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T Kiyono

Publications and source records attributed to T Kiyono.

At least 37 records · Page 2Linked to original sources
PubMed1998

Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells.

Normal human cells undergo a limited number of divisions in culture and enter a non-dividing state called replicative senescence. Senescence is accompanied by several changes, including an increase in inhibitors of cyclin-dependent kinases and telomere shortening. The mechanisms by which viral oncogenes reverse these processes are not fully understood, although a general requirement for oncoproteins such as human papillomavirus E6 and E7 has suggested that the p53 and Rb pathways are targeted. Expression of the catalytic component of telomerase, hTERT, alone significantly extends the lifespan of human fibroblasts. Here we show that telomerase activity is not sufficient for immortalization of human keratinocyte or mammary epithelial cells: we find that neither addition of hTERT nor induction of telomerase activity by E6, both of which are active in maintaining telomere length, results in immortalization. Inactivation of the Rb/p16 pathway by E7 or downregulation of p16 expression, in combination with telomerase activity, however, is able to immortalize epithelial cells efficiently. Elimination of p53 and of the DNA-damage-induced G1 checkpoint is not necessary for immortalization, neither is elimination of p19ARF.

Breast↗
PubMed1998

cDNA cloning and expression during development of Drosophila melanogaster MCM3, MCM6 and MCM7.

cDNAs encoding three Drosophila melanogaster MCM proteins, DmMCM3, DmMCM6 and DmMCM7, candidates of DNA replication-licensing factors, were cloned and sequenced. The deduced amino-acid sequences displayed 60, 59 and 68% identities with the respective Xenopus laevis homologues, XMCM3, XMCM6 and XMCM7. Six members of the D. melanogaster MCM family were found to share 31-36% identities in their amino-acid sequences, and to possess the five common domains carrying conserved amino-acid sequences as reported with X. laevis MCM proteins. DmMCM3, DmMCM6 and DmMCM7 genes were mapped to the 4F region on the X chromosome, the 6B region on the X chromosome and the 66E region on the third chromosome, respectively, by in situ hybridization. Contents of their mRNAs were proved to be high in unfertilized eggs and early embryos (0-4h after fertilization), then decrease gradually by the 12h time point, with only low levels detected at later stages of development except in adult females. This fluctuation pattern is similar to those of genes for proteins involved in DNA replication, such as DNA polymerase alpha and proliferating cell nuclear antigen, suggesting that expression of DmMCM genes is under the regulatory mechanism which regulates expression of other genes involved in DNA replication.

Amino Acid Sequence↗
PubMed1998

Cloning and characterization of human MCM7 promoter.

MCM7 is a member of the MCM protein family which has been implicated in the regulatory machinery allowing DNA to replicate only once during S phase. In quiescent cells, human MCM7 (hMCM7) mRNA is almost undetectable. Stimulation of cells to enter the cell cycle results in induction of hMCM7 expression. Here, we report cloning and characterization of the hMCM7 promoter. We isolated and sequenced a 0.5 kb genomic fragment that contains putative transcription factor binding sites including three E2F sites, three GC boxes and an E box. Several transcription start sites, which were used upon growth stimulation, were identified. The minimal promoter region required for transcription of a luciferase reporter gene was delineated, and it contained an E box and one E2F site, which were important for promoter activity. Interestingly, the cloned sequence appears to act as a promoter for mu-adaptin-related protein 2 (mu-ARP2) gene in the opposite orientation.

Base Sequence↗
PubMed1998

TGF-beta 1 induces the cyclin-dependent kinase inhibitor p27Kip1 mRNA and protein in murine B cells.

TGF-beta1 inhibits the cell cycle progression of many types of cells by arresting them in the G1 phase. This cell cycle arrest has been attributed to the regulatory effects of TGF-beta1 on both the levels and the activities of the G1 cyclins and their kinase partners. The activities of these kinases are negatively regulated by a number of proteins, such as p15INK4b, p21WAF1/Cip1, and p27Kip1, that physically associate with cyclins, cyclin-dependent kinases (Cdk), or cyclin-Cdk complexes. In epithelial cell lines, TGF-beta1 was previously shown to inhibit cell cycle progression through down-regulation of Cdk4 and/or up-regulation of p15INK4b and/or p21WAF1/Cip1. However, TGF-beta1 had little or no effect on the p27Kip1 mRNA and protein levels. In this report, we show that, in contrast to observations in epithelial cell lines, TGF-beta1 increased the p27Kip1 mRNA and protein levels in the murine B cell lines CH31 and WEHI231. This TGF-beta1-mediated induction of p27Kip1 also resulted in an increased association of p27Kip1 with Cdk2 and a decreased Cdk2 kinase activity. In contrast to epithelial cells, however, TGF-beta1 had little or no effect on the Cdk4 and p21WAF1/Cip1 protein levels in these B cells. Finally, although several studies suggested a direct role of p53 in TGF-beta1-mediated cell cycle arrest in epithelial cells, TGF-beta1 inhibited cell cycle progression in CH31 even in the absence of wild-type p53. Taken together, these results suggest that TGF-beta1 induces G1 arrest in B cells primarily through a p53-independent up-regulation of p27Kip1 protein.

Animals↗
PubMed1997

Binding of high-risk human papillomavirus E6 oncoproteins to the human homologue of the Drosophila discs large tumor suppressor protein.

In the majority of cervical cancers, DNAs of high-risk mucosotpropic human papillomaviruses (HPVs), such as type 16, are maintained so as to express two viral proteins, E6 and E7, suggesting an essential importance to carcinogenesis. The high-risk HPV E6 proteins are known to inactivate p53 tumor suppressor protein but appear to have an additional, molecularly unknown function(s). In this study, we demonstrate that these E6 proteins can bind to the second PDZ domain of the human homologue of the Drosophila discs large tumor suppressor protein (hDLG) through their C-terminal XS/TXV/L (where X represents any amino acid, S/T serine or threonine, and V/L valine or leucine) motif. This finding is similar to the interaction between the adenomatous polyposis coli gene product and hDLG. E6 mutants losing the ability to bind to hDLG are no longer able to induce E6-dependent transformation of rodent cells. These results suggest an intriguing possibility that interaction between the E6 protein and hDLG or other PDZ domain-containing proteins could be an underlying mechanism in the development of HPV-associated cancers.

Adaptor Proteins, Signal Transducing↗
PubMed1997

cca1 is required for formation of growth-arrested confluent monolayer of rat 3Y1 cells.

A novel cDNA fragment, named cca1 (confluent 3Y1 cell-associated 1), was previously isolated on the basis of preferential accumulation of the corresponding mRNA in growth-arrested confluent but not in growing subconfluent rat 3Y1 cells. The cca1 cDNA was found to consist of 5022 nucleotides with an open reading frame of 1905 nucleotides, encoding a protein of 635 amino acids. Unlike the 3Y1 cell case, cca1 mRNA was not detected in confluent 3Y1 BU, 3Y1 BU/pTK, 3Y1-16E6, or F2408 cells, whose growth patterns monitored by phalloidin staining and bromodeoxyuridine incorporation were different from those of the confluent 3Y1 cells. A restoration of the confluent 3Y1-type growth pattern was observed in the cca1 cDNA-introduced 3Y1 BU and 3Y1 BU/pTK cells after reaching confluence but not in the cDNA-introduced 3Y1-16E6 or F2408 cells. The results allow us to conclude that cca1 is required but not sufficient for formation of growth-arrested confluent monolayer of 3Y1 cells.

Amino Acid Sequence↗
PubMed1997

Isolation of a novel cDNA whose corresponding mRNA is accumulated in growth-arrested confluent but not in growing sub-confluent rat 3Y1 cells.

Four cDNA fragments, whose corresponding mRNAs were accumulated in growth-arrested confluent but not in growing sub-confluent rat 3Y1 cells, were isolated by the mRNA differential display method. Sequencing of the four cDNA fragments indicated that one of them was highly homologous to 3' untranslated DNA region of mouse growth-arrest specific1 cDNA, while the other three (named confluent 3Y1 cell-associated No. 1 (cca1), cca2 and cca3) were novel. Of the three novel cDNAs, we presented some characteristics of cca2 cDNA: the cDNA consisted of 1795 nucleotides with a deduced open reading frame (ORF) encoding a protein of 338 amino acids, which showed a 35% identity with rat type IV 3beta-hydroxysteroid dehydrogenase. The CCA2 protein, with a molecular weight of 38 kDa, was accumulated in 3Y1 cells under growth-arrest conditions.

Amino Acid Sequence↗
PubMed1997

In vivo interaction of human MCM heterohexameric complexes with chromatin. Possible involvement of ATP.

The MCM protein family, which consists of at least six members, has been implicated in the regulatory machinery causing DNA to replicate once in the S phase. Mammalian MCM proteins are present in the nucleus in two different forms, one extractable by nonionic detergents and the other resistant to such extraction. The latter is assumed to be tightly associated with nuclear structures and released at the time of initiation of replication. However, details of the mode of binding remain unclear. In the present study, we found that, in nonionic detergent-permeabilized nuclei, the association of human MCM (hMCM) proteins with them could be stabilized by the addition of ATP. The hMCMs bound to the nuclei in the presence of ATP were released by digestion with nucleases, suggesting that they are chromatin-associated. The nuclease-directed solubilization of the chromatin-bound hMCMs thus provided a means to analyze them as well as soluble hMCMs by co-immunoprecipitation. The results indicate that the six hMCM members exist as heterocomplexes, whether bound or unbound. We therefore propose that hMCM proteins may function in DNA replication as heterohexamers associated with chromatin and that ATP is possibly involved in the association. Nuclease digestion-immunoprecipitation techniques of the type described here should facilitate further elucidation of the mode of interaction between hMCMs and chromatin.

Adenosine Triphosphate↗
PubMed1996

Cloning of a cDNA encoding a human homologue of CDC47, a member of the MCM family.

A complementary DNA (cDNA) clone encoding a 719-amino acid (aa) protein was isolated, which has a 49% aa identity with budding yeast CDC47, a member of the MCM family. Antiserum raised against a C-terminal polypeptide bacterially produced from the cDNA clone detected a cellular protein about 80 kDa, which coincides with the size of the in vitro transcription/translation product of the cDNA. These results indicate that the cDNA covers the entire coding region of a human homologue of CDC47.

Amino Acid Sequence↗
PubMed1996

Serial transplantation in SCID mice of an epidermodysplasia verruciformis-associated squamous cell carcinoma without alteration of its histological and virological features.

An epidermodysplasia verruciformis-associated squamous cell carcinoma was xenografted to a SCID mouse, and the resultant tumor was transplanted through mice for 10 generations. Histological and virological features, with replication of about a thousand copies of the papillomavirus DNA, were the same in both the original carcinoma and the transplanted tumors.

Animals↗
PubMed1996

hCDC47, a human member of the MCM family. Dissociation of the nucleus-bound form during S phase.

hCDC47 is a putative human homologue of yeast CDC47 and a member of the MCM protein family, which has been implicated in the regulatory machinery causing DNA to replicate only once in the S phase. In the present study, we performed an initial characterization of hCDC47. We found that hCDC47 protein was present in the nucleus of cultured human cells in two different forms; one extractable by a non-ionic detergent and the other resistant to such extraction and tightly associated with the nucleus. The levels of the nucleus-bound form gradually diminished during S phase progression, although the total amount of nuclear hCDC47 protein remained relatively constant, suggesting that the nucleus-bound form becomes dissociated from the nuclear structure during DNA replication. This behavior of hCDC47 protein is very similar to that of other mammalian MCM proteins reported recently. We also found that expression of hCDC47 mRNA was repressed in quiescent cells but was induced at the late G1 to S phase by growth factor stimulation. Together, these findings indicate that hCDC47 protein together with other MCM proteins participates in the regulation of mammalian DNA replication.

Cell Cycle↗
PubMed1996

Inhibition of S-phase entry of human fibroblasts by an antisense oligomer against hCDC47.

The MCM protein family has been implicated in the regulatory machinery causing DNA to replicate only once in the S phase. Recently, we have identified human (h) CDC47, another member of the family, and demonstrated its unique S-phase nuclear association pattern implying participation in the regulation of DNA replication. In the present study, we applied an antisense strategy to assess the phenotype of human cells in which hCDC47 is repressed. As a result serum-induced accumulation of hCDC47 protein in human fibroblast cells was reduced by a specific antisense oligodeoxynucleotide and S-phase entry of such cells was significantly inhibited, indicating that hCDC47 protein is essential for DNA replication.

Base Sequence↗
PubMed1996

E7 proteins of four groups of human papillomaviruses, irrespective of their tissue tropism or cancer association, possess the ability to transactivate transcriptional promoters E2F site dependently.

In an experimental system in which an expression vector including the E7 gene of a given human papillomavirus (HPV), together with a luciferase reporter plasmid including the adenovirus E2 (Ad E2) promoter, was transiently transfected into cultured mouse NIH3T3 fibroblastic cells, we obtained the signal indicating that E7 proteins of HPV type 5, 12, 14, 20, 21, 25, and 47, which are associated with epidermodysplasia verruciformis (EV), can transactivate the Ad E2 promoter, as previously reported for E7 proteins of other HPVs. Because the underlying mechanism of the transactivation had not been analyzed, except for transactivation by E7 gene of cervical cancer-associated HPV-16, we compared the E7 genes of representatives of three other groups of HPVs (HPV-1, -11, and -47) with that of HPV-16 with regard to their transactivating activity toward artificially constructed promoters. The experiment with a shortened AdE2 promoter carrying only the E2F sites and TATA box provided evidence that all four E7 proteins can transactivate the shortened promoter and that this phenomenon is E2F site dependent. Further experiments with the reporter gene constructs carrying basal promoters or more complex forms with or without linked E2F sites, (a) confirmed previous finding by others that in cells producing no transactivator, the transcriptional level from promoters linked to E2F sites is rather repressed in comparison with the level of the corresponding promoters that are not linked to the E2F sites, and (b) demonstrated, for the first time, that in cells expected to produce the E7 protein of any one of the four HPVs, transcription from the promoter linked to the E2F sites was released from repression. In other words, the present results reveal that E7 proteins of any of the four HPVs can remove the E2F site-dependent repression, probably by modulating E2F complexes from repressing forms to activating ones.

Base Sequence↗
PubMed1996

Detection of human papillomavirus (HPV) type 47 DNA in malignant lesions from epidermodysplasia verruciformis by protocols for precise typing of related HPV DNAs.

Our discovery of human papillomavirus type 47 (HPV47) in benign lesions from a patient suffering from epidermodysplasia verruciformis prompted us to examine whether the viral DNA also resided in malignant lesions from the same patient. By using newly devised protocols for amplifying a group of epidermodysplasia verruciformis-associated HPV DNAs by PCR and differentially identifying them by reverse-phase dot blot hybridization, we demonstrated that HPV47 DNA, but not other HPV DNAs of the group, was abundant (about 10(3) copies per diploid amount of cell DNA) in DNAs prepared from three carcinomas. Using DNA from one of these carcinomas, we also confirmed that DNA of HPV5, HPV14, or HPV21, detected in significant amounts in DNAs from benign lesions from the patient, were present only in negligible amounts or not at all. The results suggest the involvement of HPV47 DNA in tumorigenesis. Furthermore, we demonstrated by the Southern technique that most, if not all, of the HPV47 DNA consists of either a unit (or a nongrossly deleted unit) length of the viral genome carrying no (or no gross) internal rearrangements or tandem repeats. This and other results obtained by this technique indicated that a considerable amount of the viral DNA resides as a circular monomer a unit length of the viral genome in carcinoma cells, while the remainder reside as catenanes, concatemers, or both. The concatemers were considered more likely to be replicated without integration into cellular DNA than to be integrated, because no bands for the corresponding fragments including integration sites were detected by treatment with restriction enzymes that would have produced such fragments.

Base Sequence↗
PubMed1995

The Impacts of Breast Conserving Treatment and Mastectomy on the Quality of Life in Early-stage Breast Cancer Patients.

The quality of life (QOL) in 55 early-stage breast cancer patients after surgery was prospectively assessed using a newly developed Japanese QOL questionnaire: The QOL Questionnaire for Cancer Patients Treated with Anticancer Drugs (QOL-ACD). The impacts of breast conserving treatment (BCT) (22 cases) and modified radical mastectomy (MRM) (33 cases) on the QOL in those subjects were compared. The overallQOL scores were evaluated during four periods (before surgery, 0-2, 3-12, and 13-24 months after surgery). The mean scores of the four categories of the QOL-ACD (activity, physical condition, psychological condition, and social relationships) were also compared. The results demonstrated that a significant improvement was observed in the overall QOL scores among the three periods after surgery (0-2, 3-12, and 13-24 months) only in the BCT group (p<0.05). There were no significant differences between the two groups in the overall QOL scores during any of the three periods after surgery, and the mean score of the 'psychological condition' during 0-2 months period in the BCT group was significantly lower than that in the MRM group (p<0.05). These results suggest that BCT does not always improve the patients' QOL more than MRM does, and that the patients receiving BCT require more psychological support than those receiving MRM during the early postoperative period.

Journal Article↗