was inhibited by recombinant human nm23-H1, -H2, mouse nm23-M1, and -M2 proteins. Moreover, both the mutant nm23-H2His protein and truncated nm23-H2 protein containing N-terminal (1-60) peptide, which do not have NDP kinase activity, also inhibited erythroid differentiation of HEL cells. These results suggest that (1) the differentiation inhibitory activity of I-factor/nm23 protein is not restricted to monocytic differentiation of M1 cells, (2) the inhibitory activity is exhibited without species specificity, and (3) the differentiation inhibitory activity of the nm23/NDP kinase protein is independent of its enzyme activity and requires the presence of N-terminal peptides.

Base Sequence↗
PubMed1995

Two isotypes of murine nm23/nucleoside diphosphate kinase, nm23-M1 and nm23-M2, are involved in metastatic suppression of a murine melanoma line.

A series of sublines of a murine melanoma B16 of C57BL/6 origin were established and examined regarding their metastatic capacity and expression of nm23. The number of pulmonary metastases developed by these sublines was inversely correlated with the expression of two isotypes of nm23, nm23-M1 and nm23-M2. The cDNAs of nm23-M1, nm23-M2, and a combination of both were transfected into the highly metastatic melanoma subline FE7, with low nm23 expression. FE7 transfectants of any of these cDNAs expressed transfected genes, and their metastatic capacity was suppressed when compared with parental FE7 or FE7 transfected with a control neo gene. These cell lines, however, did not change in terms of in vitro growth in the presence of 3 or 10% fetal bovine serum and in vivo growth when injected s.c. into C57BL/6-nu/nu mice. Similar experiments were also performed using FE7 transfectants of human nm23 genes. Transfectants of nm23-H1, nm23-H2, and their combination did not present altered metastatic potential. These findings indicated that two murine isotypes of nm23 but not those of humans are intimately related with the suppression of metastasis in the murine body.

Animals↗
PubMed1995

A new function of Nm23/NDP kinase as a differentiation inhibitory factor, which does not require it's kinase activity.

We recently identified a differentiation inhibiting factor (I-factor) in mouse myeloid leukemia M1 cells as a murine homolog of nm23-H2/nucleoside diphosphate kinase (NDPK)-B gene product. We examined the I-factor activities of several authentic nm23/NDPK proteins, i.e. recombinant rat NDPK alpha and beta, recombinant mouse nm23-M1 and -M2, and recombinant human nm23-H1 and -H2 containing a mutant nm23-H2His protein lacing NDPK activity. Almost all these nm23/NDPK proteins showed I-factor activity. Moreover, to understand the active domain exhibiting I-factor activity of nm23-H2 protein lacking NDPK activity, we have investigated the I-factor activities of some truncated nm23-H2 proteins. The truncated nm23-H2 protein containing N-terminal peptide 1-60 retained the I-factor activity. These results provide the first evidence for a function of nm23/NDPK as a differentiation inhibiting factor in leukemic cells, that is independent of its NDPK activity and dependent on the presence of N-terminal peptide.

Animals↗
PubMed1995

Substrate specificity of beta 1,4-N-acetylgalactosaminyltransferase in vitro and in cDNA-transfected cells. GM2/GD2 synthase efficiently generates asialo-GM2 in certain cells.

The substrate specificity of beta 1,4-N-acetylgalactosaminyltransferase has been analyzed using a fusion enzyme which consisted of the catalytic domain of the enzyme and the IgG binding domain of protein A, and also by extracts from cDNA transfectants. Both enzyme sources were capable of producing not only GM2 and GD2, but also asialo-GM2, GalNAc-sialylparagloboside, and Gal-NAc-GD1a from appropriate acceptors, although the efficiencies were at most 1-3% of those of GM2/GD2. The biological significance of these low specificities was studied with transient and stable transfectant cells. From the results of transient expression of the cDNA, asialo-GM2 expression appeared to inversely correlate with GM2 synthase levels in those lines. Consequently, GM2 seemed to be preferentially synthesized when both GM3 and lactosylceramide are available, and asialo-GM2 is synthesized in the absence of GM3 synthesis. However, the results of double immunostaining of CHO transfectants with anti-GM2 and anti-asialo-GM2 antibodies indicated that another factor may be involved in asialo-GM2 synthesis. From the in vitro assay using mixed acceptors, it was concluded that the presence of certain levels of GM2 might enhance the asialo-GM2 synthesis. These results suggest that even acceptors showing low efficiencies in vitro might be used in certain cells depending on the availability of precursors, expression levels of other gangliosides, as well as the kinetic properties of the enzyme, and the compartmentation of the glycosylation machineries in the cells.

Animals↗
PubMed1995

Translocation of protein phosphatase 1 catalytic subunits during 1,25-dihydroxyvitamin D3-induced monocytic differentiation of HL-60 cells.

To elucidate the roles of protein phosphatases type 1 (PP1) and type 2A (PP2A) in 1,25-dihydroxy-cholecalciferol [1,25(OH)2D3]-induced differentiation of HL-60 cells into monocytes, we examined the enzyme activity and the protein and gene expressions of PP1 and PP2A in these cells. Calyculin-A augmented the 1,25(OH)2D3-induced differentiation of the cells. Treatment of the cells with 1,25(OH)2D3 led to a decrease in PP1-like activity in the cytosol fraction, with a concomitant increase in the membrane and nuclear PP1-like activity, as determined when protein phosphatase activity was assayed using myosin light chain as substrate in the presence of 5 nM okadaic acid. Western blot analysis with antibodies specific for PP1 catalytic subunit isozymes (PP1 alpha, PP1 gamma, and PP1 delta) showed that all three PP1 isozymes were expressed but were differentially distributed in each cellular fraction. Subcellular redistribution of PP1-like activity during 1,25(OH)2D3-induced differentiation was mainly attributed to PP1 gamma and PP1 alpha proteins. In contrast, the localizations of PP1 delta and PP2A catalytic and regulatory subunits were not significantly affected by 1,25(OH)2D3 treatment. The gene expressions of PP1 alpha and PP1 gamma appeared to be constant during processes of monocytic differentiation. The correlation between phenotypic and functional changes of HL-60 cells on the one hand and subcellular redistribution of PP1-like activity on the other suggest that the translocations of PP1 alpha and PP1 gamma isozymes may contribute to the 1,25(OH)2D3-induced monocytic differentiation of HL-60 cells.

Calcitriol↗
PubMed1995

Induction of murine gamma delta T cells cytotoxic for xenogeneic rat cells.

C57BL/6 mice deprived or nondeprived of CD4+ and CD8+ T cells by mAbs were challenged with a rat T cell line, W7TM-1. Spleen cells obtained from CD4- and CD8-depleted animals rejecting W7TM-1 were examined by cytofluorometry, which demonstrated the presence of highly increased gamma delta type CD4-CD8- T cell population (30 to 50% of entire T cells). In vitro sensitization of these spleen cells with W7TM-1 generated a mixture of gamma delta and alpha beta type CD4-CD8- CTL for W7TM-1. Repeated stimulation of these cells with W7TM-1 resulted in a gamma delta-type T cell population with more than 95% purity by day 45. In contrast, alpha beta type CD8+ CTL for W7TM-1 were induced from mice nondeprived of CD4+ and CD8+ T cells. Both gamma delta-type CD4-CD8- CTL and alpha beta type CD8+ CTL were cytotoxic for rat cells in a species-specific manner. However, only reactivity of gamma delta type CD4-CD8- CTL, but not alpha beta type CTL, was inhibited by a mAb for TCR-gamma delta. The gamma delta type CD4-CD8- CTL clones were also prepared from spleen cells derived from CD4- and CD8-depleted mice. They were also reactive for xenogeneic cells in a species-specific manner. Spleen cells derived from CD4- and CD8-depleted mice rejecting the whole-layer rat skin grafts were in vitro sensitized with rat spleen cells, which also generated gamma delta type CD4-CD8- CTL specific for rat cells. V gamma 1 was detected as a major V gamma gene expressed in this gamma delta population by reverse transcriptase-PCR. Cytotoxicity for xenogeneic cells may represent one of major function of gamma delta T cells.

Animals↗
PubMed1995

T cell receptor-mediated stimulation of mouse thymocytes induces up-regulation of the GM2/GD2 synthase gene.

cDNA clones of the mouse GM2/GD2 synthase (EC 2.4.1.92) gene were isolated, and their analyses revealed that the protein has a type II transmembrane structure with 533 amino acids, which was very similar to the human homolog except for the mRNA size. The mRNA level in thymocytes dramatically increased after treatment with anti-CD3 monoclonal antibody, whereas it was not elevated when treated with prostaglandin E2. In situ hybridization showed an elevation of mRNA levels in medullar thymocytes, suggesting that T cell receptor-mediated signaling induces up-regulation of the GM2/GD2 synthase gene in mature thymocytes.

Amino Acid Sequence↗
PubMed1995

Expression of alpha 2,8-sialyltransferase (GD3 synthase) gene in human cancer cell lines: high level expression in melanomas and up-regulation in activated T lymphocytes.

GD3 Synthase (alpha 2,8sialyltransferase) (EC 2.4.99.8) cDNA has been cloned by eukaryotic cell expression cloning. Using this cDNA as a probe, the expression level of the gene in human cancer cell lines was analysed by Northern blotting and RT-PCR, then correlated with the ganglioside expression and enzyme activity. Melanoma cell lines showed extremely strong bands in Northern blot and RT-PCR/Southern analysis. The enzyme activity was also very high in melanomas as expected. Neuroblastoma and astrocytoma lines showed relatively low levels of the gene expression, whereas they expressed high levels of GD2. Although the mRNA level of the GD3 synthase gene and enzyme activity in individual cell lines correlated positively, some cell lines showed much higher activity than expected from the mRNA level. Among leukaemia lines, adult T cell leukaemia-associated (HTLV-I+) lines showed fairly high levels of the mRNA. On the other hand, T-ALL lines showed very low levels. In addition, GD3 and GD2 expression and mRNA level of the gene during T lymphocyte activation were analysed. Only GD3 expression was induced by any of the stimulatory reagents used, and corresponding up-regulation of the GD3 synthase gene was shown in RT-PCR/Southern analysis.

Base Sequence↗
PubMed1995

Diverse expression of beta 1,4-N-acetylgalactosaminyltransferase gene in the adult mouse brain.

Among various tissues of mouse, beta 1,4-N-acetylgalactosaminyltransferase (GM2/GD2 synthase) gene is expressed predominantly in the brain. Further analysis of the gene expression in the mouse CNS was performed by northern blotting and by enzyme assays using extracts from various parts of the CNS. In situ hybridization was also done to investigate the distribution of cells generating GM2/GD2 synthase. In northern blots, diverse levels of the gene expression were observed, depending on the regions examined. By in situ hybridization, pyramidal cells in the hippocampus, granular cells in dentate gyrus and cerebral cortex, Purkinje cells in cerebellum, and mitral cells in the olfactory bulb expressed high levels of the mRNA; these results corresponded to the results obtained by northern blot. Enzyme levels in these sites were accordingly high. However, enzyme levels in certain areas with low mRNA intensities, such as thalamus and pons medulla, were higher than expected from the results of northern blotting. The significance of the high gene expression in certain areas for brain function and the reason for the discrepancy between mRNA level and enzyme activity in some regions are discussed.

Animals↗
PubMed1995

Tissue factor expression in endothelial cells in health and disease.

Plasma tissue factor (TF) antigen can be detected in healthy volunteers and may be significantly increased in patients with disseminated intravascular coagulation (DIC). Plasma TF antigen level in patients with DIC was significantly reduced after therapy. The TF activity of human umbilical vein endothelial cells (HUVEC) cultured with lipopolysaccharide (LPS), cytokines and the medium of cultured mononuclear cells (MNC) was significantly increased. TF expression was induced in HUVEC and MNC by incubation with lipoproteins, suggesting that hyperlipidaemia is a direct risk factor in thrombotic disease. TF activity in HUVEC was significantly increased in the presence of plasma and this activation was higher in patients with thrombotic thrombocytopenic purpura (TTP) and DIC. Enhanced TF production by endothelial cells may be important in the pathogenesis of thrombotic diseases.

Cells, Cultured↗
PubMed1995

Expression of CD44 variant exons 8-10 in colorectal cancer and its relationship to metastasis.

Splice variants of CD44 are overexpressed in human lung, breast, and colon carcinoma cell lines. This study was conducted to clarify the association between the expression of CD44 variant exons 8-10 and metastatic potential in human colorectal cancer. We found that the expression of a CD44 splice variant containing exons v8-10 was increased in all of 60 colorectal cancer specimens examined compared with matched normal colerectal mucosa, as determined by Northern blotting. Expression of CD44 variant exons 8-10 did not significantly correlate with histological type, depth of tumor invasion, lymphatic invasion, venous invasion, or lymph node metastasis. However, the level of CD44 variant exon 8-10 expression was significantly higher in carcinomas associated with liver metastasis than in those without liver metastasis. In addition, expression of CD44 variant exons 8-10 in the liver metastases was more intense than that in the primary colorectal cancers. These findings indicated that this domain of the CD44 glycoprotein encoded by exons v8-10 may play an important role in tumor hematogenous metastasis of human colorectal cancer.

Blotting, Northern↗
PubMed1995

Expression of CD44 variant exons 8-10 in gastric cancer.

The expression of CD44 variant containing variant exons 8-10 product (CD44v8-10) was studied by western blot analysis and immunohistochemistry in gastric cancers using a monoclonal antibody, 44-1V. On western blots, a single band of 130 kD was recognized in stomach cancer cell lines. CD44v8-10 expression, with reactivity localized in the cell membrane, was found in 65 (33.5%) of the 194 advanced gastric cancers. There was no correlation between CD44v8-10 immunoreactivity and serosal, lymphatic, or lymph node invasion. However, there was significant correlation with CD44v8-10 immunoreactivity and venous invasion. CD44v8-10-positive cancers were more frequently associated with hematogenous metastasis than those which were immunonegative. There was an inverse association between CD44v8-10 immunoreactivity and peritoneal dissemination, especially in diffuse type adenocarcinomas. These observations indicate that CD44v8-10 may play a role in the metastasis of gastric cancer.

Adenocarcinoma↗
PubMed1995

Decreased expression of protein phosphatase type 2A in HL-60 variant (HL-60RAr) cells resistant to induction of cell differentiation by all-trans retinoic acid.

To evaluate the molecular basis for susceptibility of the cell differentiation induced by all-trans retinoic acid (ATRA), we examined biochemical activities and expression of protein phosphatases type 1 (PP1) and type 2A (PP2A) from HL-60 cells that are susceptible to differentiation induced by ATRA and HL-60RAr cells, HL-60 variant cells that are resistant to such induction. One nM of calyculin-A (CAL-A) achieved the enhancement of granulocytic differentiation in ATRA-treated HL-60 (1 microM) cells. ATRA exerted no differential action in HL-60RAr cells, but when used in combination with CAL-A, the differential activity was partly resumed at functional and phenotypic levels without change in morphology. The phosphatase activity in the cytosol from HL-60RAr cells was 50% of that from parental HL-60 cells, but the enzyme activities in either membrane or nuclear fractions showed similar values. The decreased phosphatase activity in the cytosol of HL-60RAr cells was mainly due to the decreased expression of the PP2A catalytic subunit. This low level of PP2A protein was reflected at a relative deficiency in expression of the PP2A beta gene in HL-60RAr cells. The exposure to 1 microM ATRA resulted in downregulation of PP2A catalytic subunit protein in HL-60 cells, but ATRA did not affect PP2A expression in HL60RAr cells. Both cell lines expressed the proteins of each PP1 catalytic subunit isozyme (i.e., PP1 alpha, PP1 gamma, and PP1 delta) at comparable levels. ATRA treatment had no effect on the levels of PP1 isozymes. Our results show a correlation between the extent of PP2A expression and the response of HL-60 and HL-60RAr cells to the differentiative effects of ATRA.

Blotting, Northern↗
PubMed1995

Preferential reduction of nm23-H1 gene product in metastatic tissues from papillary and follicular carcinomas of the thyroid.

The nm23 gene product is a possible mediator of cancer invasion and metastasis, and has been divided into two distinct gene products, NM23-H1 and NM23-H2. It has been shown previously that expression of nm23-H1 messenger RNA is reduced in metastatic lymph nodes from patients with papillary carcinoma of the thyroid (Am J Pathol 142:1938-1944, 1993). Since mAb H1-229 specific for NM23-H1 is now available, we further extended our study to examine the expression of nm23-H1 gene product in various types of thyroid cancer (37 cases of papillary carcinoma, 42 cases of follicular carcinoma, 17 cases of anaplastic carcinoma, and 3 cases of medullary carcinoma). NM23-H1 was expressed in primary papillary and follicular carcinomas (14/15 and 21/22), but weakly or hardly expressed in metastatic lymph nodes and metastatic bone marrow (6/23 and 1/4) (P < 0.01). Expression of NM23-H1 was low or absent in anaplastic and medullary carcinoma (3/17 and 0/3) (P < 0.01).

Adenocarcinoma, Follicular↗
PubMed1995

[The role of CD44 adhesion molecules].

CD44 is a transmembrane glycoprotein involved in the interaction between cells and extracellular matrix. CD44 is ubiquitously expressed on cells, and has been thought to be a cell adhesion molecule with proposed functions in extracellular matrix binding, cell migration and lymphocyte homing. In 1991, Stamenkovic et al. showed the existence of two forms of CD44: a hematopoietic (standard) and an epithelial form which was highly expressed by carcinomas. The alternative splicing of 10 variant exons encoding the membrane proximal portion of the external domain of CD44, in particular, results in many variant isoforms. These may play a critical role in malignant behavior and in determining organ specificity in metastasis.

Humans↗
PubMed1995

Decreased plasma tissue factor pathway inhibitor levels in patients with thrombotic thrombocytopenic purpura.

We measured plasma levels of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) in patients with thrombotic thrombocytopenic purpura (TTP) and disseminated intravascular coagulation (DIC) to examine the relationship between TFPI and vascular endothelial cell injury. TF antigen was detected in the plasma of healthy volunteers, and the levels were significantly increased in the patients with DIC, but decreased slightly in those with TTP. Plasma TFPI levels were significantly decreased in patients with TTP compared with those in healthy volunteers. The concentration of plasma thrombomodulin (TM) antigen was significantly higher in those with TTP than in normal volunteers. One month after treatment, TTP patients showed a significant decrease in plasma TM levels, and a significant increase in plasma TFPI levels, but plasma levels of TF antigen were not significantly increased. As plasma TFPI/TF ratio was significantly increased after treatment, the hypercoagulable state was therefore improved after treatment. There was no significant difference in plasma TF and TFPI levels between those who achieved complete remission (CR) and those who died. However, plasma TM levels were significantly higher in those who died than in those who achieved CR. Plasma TFPI levels might reflect injury of vascular endothelial cells as do plasma TM levels, and decreased plasma TFPI/TF ratio and vascular endothelial cell injuries might play causative roles in TTP.

Adolescent↗