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Biomedical subjects

H Shiku

Publications and source records attributed to H Shiku.

At least 217 records · Page 12Linked to original sources
PubMed1993

A human T lymphotropic virus type I (HTLV-I) long terminal repeat-directed antisense c-myc construct with an Epstein-Barr virus replicon vector inhibits cell growth in a HTLV-I-transformed human T cell line.

A panel of EB virus replicon-based vectors was constructed to examine the relative utility of four distinct eukaryotic promoters for high-level gene expression in a HTLV-I-transformed human T cell line, HUT102. We found that HTLV-I LTR, which is trans-activated by the viral tax protein, was most suited for EBV vector-based stable gene expression in it. We prepared a HTLV-I LTR-directed antisense c-myc construct with an EBV vector. This antisense plasmid suppressed c-myc expression and inhibited growth of HUT102 cells in vitro with unaltered expression of tax. Non-specific plasmid toxicity was excluded by showing that the antisense construct had little effect on growth and c-myc expression of HTLV-I-negative Jurkat T cells, in which the viral LTR is expected to be less active. Our results indicate that c-myc may play an important role in the deregulated growth of HTLV-I-transformed T cells.

Actins↗
PubMed1993

GD2 ganglioside on human T-lymphotropic virus type I-infected T cells: possible activation of beta-1,4-N-acetylgalactosaminyltransferase gene by p40tax.

Ganglioside expression on adult T-cell leukemia (ATL) and human T-cell lymphotropic virus type I (HTLV-I)-infected cells was determined by using a panel of monoclonal antibodies. ATL lines and HTLV-I-infected cells specifically expressed GD2. Leukemia cells from ATL patients generally expressed low levels of GD2 but the percentage of GD2+ cells increased up to 40-70% after in vitro culture in the presence of interleukin 2 for about a week. No other type of leukemia cells and normal peripheral T cells expressed GD2 during in vitro culture under the same conditions. The appearance of GD2 in the cultured ATL cells corresponded with the expression of p40tax, a product of the HTLV-I gene. Peripheral lymphocytes infected with a p40tax-expressing retroviral vector expressed high levels of GD2 in comparison with control lymphocytes containing the neomycin-resistance gene alone. The apparently increased levels of beta-1,4-N-acetylgalactosaminyltransferase (GM2/GD2 synthase) mRNA in these cells were demonstrated by reverse transcription-polymerase chain reaction analysis. Concordance between mRNA expression for the HTLV-I tax1/rex1 genes and the beta-1,4-N-acetylgalactosaminyltransferase gene was also observed in uncultured ATL cells. These results suggest that high GD2 expression was due to neosynthesis from precursor GD3 by increased expression of this enzyme induced by p40tax in vitro and in vivo.

Base Sequence↗
PubMed1993

Up-regulation of small GTP-binding proteins smg P21A and ras P21S during TPA-induced differentiation of human leukemia cell lines.

The protein smg p21A/Krev-1/rap 1A was identified as a ras p21-like small G-protein, having the ability to revert v-Ki-ras transformed NIH 3T3 fibroblasts. The expression level of smg p21A and ras p21s during phorbol ester-induced differentiation of HL-60 and MEG-01 cell lines was analyzed by immuno- and Northern blotting. In both cell lines, levels of smg p21 and ras p21s increased quickly in early phase of differentiation along with the appearance of differentiation phenotypes. They increased 3-4 fold on days 1-2, then decreased gradually. The increasing smg 21 mRNA levels also corresponded with that of products. Among ras mRNAs, Ha-ras and N-ras transcripts increased somewhat faster than smg 21. These small G-proteins may play closely related roles in the differentiation of these leukemia cell lines.

Cell Differentiation↗
PubMed1993

Inverse association of nm23-H1 expression by colorectal cancer with liver metastasis.

The expression of nm23-H1 mRNA and protein was studied in colorectal cancers by Northern blotting and immunohistochemistry. All 21 colorectal cancers studied by Northern blotting had increased levels of nm23-H1 mRNA relative to the adjacent normal colonic mucosa. Increased nm23-H1 protein expression was also observed in all 36 colorectal cancer cases including those studied by Northern blotting. There was no significant correlation between nm23-H1 expression and tumour histology, serosal invasion, lymphatic invasion, venous invasion, or lymph node metastasis. However, the expression of both mRNA and protein was significantly lower in tumours associated with liver metastasis than in those without such metastasis. These observations indicate that the nm23 gene may play a role in the suppression of liver metastasis of colorectal cancer.

Antibodies, Monoclonal↗
PubMed1993

Detection of point mutations in the Kirsten-ras oncogene provides evidence for the multicentricity of pancreatic carcinoma.

It has been reported that multicentricity of pancreatic carcinomas extending beyond the pancreatic duct occur in 15% to 40% of patients. This has been difficult to confirm, however, with currently available histologic techniques. Mutations in the Kirsten (Ki)-ras oncogene, which can be detected frequently in pancreatic carcinomas using the polymerase chain reaction (PCR), may serve as a potential clonal marker of the cancer cells. Fifty-three patients with a histopathologic diagnosis of pancreatic carcinoma were selected for the determination of certain Ki-ras mutations through PCR. The authors identified mutations in the Ki-ras codon 12 in 46 of 53 tumors. Two of these 46 tumors had two different mutations to aspartic acid (GAT) and to valine (GTT) in Ki-ras codon 12. Another isolate had an additional mutation in Ki-ras codon 13. The detection of different mutations in the same tumor suggests that there may be multicentricity in pancreatic carcinomas and that its frequency may be as low as 6% of the carcinomas. These results imply that total pancreatectomy for eliminating tumor recurrence due to multicentricity may not be warranted.

Adenocarcinoma↗
PubMed1993

Effect of accessory cells on stimulation of murine T-cell leukemia with antibodies to the CD3/T cell antigen receptor complex.

Stimulation of EL4 and RL male 1 leukemia cells in vitro with immobilized anti-CD3 epsilon monoclonal antibody (mAb) (145-2C11) or anti-TCR beta mAb (H57-597) in the absence of accessory cells induced interleukin-2 (IL-2) production, and caused growth inhibition. The growth inhibition was, however, transient and the tumors started to grow again within 5 days in immobilizing plates treated with antibodies at concentrations of 2.5-100 micrograms/ml. Addition of mitomycin C-treated accessory cells to the culture inhibited IL-2 production and resulted in augmented and persistent growth inhibition. No recovery of tumor growth was observed. Furthermore, DNA from EL4 and RL male 1 leukemia cells stimulated with anti-CD3/TCR mAbs was fragmented even in the absence of accessory cells, but fragmentation was much greater in the presence of accessory cells. Marginal and high expression of the bcl-2 gene were observed in EL4 and RL male 1, respectively, indicating that apoptosis of these leukemias mediated by signalling through the CD3/TCR complex has no direct relationship with expression of the bcl-2 gene.

Animals↗
PubMed1993

Expression of nm23-H1 and nm23-H2 proteins in prostate carcinoma.

The nm23 gene products/nucleoside diphosphate (NDP) kinase expression in prostate carcinomas and benign hyperplasias was evaluated immunohistochemically. Monoclonal antibodies against nm23-H1 and nm23-H2 proteins were prepared using the corresponding proteins fused with glutathione S-transferase as immunogens. Of the 80 cases of nonmetastatic prostate carcinoma examined, 74% (59/80) and 60% (48/80) were immunoreactive for nm23-H1 or nm23-H2 protein, respectively. Negative staining for nm23-H1 occurred in 83% of metastatic lesions, while 34% were negative for nm23-H2. All primary tumors corresponding to the metastases examined showed positive immunostaining for nm23-H1, indicating an inverse relationship between expression of this protein and metastatic status. nm23-H2 protein was detected in 83% of primary tumors and its expression appeared to be significantly correlated to the degree of histological differentiation. In contrast, all cases of benign prostatic hyperplasia showed elevated levels of both nm23-H1 and nm23-H2 expression. These data suggest that the nm23/NDP kinase may play a role in suppressing the expression of malignant potential in prostate carcinomas.

Adenocarcinoma↗
PubMed1993

Correlation of p53 expression and proliferative activity in gastric cancer.

We analyzed immunohistochemically the association between p53 tissue status and prognostic parameters of 357 gastric cancer patients, using endoscopically obtained biopsy materials, embedded in paraffin. Using PAb1801, an anti-p53 monoclonal antibody p53 immunoreactivity was detected in the nuclei of cancer cells in 113 cases (32%). The nuclear p53 immunoreaction was closely associated with positive lymph node metastasis, serosal invasion, and liver metastasis. For the estimation of proliferative activity, proliferating cell nuclear antigen (PCNA) labeling rates (LRs) of biopsy specimens were immunohistochemically measured. A significant positive correlation was found between PCNA LRs and p53 tissue status. In addition, a positive nuclear p53 immunoreaction was found to be significantly associated with shorter overall survival. Especially, in the group of patients with stages III and IV, the prognosis of patients with p53-positive tumours was significantly poorer than that of patients with p53-negative tumours. These results indicate that immunohistochemical staining for nuclear p53 in biopsied materials may be useful in deciding the therapeutic schedule of patients with gastric cancer, preoperatively.

Antibodies, Monoclonal↗
PubMed1993

Expression of nm23/NDP kinase proteins on the cell surface.

We determined whether proteins encoded by the nm23/nucleoside diphosphate (NDP) kinase gene, a potential metastasis-suppressor gene, are expressed on the cell surface. Monoclonal antibodies (mAb) specific for nm23-H1 or H2 proteins were prepared using the corresponding fusion proteins with glutathione S-transferase (GST) as immunogens. mAb H1-229 was specifically reactive with nm23-H1 protein, whereas mAb H2-439 was specific for nm23-H2 protein in immunoprecipitation and immunoblotting. mAb H1-229 was reactive with most human hematopoietic and some non-hematopoietic cell lines in flow cytometry. On the other hand, mAb H2-439 was reactive with only a limited number of cell lines. Based upon the surface expression of nm23/NDP kinase, cells were classified as nm23-H1+H2-, nm23-H1+H2+ or nm23-H1-H2-. No cell lines with nm23-H1-H2+ were found among those examined. The specificity of flow cytometry analysis was confirmed in the murine myeloma line NS-1 transfected with either the nm23-H1 or H2 genes. Both mAbs were reactive only to NS-1 transfected with the corresponding nm23 genes. Immunoprecipitation and SDS-PAGE analysis identified 20.5- and 18-kDa proteins with mAb H1-229 or H2-439, respectively, in cellular extracts of 125I-surface labeled NS-1 transfected with the corresponding genes. The presence of nm23/NDP kinase on the cell surface indicates an extracellular role for these proteins in addition to their reported intracellular functions.

Animals↗
PubMed1992

Molecular cloning and functional expression of the second mouse nm23/NDP kinase gene, nm23-M2.

A new murine cDNA of nm23/NDP kinase was isolated. A RT-PCR product was obtained from the normal mouse liver mRNA with primers designed for the human nm23-H2 gene. The product was used as a probe to screen a cDNA library from the murine melanoma cell line, B16, and two clones containing the entire open reading frame were obtained. It was predicted that the DNA sequence encoded 152 amino acids which was 98% identical to the nm23-H2 protein. The entire nm23-M1 and -M2 gene-coding regions were translated as fusion proteins with a glutathione S-transferase. These fusion proteins displayed NDP kinase activities.

Amino Acid Sequence↗
PubMed1992

Expression cloning of beta 1,4 N-acetylgalactosaminyltransferase cDNAs that determine the expression of GM2 and GD2 gangliosides.

GM2 and GD2 gangliosides are sialic acid-containing glycosphingolipids expressed in some normal tissues such as brain and in various tumors such as neuroblastomas, astrocytomas, and malignant melanomas. We used a eukaryotic cell transient expression system to isolate cDNA clones that determine GM2 expression. We developed a new cell line from murine melanoma line B16 by transfecting with the polyoma T antigen gene that was suitable for this purpose. Two cDNA clones, both of which have a continuous open reading frame of 1683 base pairs, were isolated. Although the cloned cDNAs had no primary sequence similarity to reported glycosyltransferases, the deduced amino acid sequence predicted a type II transmembrane protein with an overall structure similar to other glycosyltransferases. The cDNA clones, when stably transfected, determined the expression of GM2 in B16 cells and GM2 and GD2 in the human melanoma line MeWo. Northern blot analysis revealed two transcripts in all cells that expressed either GM2 or GD2 or both. These findings indicate that the cDNAs catalyze the transfer of GalNAc onto GM3 and GD3 by a beta 1,4 linkage, resulting in the synthesis of GM2 and GD2, respectively. Namely they suggest that these cDNAs derive from the UDP-GalNAc: GM3/GD3 beta 1,4 N-acetylgalactosaminyltransferase (EC 2.4.1.92) gene.

Amino Acid Sequence↗
PubMed1992

Preferential usage of JH2 in D-J joinings with DQ52 is determined by the primary DNA sequence and is largely dependent on recombination signal sequences.

Rearrangement of DQ52 with JH in IgH genes takes place in a large part of lymphocytes at a very early stage of differentiation although usage of DQ52 seems to be limited in the functional IgH genes of mature B cells. A possible and likely explanation for this, is secondary D-JH joinings occurring after the initial DQ52-JH joinings, which becomes possible only when DQ52 segments join 5'-located JH segments. Previously we reported the non-random use of JH in joinings with DQ52, in which DQ52-JH2 joinings are dominant. We questioned how the preferential usage of JH2 in DQ52-JH joinings is determined by utilizing extrachromosomal DNA substrates. Extrachromosomal DNA substrates with a combination of DQ52 and multiple JH segments were prepared. These plasmids with a polyoma-derived replication system were transfected into pre-B cell lines and recombination profiles of recovered plasmids were analyzed semi-quantitatively. Experiments with plasmids containing all four JH segments in normal configuration showed an apparent high frequency of JH2 usage, similar to the rearrangement previously observed in thymocyte genomes. In plasmids containing two JH segments, when one was JH2, it was rearranged more frequently than the other, independent of its proximity to DQ52. The JH3 with recombination signal sequences (RSS) substituted for JH2 was rearranged more frequently than the naive JH2 itself indicating the importance of RSS. A JH3 hybrid with the 200 bp upstream sequence of JH2 but excluding RSS, however, failed to increase JH3 use. The addition of one nucleotide to the 22-bp spacer of JH1 RSS increased the frequency of JH1 usage. These observations suggest that the preferential usage of JH2 is determined by the primary DNA sequence and is largely dependent on RSS.

Animals↗
PubMed1991

Alternatively spliced mRNA of the pX region of human T lymphotropic virus type I proviral genome.

The pX region of human T lymphotropic virus type I (HTLV-I) is believed to be expressed as a consequence of a 2 step splicing. It is conceivable, however, that a donor site of the 1st splicing and an acceptor site of the 2nd splicing results in the production of an alternatively spliced mRNA which is capable of coding p21X-III. This possibility was examined by amplifying cDNA derived from HTLV-I+ cells between the 5' LTR and pX region. Bands of 2 different sizes were consistently observed. Sequencing of the longer band corresponded to a cDNA derived from a double-spliced pX mRNA as previously reported. The shorter band was derived from a single-spliced mRNA. HTLV-I+ cell lines had both mRNAs to a varying degree. Expression of p40tax and p21X-III seem to be well correlated with a double-spliced and a single-spliced mRNA, respectively.

Base Sequence↗
PubMed1991

Semiquantitative analysis of integrated genomes of human T-lymphotropic virus type I in asymptomatic virus carriers.

A semiquantitative estimation of human T-lymphotropic virus type I (HTLV-I) integration by peripheral blood mononuclear cells (PBMC) was performed. Genomic DNA samples derived from 134 HTLV-I carriers were subjected to 40 or 60 cycles of the polymerase chain reaction to amplify the pol region of HTLV-I. The HTLV-I genome was detected by dot hybridization using a 32P-labeled oligonucleotide probe for the pol region. The radioactivity of hybridized dot membranes was then counted with an RI Imaging System (Ambis Inc, San Diego, CA) and the HTLV-I genome dose was determined by comparison with standard curve for serially diluted HTLV-I genome-positive DNA. A wide range of variation of HTLV-I genome integration was observed. When the integrated genome dose was calculated as the number of HTLV-I copies per 100 PBMC, 7 carriers (5%) had more than 10 copies, 56 (42%) had 1 to 10 copies, 46 (34%) had 0.1 to 1 copy, and 24 (18%) had less than 0.1 copy. In one sample, the HTLV-I genome was undetectable, which may indicate that the integrated genome was present at less than 0.01 copies per 100 PBMC. Age- or sex-related variations in the distribution of individuals with different HTLV-I genome were rather limited. However, carriers with a high level of the HTLV-I genome were always more than 30 years old and were predominantly male (six of seven).

Aging↗
PubMed1991

CD4-CD8- T cell receptor alpha beta T cells: generation of an in vitro major histocompatibility complex class I specific cytotoxic T lymphocyte response and allogeneic tumor rejection.

The generation of an in vitro major histocompatibility complex class I specific response of CD4-CD8- T cell receptor (TCR) alpha beta cytotoxic T lymphocytes (CTL) and their allogeneic tumor rejection were investigated. Inocula of BALBRL male 1 were rejected in C57BL/6 (B6) mice treated with minimum essential medium (MEM) (control), anti-L3T4 (CD4) monoclonal antibody (mAb) or anti-Lyt-2.2 (CD8) mAb and CTL against the tumor were generated in vitro. No rejection and no induction of CTL were observed in B6 mice treated with anti-L3T4 (CD4) plus anti-Lyt-2.2 (CD8) mAb. CTL with the classical Thy-1+ CD3+CD4-CD8+ TCR alpha beta phenotype were generated in mixed lymphocyte tumor cell culture (MLTC) spleen cells from B6 mice treated with MEM (control) or anti-L3T4 (CD4) mAb, whereas CTL with an unusual Thy-1+CD3+CD4-CD8- TCR alpha beta phenotype were generated in MLTC spleen cells from anti-Lyt-2.2 (CD8) mAb-treated B6 mice. Both types of CTL were reactive with both H-2Kd and Dd (Ld) class I antigen. These findings suggest that when CD4+ cells were blocked by anti-L3T4 (CD4) mAb, CD8+ CTL mediated rejection, and when CD8+ cells were blocked by anti-Lyt-2.2 (CD8) mAb, CD4+ cells were capable of mediating rejection, although less efficiently than CD8+ cells, by inducing CD4-CD8- TCR alpha beta CTL. The finding that adoptive transfer of CD4 and CD8-depleted MLTC spleen cells, obtained from anti-Lyt-2.2 (CD8) mAb-treated B6 mice that had rejected BALBRL male 1, resulted in rejection of BALBRL male 1 inoculated into B6 nu/nu mice confirmed the above notion. CTL clones with the CD4-CD8- TCR alpha beta phenotype specific for Ld were established.

Animals↗
PubMed1991

Induction of in vitro and in vivo anti-tumor responses by sensitization of mice with liposomes containing a crude butanol extract of leukemia cells and transferred inter-membranously with cell-surface proteins.

Generation of cytotoxic T lymphocytes (CTL) in vitro and tumor-rejection responses by sensitization of semi-syngeneic mice with tumor-antigen-reconstituted liposomes were investigated. Liposomes were prepared from a crude butanol extract (CBE) of BALBRVD leukemia cells and egg phosphatidylcholine (PC): 1,2-dimyristoylamido-1,2-deoxyphosphatidylcholine (DDPC) (3:2) or dimyristoylphosphatidylcholine (DMPC):DDPC (1:4). Inter-membrane protein transfer (IMPT) liposomes were prepared by incubating BALBRVD cells with DMPC:DDPC (1:4) liposomes. Sensitization of male CB6F1 mice with CBE or IMPT liposomes induced a level of cytotoxicity similar to that on sensitization with mitomycin-C(MMC)-treated BALBRVD against BALBRVD target cells after in vitro sensitization with the tumor cells. Sensitization with CBE alone resulted in only marginal cytotoxicity. The cytotoxic effector cells induced by either mode of sensitization were CD8+ T-cells whose recognition was Kd-restricted. No difference in specificity was observed with the different modes of sensitization. Two in vivo immunizations with CBE or with CBE liposomes at a dose of 25 micrograms of protein (equivalent to 2.5 x 10(7) cells) cause moderate inhibition of BALBRVD tumor growth in male CB6F1 mice and immunization with IMPT liposomes at a dose of 1 microgram of protein result in efficient protection.

1-Butanol↗