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K Shiroki

Publications and source records attributed to K Shiroki.

At least 37 records · Page 2Linked to original sources
PubMed1990

Identification of sequences responsible for positive and negative regulation by E1A in the promoter of H-2Kbm1 class I MHC gene.

The mechanism of transcriptional regulation of the H-2Kbm1 major histocompatibility complex (MHC) class I gene by adenovirus type 12 E1A (Ad12-E1A) was studied in transfected rat embryonal fibroblasts. Results of long-term expression of the chloramphenicol acetyl transferase (CAT) gene placed under the control of the 5'-flanking region of the mouse MHC class I gene. H-2Kbm1, and the results of nuclear run-on transcription assays, yield evidence for both positive and negative regulation of H-2Kbm1 by E1A gene product. Deletion studies in the H-2Kbm1 promoter region revealed that a proximal 58 bp upstream sequence (-194 to -136, relative to the cap site) and a distal 316 bp sequence (-1837 to -1521) respectively contribute to positive and negative regulation mediated by the E1A gene product. Both regulatory elements of MHC class I gene promoter region are responsible for the differential expression of the H-2Kbm1 gene in Ad12 transformed cells. A nuclear factor binding to the negative element has been detected only in extracts derived from cells expressing Ad12-E1A.

Adenovirus Early Proteins↗
PubMed1990

Transfection with fragments of the adenovirus 12 gene induces tumorigenicity-associated alteration of N-linked sugar chains in rat cells.

N-linked sugar chains of rat 3Y1 cells and their poorly tumorigenic (E1Y cells) and highly tumorigenic (CY1 cells) transformants carrying various adenovirus 12 gene fragments were quantitatively released as oligosaccharides from their membrane preparations by hydrazinolysis. After being fractionated by a series of immobilized lectin column chromatography, structures of oligosaccharides in each fraction were studied by sequential glycosidase digestion in combination with methylation analysis. All cells contain bi-, tri-, and tetraantennary complex-type oligosaccharides as well as high mannose-type oligosaccharides in different molar ratio. Expression of 2,4-branched triantennary oligosaccharides increased in both transformed cells. However, their contents were rather higher in poorly tumorigenic E1Y cells than in highly tumorigenic CY1 cells. In contrast, the increase of 2,6-branched triantennary and tetraantennary oligosaccharides was positively correlated to the tumorigenic potential of the transformed cells. The data indicate that glycosylation of cellular proteins is differently affected by the expression of specific regions of the adenovirus genome, and the combined action of E1 and E4 gene products is important for the expression of the GlcNAc beta 1----6Man group associated with tumorigenicity of the cells.

Adenoviridae↗
PubMed1990

Tandemly repeated hexamer sequences within the beta interferon promoter can function as an inducible regulatory element in activation by the adenovirus E1B 19-kilodalton protein.

The expression of the human beta interferon (IFN-beta) gene was activated by the adenovirus E1B-19K protein. The sequence within the IFN-beta promoter which is related to the activation was analyzed by chloramphenicol acetyltransferase (CAT) assay. The repeated hexamer units, present within the region between -109 and -65 relative to the cap site, were required for the activation of the IFN-beta gene by the E1B-19K protein. The (AAGTGA)8 region, as a typical hexamer of the consensus sequences, was tested for function in the activation by the E1B-19K protein. When the hexamer (AAGTGA)4-8 was inserted upstream of several reporter genes (such as p55cat, pdlE1A-CAT, and pE1B-CAT) which were inefficiently stimulated, the CAT activities of these fusion genes were efficiently stimulated by the E1B-19K protein. These results show that the tandemly repeated hexamer sequences within the IFN-beta promoter can function as an inducible regulatory element in the activation by the adenovirus E1B 19K protein.

Adenovirus Early Proteins↗
PubMed1990

Nonlethal G0-ts mutant tsJT60 becomes lethal at the nonpermissive temperature after transformation: a hint for new cancer chemotherapeutics.

tsJT60 is a nonlethal temperature-sensitive (ts) mutant of a Fischer rat cell line (3Y1) classified as a G0 mutant; i.e., the ts defect is not expressed within the cell growth cycle but is expressed only between the G0 and S phase. tsJT60 clones transformed with oncogenes such as adenovirus E1A, polyoma large T, polyoma middle T, v-Ki-ras, and LTR activated c-myc, or with a chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine, grew well at 34 degrees C. However, most of these clones grew slowly at 40 degrees C, producing many floating dead cells, and some clones were killed at 40 degrees C. When they were cultured under conditions inadequate for growth of untransformed cells, such as high cell density or serum restriction, they were killed at 40 degrees C. These and previous results from SV40- and adenovirus-transformed tsJT60 clones favour the idea that transformed tsJT60 cells occasionally enter the G0 phase and are metabolically imbalanced at 40 degrees C during self-stimulation from the G0 to S phase. We propose that a drug which exclusively block, G0-G1 transition would be cytocidal to transformed cells but cytostatic to normal cells.

Adenovirus Early Proteins↗
PubMed1989

The high sensitivity of cells transformed by E1A gene of adenovirus type 12 to diacylglycerol-mediated cell killing.

Viability of rat 3Y1 fibroblasts transformed by adenovirus type 12 (Ad12) was markedly impaired by the administration of dilinoleoylglycerol (DLG) to the culture medium. To identify the gene(s) of Ad12 responsible for the high sensitivity to DLG, we established several transformed sublines of 3Y1 induced by the viral E1A gene or by the mutants of Ad12 which have mutations in the E1A region. All of the transformed sublines of 3Y1 expressing either the 12S or the 13S message from the E1A region were highly sensitive to the cytotoxicity of DLG. We propose that the high sensitivity of Ad12-transformed cells to the DLG mediated cytotoxicity is attributable to the common function of E1A-12S and E1A-13S mRNA products.

Adenoviridae↗
PubMed1989

A variant Schmidt-Ruppin strain of Rous sarcoma virus with increased affinity for mammalian cells.

SR-RSV-D(H), a variant virus with extremely high tropism for mammalian cells, was isolated by passage of the Schmidt-Ruppin strain of Rous sarcoma virus of subgroup D (SR-RSV-D) through hamster cells. This variant virus has acquired an altered envelope glycoprotein, encoded by the env gene, that has high affinity for receptors on the surface of mammalian cells. The variant virus transforms rat cells at about 100 times the efficiency of the parental virus, SR-RSV-D(S), as assayed by focus formation. Addition of amphotericin B (Fungizone) to the medium at a concentration of 0.2 micrograms/ml completely inhibited rat cell transformation by SR-RSV-D(H), possibly by blocking virus penetration into the cells, whereas the drug showed no inhibitory effect on transformation of chick embryo fibroblast (CEF) cells by the variant virus or on transformation of rat cells by the parental virus. The efficiency of transformation of rat cells by the variant virus was much less than its efficiency of transformation of CEF cells. Analysis of infection of rat cells suggested that the virus can infect rat cells as efficiently as CEF cells but that rat cells were not transformed by the virus as fully as CEF cells because of inefficiency of some post-penetrational step involved in viral gene expression. The finding that E1AY cells, rat cells expressing adenovirus E1A gene, were transformed by SR-RSV-D(H) as efficiently as CEF cells supports this conclusion and suggests that expression of the E1A gene in rat cells may overcome the defect in the transforming step(s) in rat cells.

Amphotericin B↗
PubMed1988

Analysis of cellular expression of gangliosides by gene transfection. I: GD3 expression in myc-transfected and transformed 3Y1 correlates with anchorage-independent growth activity.

Transfection of c-myc DNA into rat fibroblastic 3Y1 cell line resulted in the neosynthesis of GD3 ganglioside, as has been previously shown to occur after transfection of 3Y1 cells with adeno El (Nakakuma, H., Sanai, Y., Shiroki, K., and Nagai, Y. (1984) J. Biochem. 96, 1471-1480); in both cases, the products are expressed intranuclearly. Moreover, a clear correlation was identified between levels of GD3 expression and colony-forming activity (in soft agar) of myc-transformed 3Y1 cells, implying that GD3 plays some specific role in myc-induced transformation of 3Y1 cell line.

Animals↗
PubMed1988

A cell cycle G0-ts mutant, tsJT60, becomes lethal at the nonpermissive temperature after transformation with adenovirus 12 E1B 19K mutant.

tsJT60, a temperature-sensitive (ts) cell-cycle mutant of Fischer rats, is viable at both the permissive (34 degrees C) and nonpermissive (40 degrees C) temperatures. The cells grow normally in exponential growth phase at both temperatures, but when stimulated with serum from G0 phase they enter S phase at 34 degrees C but not at 40 degrees C. tsJT60 cells transformed with human adenovirus (Ad) 12 dl205, which lacks the E1B 19-kDa polypeptide gene, were lethal at 40 degrees C, whereas tsJT60 cells transformed with Ad12 wt, dl207, which lacks E1B 58-kDa protein gene, or in206B, which produces 19- to 58- kDa fused protein, were viable. Degradation of cell DNA occurred in dl205-transformed tsJT60 cultured at both 34 degrees C and 40 degrees C. Neither cytocidal phenotype nor degradation of DNA occurred in 3Y1 cells (a parental line of tsJT60) transformed with dl205. These results suggest that the lethal phenotype and degradation of DNA are related to the ts mutation in tsJT60 and also to the lack of Ad12 E1B 19kDa polypeptide.

Adenoviridae↗
PubMed1988

Suppression of block to entry into S phase in cell-cycle mutants of rat 3Y1 fibroblasts after transformation by adenovirus type 12.

Four temperature-sensitive (ts) mutants of rat 3Y1 fibroblasts, belonging to separate complementation groups, cease to proliferate in the G1 phase of the cell cycle at a restrictive temperature (39.8 degrees). These ts mutants were transformed at a permissive temperature with adenovirus type 12 (Ad12), its E1A gene, or in203S mutant of Ad12 which has a mutation in the E1A 13 S mRNA unique region. We examined whether the proliferation of the transformed cells would be blocked in the G1 phase, at 39.8 degrees. One mutant did not cease to proliferate at 39.8 degrees after transformation with either Ad12 or E1A. In two other mutants, Ad12-transformed cells did not cease to proliferate at 39.8 degrees, whereas E1A-transformed cells did not survive at 39.8 degrees, though they did continue to enter the S phase. Analysis of transcription of the viral early genes in the transformants of one of the latter two mutants suggests that the expression of other viral early genes, in addition to E1A, is required for cell proliferation, in addition to entry into S phase. In the fourth mutant, both Ad12- and E1A-transformed sublines did not cease to enter the S phase but cells readily detached from the dishes. These results suggest that (1) function(s) of the E1A gene alone is sufficient for Ad12 to suppress the inhibition of the initiation of cellular DNA synthesis caused by four different cellular ts defects, (2) functions of Ad12 early genes other than, or in addition to, E1A are necessary for suppression of the inhibition of cell proliferation (and/or for survival) in two of the four ts mutants, and (3) in the case of one other ts mutant, E1A alone overcomes the ts defect and allows for the entire cell proliferation.

Adenoviridae↗
PubMed1988

Activation of the human beta interferon gene by the adenovirus type 12 E1B gene.

The transcription of endogenous beta interferon mRNA was activated in human embryo kidney (HEK) cells infected with adenovirus type 12 (Ad12) but was activated only inefficiently or not at all in HEK cells infected with Ad5 and rc-1 (Ad5 dl312 containing the Ad12 E1A region). The analysis with Ad12 mutants showed that Ad12 E1B products, especially the 19K protein, were important for the expression of the endogenous beta interferon gene and Ad12 E1A products were not involved in the expression. The expression of exogenously transfected pIFN-CAT (a hybrid plasmid having the human beta interferon promoter fused with the CAT gene) was activated in HEK and chicken embryo fibroblast (CEF) cells infected with either Ad12 or Ad5. The analysis of cotransfection of CEF cells with pIFN-CAT and plasmids containing fragments of Ad12 or Ad5 DNA showed that Ad12 or Ad5 E1B (possibly the 19K protein) was and E1A was not involved in the expression of the exogenous pIFN-CAT.

Adenoviridae↗
PubMed1987

Induction of cellular DNA synthesis in G0-specific ts mutant, tsJT60, following infection with SV40 and adenoviruses.

tsJT60 cells, a temperature-sensitive G0 mutant of a Fischer rat cell line, grew normally in an exponential growth phase at both permissive (34 degrees C) and nonpermissive (39.5 degrees C) temperatures, but when stimulated with fetal bovine serum in the growth-arrested state (G0 phase) they entered S phase at 34 degrees C but not at 39.5 degrees C. Infection of G0-arrested tsJT60 cells with SV40, adenovirus (Ad) 5 wild type and its E1B mutant dl313, and Ad12 wild type and its E1B mutants in205B, in205C, dl205, and in206B induced DNA synthesis at both temperatures. The DNA synthesized after virus infection was shown to be cellular by Hirt separation of DNA from SV40-infected cells and by CsCl equilibrium density gradient centrifugation of DNA from Ad5-infected cells.

Adenoviruses, Human↗
PubMed1987

tsJT60, a cell cycle G0-ts mutant, becomes lethal at non-permissive temperature by transformation with adenovirus 5 when the expression of E1B gene is lacking.

tsJT60, a temperature-sensitive (ts) mutant cell line of Fischer rat, is viable at both permissive (34 degrees C) and non-permissive (39.5 degrees C) temperatures. The cells grow normally in exponential growth phase at both temperatures, but when stimulated with fetal bovine serum (FBS) from G0 phase they re-enter S phase at 34 degrees C but not at 39.5 degrees. When tsJT60 cells were transformed with adenovirus (Ad) 5 wild type, they grew well at both temperatures, expressed E1A and E1B genes, and formed colonies in soft agar. When tsJT60 cells were transformed with Ad5 dl313, that lacks E1B gene, the transformed cells grew well at 34 degrees C but failed to form colony in soft agar. They died very soon at 39.5 degrees C. 3Y1 cells (a parental line of tsJT60) transformed with dl313 grew well at both temperatures, although neither expressed E1B gene nor formed colonies in soft agar. The phenotype of being lethal at 39.5 degrees C of dl313-transformed tsJT60 cells was complemented by cell fusion with 3Y1BUr cells (5-BrdU-resistant 3Y1), but not with tsJT60TGr cells (6-thioguanine resistant tsJT60). These results indicate that the lethal phenotype is related to the ts mutation of tsJT60 cells and also to the deletion of E1B gene of Ad5.

Adenoviridae↗
PubMed1987

Induction of cellular DNA synthesis by adenovirus type 12 in a set of temperature-sensitive mutants of rat 3Y1 fibroblasts blocked in G1 phase.

Four temperature-sensitive (ts) mutants of rat 3Y1 fibroblasts, which represent separate complementation groups, cease to proliferate predominantly with a 2C DNA content, either at 39.8 degrees (temperature arrest), or at 33.8 degrees at a confluent cell density (density arrest). When infected at 39.8 degrees with adenovirus type 12 (Ad12), cells of all four ts mutants in both arrest states entered the S phase, thereby suggesting that Ad12 overcomes the four independent functional blocks to cellular entry into S phase. Results of experiments using Ad12 E1-region mutants suggest that the E1A gene product(s) is indispensable to overcoming the ts block, whereas the E1B product(s) may be dispensable. The cell killing observed in 3Y1 cells infected with wild-type Ad12 did not occur in infection with one of the E1-region mutants with a 6-bp insertion in the E1A 13 S mRNA unique region. When infected with this mutant at 39.8 degrees, two ts mutants of 3Y1 (3Y1tsF121 and 3Y1tsG125) in both arrested states proliferated through at least one generation. Another mutant (3Y1tsD123) was accelerated to die following entry into the S phase. In the other mutant (3Y1tsH203), the cell number was either unchanged (temperature arrest) or was increased less than twofold and then decreased (density arrest). The findings with the latter two mutant lines suggest that induction of cellular DNA synthesis is not sufficient for the subsequent proliferation of the infected cells, and that the Ad12 gene function(s) does not directly rescue the primary lesions in these ts mutants but does overcome some of the blocks to concomitantly occurring events. In the former two mutant lines, however, Ad12 gene function(s) may directly rescue the ts lesions. We propose that the Ad12 gene product(s) can overcome blocks to the initiation of cellular DNA synthesis but cannot overcome blocks to events related to cell survival.

Adenoviridae↗
PubMed1987

Interferon induction by adenovirus type 12: stimulatory function of early region 1A.

Adenoviruses are generally weak interferon inducers, triggering chicken embryo fibroblast cells by a UV-resistant viral component, probably the capsid or capsid elements, to produce 50 to 100 IU of interferon per ml. Adenovirus types 12, 18, and 31, however, can induce by a UV-sensitive mechanism 10 to 20 times more interferon than other types do. By using mutant and recombinant adenoviruses, we demonstrated that early region 1A was responsible for the enhanced interferon production of chicken cells infected with adenovirus type 12.

Adenoviridae↗
PubMed1986

An insertion mutation in the adenovirus type 12 early region 1A 13S mRNA unique region.

An adenovirus type 12 mutant, in203S, was constructed to contain an insertion of two amino acids in the early region 1A (E1A) 13S mRNA-coding region and in the E1A 12S mRNA intron. in203S could not grow in HeLa and KB cells. Virus DNA replication was scarcely detected at a low multiplicity of infection, but was detected at a high multiplicity of infection. The transcription of early genes other than E1A was not detected at 13 h after infection, but became detectable after longer incubation. The transcription of the E1A gene was also reduced to about one-fifth of the wild-type level. The mutant induced fewer foci of smaller sizes than the wild type in rat 3Y1 and secondary rat kidney cells. The induction of cellular DNA synthesis was reduced in rat 3Y1 cells infected with in203S as compared with that in wild type-infected cells. These results show that the E1A 13S mRNA-derived polypeptide of adenovirus type 12 is required for activation of early genes, cell transformation, and induction of cellular DNA synthesis.

Adenovirus Early Proteins↗
PubMed1986

The adenovirus type 12 early-region 1B 58,000-Mr gene product is required for viral DNA synthesis and for initiation of cell transformation.

An E1B 58K mutant of adenovirus type 12 (Ad12), dl207, was constructed by the deletion of 852 base pairs in the E1B 58K coding region. The mutant could grow efficiently in 293E1 cells but not in HeLa, KB, or human embryo kidney (HEK) cells. Viral DNA replication of dl207 was not detected in HeLa and KB cells and was seldom detected in HEK cells. Analysis of viral DNA synthesis in vitro showed that the Ad12-DNA-protein complex replicated by using the nuclear extract from Ad12 wild-type (WT)-infected HeLa cells but not by using the nuclear extract from dl207-infected cells. In dl207-infected HeLa and KB cells, early mRNAs were detected, but late mRNAs were not detected. The mutant induced fewer transformed foci than the WT in rat 3Y1 cells. Cells transformed by dl207 could grow efficiently in fluid medium, form colonies in soft agar culture, and induce tumors in rats transplanted with the transformed cells at the same efficiency as WT-transformed cells. Tumors were induced in hamsters injected with WT virions but were not induced in hamsters injected with dl207 virions. The results indicate that the E1B 58K protein is required both for viral DNA replication in productive infection and for initiation of cell transformation, but not for maintenance of the transformed phenotype.

Adenovirus Early Proteins↗
PubMed1986

Mitogenic activity of the adenovirus type 12 E1A gene induced by hormones in rat cells.

Several lines of rat 3Y1 cells in which expression of the adenovirus type 12 E1A gene can be regulated by dexamethasone were established by introduction of recombinant vector DNA containing the adenovirus type 12 E1A gene placed downstream of the hormone-inducible promoter of mouse mammary tumor virus. These cell lines (gMA cells) produced low basal levels of the E1A transcripts and proteins in normal medium and much higher levels upon addition of dexamethasone to the medium. When dexamethasone was added to density-arrested cells, DNA synthesis was induced in 10 to 40% of the cells, the percentage depending on the cell line. DNA synthesis was increased to up to 60% of the cell population by further addition of epidermal growth factor. Indirect immunofluorescence detection of E1A proteins in gMA cells treated with dexamethasone indicated that the intensity of fluorescence in cells varied and that the proportion of cells synthesizing DNA was correlated with the proportion that exhibited strong fluorescence. These results indicate that the E1A gene has a function to trigger the synthesis of cellular DNA.

Adenovirus Early Proteins↗