Biomedical subjects

K Yoshiike

Publications and source records attributed to K Yoshiike.

At least 37 records · Page 2Linked to original sources

PubMed1992

The E7 functions of human papillomaviruses in rat 3Y1 cells.

Among more than 60 human papillomavirus (HPV) genotypes, several HPVs are believed to be high risk because they are found in close association with cervical carcinoma. We compared the E7 genes from HPVs 1, 6b, 16, 18, and 33 for their transactivating, transforming, and mitogenic functions in a single cell line rat 3Y1. Whereas both the low-risk (1 and 6b) and the high-risk (16, 18, and 33) HPVs were transactivating for the adenovirus E2 promoter, only the high-risk HPVs were capable of focal transformation as assayed by an efficient method using the SR alpha-promoter and in conjunction with the HPV 16 E6 gene. The putative oncogenicity of HPVs appears to be reflected in vitro by the focal transformation, but not by the transactivation. Transient expression of the E7 genes controlled by the dexamethasone-responsive MMTV-LTR showed that the HPV 16 mutant E7s only with residual transforming activity were not mitogenic, but that, although the low-risk HPV E7s were less efficient, both the low-risk and high-risk HPV E7s were capable of inducing cellular DNA synthesis. Probably, the capability to induce cell DNA synthesis is necessary but not sufficient for the E7-mediated focal transformation.

Animals↗

PubMed1992

Changing the spacing between metal-binding motifs decreases stability and transforming activity of the human papillomavirus type 18 E7 oncoprotein.

Spacing composed of 29 amino acids (AAs) between a pair of metal-binding motifs, Cys-X-X-Cys, is common to human papillomavirus (HPV) zinc-binding oncoproteins E6 and E7 from various HPV types. From the HPV 18 E7 gene encoding a 105-AA protein with the motifs at AAs 65-68 and 98-101, we constructed expression plasmids for two mutants, 18del73 and 18ins84, with varied spacing between the motifs. Mutant proteins 18del73 and 18ins84 had a deletion from AAs 74 to 88 and an insertion of three AAs substituting for AA 85, respectively. The mutations lowered the efficiency of E7-mediated focal transformation of rat 3Y1 cells, approximately parallel to the reduced level of steady-state E7 expressed in COS-1 cells. It is likely that, besides the presence of the motifs, the conserved spacing between the motifs is important for a stable structure of E7, but is not essential for the E7-transforming activity.

Amino Acid Sequence↗

PubMed1991

Human papillomavirus type 16 E6 proteins with glycine substitution for cysteine in the metal-binding motif.

The human papillomavirus type 16 (HPV 16) E6 is a 151 amino acid protein containing four metal-binding motifs, Cys-X-X-Cys. We constructed and characterized three mutants with Gly substitutions for Cys within the motif; for Cys-66, for Cys-136, and for both, respectively. Zinc binding to bacterially expressed E6 was markedly reduced by the substitution for Cys-66, but DNA binding was unaffected by any of these mutations. Immunofluorescence staining showed that, whereas the E6 expressed in monkey COS-1 cells appeared mostly nuclear, the Cys-66 mutant appeared cytoplasmic. Subcellular fractionation followed by immunoprecipitation showed that the E6 in COS-1 cells was located in the membrane, nuclear, and nuclear-wash fractions, but not in the soluble cytoplasmic fraction, and that the nuclear Cys-66 protein was markedly reduced. The mutant proteins in COS-1 cells appeared to be less stable than the wild type, because the immunofluorescent cells were fewer and because the E6 bands in autoradiograms were less dense. The substitution mutants lost their capacity to enhance HPV 16 E7 transformation of rat 3Y1 cells. The data indicate that Cys-66 plays a crucial role for zinc binding and nuclear localization of E6 and that both Cys-66 and Cys-136 are required for a stable or functional structure of E6.

Amino Acid Sequence↗

PubMed1991

Two immunodominant regions of the human papillomavirus type 16 E7 protein are masked in the nuclei of monkey COS-1 cells.

The human papillomavirus type 16 E7 protein is a 98-amino acid (AA)-long nuclear oncoprotein. We established eight independent mouse hybridoma cell lines producing monoclonal antibodies (MAbs) specific for the E7 protein and characterized the MAbs by competitive binding analysis with the bacterially expressed, nonfusion E7 protein and synthesized oligopeptides. The MAbs were classified into two groups; three and five MAbs recognizing epitopes in probable immunodominant regions AAs 8 to 22 (region I) and AAs 39 to 54 (region II), respectively. These MAbs were capable of immunoprecipitating the E7 protein transiently expressed in monkey COS-1 cells. By immunofluorescence staining of the acetone-fixed E7-producing COS-1 cells, the MAbs for regions I and II detected no E7 protein and only cytoplasmic E7 protein, respectively, whereas a rabbit polyclonal antiserum (anti-lac-E7) detects both nuclear and cytoplasmic E7 proteins. Like anti-lac-E7, the MAbs of the two groups stained both nuclear and cytoplasmic E7 proteins in the COS-1 cells that were denatured with formaldehyde. The results show that two immunodominant regions of the HPV 16 E7 protein are masked in the COS-1 nuclei for binding with the corresponding MAbs. The masking of the intranuclear E7 protein may result from complex formation with cellular proteins (or structures), polymerization, or self-folding.

Amino Acid Sequence↗

PubMed1991

Detection of human antibody against the human papillomavirus type 16 E7 protein.

We examined 500 human sera for the presence of antibody against the human papillomavirus type 16 E7 protein by enzyme-linked immunosorbent assay with bacterially expressed fusion protein lac-E7, and by radioimmunoprecipitation and immunofluorescence assays with the E7 protein expressed in monkey COS-1 cells. The anti-E7 antibody was detectable in 6 out of 54 cases of cervical carcinoma, but in none of the others, including patients with other gynecologic cancers, those with sexually transmitted diseases, and healthy adults. The data indicate that expression of the E7 protein is closely related to cervical carcinoma.

Animals↗

PubMed1990

Mutational analysis of human papillomavirus type 16 E7 functions.

The human papillomavirus type 16 E7 gene encodes a nuclear oncoprotein (98 amino acids [AAs] long) consisting of three regions: regions 1 (AAs 1 to 20) and 2 (AAs 21 to 40), which show high homology to the sequences of conserved domains 1 and 2, respectively, of adenovirus E1A; and region 3 (AAs 41 to 98) containing two metal-binding motifs Cys-X-X-Cys (AAs 58 and 91 to 94). We constructed AA deletion (substitution) mutants and single-AA substitution mutants of E7 placed under the control of the simian virus 40 promoter and examined their biological functions. Stable expression of E7 protein in monkey COS-1 cells required almost the entire length of E7 and was markedly lowered by the mutations in region 3. Transactivation of the adenovirus E2 promoter in monkey CV-1 cells was lowered by the mutations. It was abolished by changing Cys-24 to Gly and markedly decreased by a mutation at His-2 or at the metal-binding motifs in region 3. Focal transformation of rat 3Y1 cells by E7 was eliminated by changing His-2 to Asp or Cys-24 to Gly and was greatly impaired by changing Cys-61 or Cys-94 to Gly. The transforming function survived mutations at Leu-13 and Cys-68 and deletion of Asp-Ser-Ser (AAs 30 to 32). The data suggest that regions 1 to 3 are required for its functions and that the meta-binding motifs in region 3 are required to maintain a stable or functional structure of the E7 protein.

Adenoviruses, Human↗

PubMed1990

[A study on the therapy and prognosis of pemphigus vulgaris].

The treatment and, in particular, the prognosis of 32 patients with pemphigus were studied. All cases were classified according to treatment, for example, corticosteroid only, corticosteroid + immune-suppressive agents, plasma exchange + corticosteroid and/or immune-suppressive agents, and other treatments. Results showed that with the plasma exchange group skin lesions disappeared the quickest, the percentage of disappearance was the highest and a long standing remission was able to be maintained. With the plasma exchange group, many cases were severe and had complications which were not able to be resolved by corticosteroid or immune-suppressive agents. Therefore, plasma exchange may be considered as a very significant form of treatment of pemphigus vulgaris.

Adrenal Cortex Hormones↗

PubMed1989

Inefficient expression in rat cells of transforming gene of BK virus with 68-bp tandem repeats in the viral promoter-enhancer.

Large-plaque BK virus (wt-501), which has three 68-bp tandem repeats in the viral promoter-enhancer, is not transforming for rat cells, whereas the minute-plaque mutant (dl-504) with only one 68-bp element is. We tested the BKV wt-501 and dl-504 promoter-enhancers for their ability to activate the heterologous and homologous genes in rat 3Y1 cells. The 501 promoter-enhancer activated expression of the chloramphenicol acetyltransferase gene and the neomycin-resistance gene more efficiently than the 504 promoter-enhancer. The 501 enhancer could not allow the efficient expression of BK virus T-antigen gene, whereas the 504 enhancer could. The wt-BKV enhancer with three 68-bp repeats seemed to function positively on the heterologous genes but negatively on the homologous transforming gene in rat cells. The sequence causing the suppressive effects by duplication was confined within the 29-bp segment containing two SV40 enhancer core homologies in the 68-bp element. The results show that inability of wt-BKV to transform rat cells is ascribable to inefficient T-antigen gene expression caused by the duplication in the enhancer and by the surrounding or downstream DNA sequence.

Animals↗

PubMed1989

Host range bias of the JC virus mutant enhancer with DNA rearrangement.

JC-HEK, a JC virus (JCV) host range mutant adapted to growth in human embryonic kidney cells (HEK), has DNA rearrangement in the control region for early transcription. The 822-bp rearranged segment of JC-HEK (containing one authentic promoter-enhancer unit of 98 bp and truncated coding region of T-antigen and VP-1 genes) was inserted into pSV0cat vector and examined for expression of chloramphenicol acetyltransferase (CAT). The CAT activity induced by the mutant was comparable to that by the SV40 promoter-enhancer (pSV2cat) and four times as high as that by the prototype JCV (Mad-1) in HEK cells, whereas the two JCV promoter-enhancers were equally efficient in a permissive neuroblastoma cell line. On the other hand, both JC-HEK DNA with three DNA replication origins and Mad-1 DNAs replicated after transfection into HEK cells at a similar efficiency in the presence of JCV T-antigen. The elevated promotor-enhancer activity caused by the DNA rearrangement seems to play a major role in adaptation of JCV to growth in HEK cells.

Antigens, Polyomavirus Transforming↗

PubMed1989

Human papillomavirus type 16 E7 protein expressed in Escherichia coli and monkey COS-1 cells: immunofluorescence detection of the nuclear E7 protein.

Human papillomavirus type 16 E7 protein, expressed in Escherichia coli as fusion protein lac alpha peptide-E7 (lac-E7), was purified by electrophoresis and used to immunize rabbits to raise antisera. The anti-lac-E7 sera recognized another bacterially expressed fusion protein trpE-E7 by the Western blot method. The antisera were used to identify E7 protein transiently expressed in monkey COS-1 cells from an SV40-derived expression plasmid containing E7 gene. Like the E7 protein from CaSki cells, the majority of 19K E7 protein in the transfected COS-1 cells was found by immunoprecipitation in the soluble cytoplasmic fraction. By immunofluorescence staining, however, the E7 protein was detectable in the nuclei of the transfected COS-1 cells. The results suggest that the E7 protein expressed in monkey cells is nuclear, although it is readily released from nuclei when the cell structure is broken.

Animals↗

PubMed1989

Expression of human papillomavirus type 16 E7 gene induces DNA synthesis of rat 3Y1 cells.

Human papillomavirus type 16 (HPV 16) open reading frames (ORF) E6, E7, and E6E7, placed under the control of dexamethasone-inducible mouse mammary tumor virus long terminal repeat, were introduced into rat 3Y1 cells, an immortalized fibroblast line, with the aid of neomycin-selection. The cell clones containing inducible HPV 16 ORFs were selected and examined for DNA synthesis. Following induction of HPV mRNA synthesis by the hormone, DNA synthesis was stimulated in the cells containing E7 or E6E7 ORFs. The data indicate that expression of the HPV 16 E7 gene is mitogenic for rat 3Y1 cells.

Animals↗

PubMed1989

Human papillomavirus type 16 transformation of primary human embryonic fibroblasts requires expression of open reading frames E6 and E7.

Human papillomavirus 16 (HPV-16) early genes were tested for their ability to transform primary human embryonic fibroblasts (WI38) or to extend the life span of the fibroblasts. The expression plasmids containing the transcriptional unit for the HPV-16 subgenomic DNA fragments controlled by the simian virus 40 promoter were introduced into WI38 cells with the aid of neomycin selection, and the drug-resistant cells that survived long-term culture were characterized. The fragments containing E6E7 open reading frames (ORFs) intact were found to be capable of extending the life span of WI38 cells. From the results obtained with frameshift mutations in either ORF and from those obtained by cotransfection with the separate plasmids containing E6 and E7 ORFs, it was concluded that HPV-16 transformation (extension of the life span) of human fibroblasts requires expression of genes E6 and E7.

Cell Survival↗

PubMed1988

Transformation of rat 3Y1 cells by human papillomavirus type-18 DNA.

Rat 3Y1 cell cultures transfected with human papillomavirus type-18 (HPV 18) DNA produced foci of morphologically transformed cells capable of anchorage-independent growth. Analyses of the transformed cell clones obtained after transfection with EcoRI-cleaved linear HPV 18 DNA showed that, although the viral DNAs integrated in the cell DNA had rearrangements (probably around the sites of integration), they appeared to retain the putative transcriptional control region and the downstream area containing E6E7 open reading frames (ORF) intact, and to be transcriptionally active. The expression plasmids constructed from an SV40-derived expression vector (pSV2-0) and HPV 18 DNA fragments containing either E6 or E7 ORF intact were examined for their transforming capacity. HPV 18 E7 ORF was found to encode a transforming gene for rat 3Y1 cells.

Cell Line↗

PubMed1988

Immortalization of primary rat cells by human papillomavirus type 16 subgenomic DNA fragments controlled by the SV40 promoter.

We tested the human papillomavirus 16 (HPV 16) early genes for their ability to immortalize or transform primary rat brain cells, using the expression plasmids that contain the neomycin-resistance-inducing unit (pSV2neo) and the transcriptional unit for the HPV 16 subgenomic DNA fragments controlled by the SV40 early promoter. After transfection, drug-resistant colonies were maintained by refeeding and replating for characterization. The E7 gene alone was found to be capable of immortalizing and morphologically transforming primary rat cells, and the transformed cells showed anchorage-independent growth. Although its activity was lower than that of the E7 gene, the E6 gene also immortalized primary rat cells. The immortalized or transformed cells contained HPV 16-specific DNA and mRNA.

Animals↗

PubMed1988

Transformation of rat 3Y1 cells by a deletion DNA of human papillomavirus type 16 molecularly cloned from genomic DNA of a cervical carcinoma.

Human papillomavirus type 16 (HPV 16) DNA integrated within the cell DNA in cervical carcinomas is frequently defective, but commonly retains its putative transcriptional control region and E6 and E7 open reading frames (ORFs) intact. One clone of such a partially deleted HPV 16 DNA was tested and found to be transforming for rat 3Y1 cells. The results indicates that HPV 16 DNA containing E6 and E7 ORFs with the homologous promoter is sufficient for inducing focal transformation of immortalized rat fibroblasts.

Animals↗

PubMed1988

Human papillomavirus type 16 open reading frame E7 encodes a transforming gene for rat 3Y1 cells.

Human papillomavirus type 16 (HPV 16) DNA is capable of morphologically transforming rat 3Y1 cells. The expression plasmids, constructed from the simian virus 40-based expression vector pSV2-0 and specific DNA fragments from the putative early region of the HPV 16 genome, were tested for their transforming capacity. Among the various pSV2 plasmids, only those containing the intact E7 coding region were found to produce foci of the transformed rat cells which could grow in a soft-agar medium. The data indicate that expression of the HPV 16 E7 open reading frame is sufficient to induce focal transformation of rat cells.

Animals↗

PubMed1987

Human papillomavirus type 16 transformation of rat 3Y1 cells.

The molecularly cloned, prototype human papillomavirus (HPV) 16 DNA has a single-base deletion in the El gene. We reconstructed a putative non-defective HPV 16 genome with an uninterrupted El gene, and examined its biological functions. The reconstructed HPV 16 DNA formed foci of morphologically transformed cells in transfected rat 3Y1 cell cultures (an immortalized normal cell line). The transformed rat cells contained transcriptionally active HPV 16 DNA, which appeared to be integrated within the cell DNA, and formed colonies in soft agar.

Animals↗

PubMed1986

Cloning of monomeric human papillomavirus type 16 DNA integrated within cell DNA from a cervical carcinoma.

We have molecularly cloned and characterized monomeric human papillomavirus type 16 DNA with flanking cell DNA sequences from a cervical carcinoma. Determination of nucleotide sequence around the junctions of human papillomavirus and cell DNAs revealed that at the site of integration within cell DNA the cloned viral DNA had a deletion between nucleotides 1,284 and 4,471 (numbering system from K. Seedorf, G. Krämmer, M. Dürst, S. Suhai, and W. G. Röwekamp, Virology 145:181-185, 1985), which includes the greater part of E1 gene and the entire E2 gene. In the remaining part of the E1 gene, three guanines were found at the location where two guanines at nucleotides 1,137 and 1,138 have been recorded. This additional guanine shifted the reading frame and erased an interruption in the E1 gene described by Seedorf et al. The data strongly suggest that, like other papillomaviruses, human papillomavirus type 16 has an uninterrupted E1 gene.

Base Sequence↗