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K Fujinaga

Publications and source records attributed to K Fujinaga.

At least 19 recordsLinked to original sources
PubMed1997

Cloning and characterization of the rat p130, a member of the retinoblastoma gene family.

A cDNA clone encoding rat p130, a member of the retinoblastoma (Rb) gene family, was isolated based on the sequence homology of the E1A-binding domain. The 4.87 kb cDNA contained an 1135-amino acid open reading frame with high homologies to the human and mouse p130 and a partial homology to the pRb protein. p130 showed difference in distribution of potential phosphorylation sites from pRb in the N-terminal and the B pocket regions. p130 mRNA was detected in most rat tissues. The p130 gene was mapped to rat chromosome 19p11-13 by fluorescence in situ hybridization.

Amino Acid Sequence↗
PubMed1997

Activation of the p21(Waf1/Cip1) promoter by the ets oncogene family transcription factor E1AF.

p21(Waf1/Cip1) is one of the key regulatory proteins in cell cycle, terminal differentiation, and apoptosis. Its promoter was shown to be transactivated by the wild-type p53 protein as well as in a p53-independent manner. In this report, we demonstrate that E1AF, an ets-related transcription factor, activates the human p21(Waf1/Cip1) promoter by interacting with the ets-binding sites located close to the two previously identified p53-responsive elements. Northern blot analysis revealed that p21(Waf1/Cip1) and E1AF were correlatively upregulated in response to cisplatin treatment in SiHa cells. Transient expression assays demonstrated that E1AF can activate the p21(Waf1/Cip1) promoter-driven luciferase reporter gene in SiHa cells. The p21(Waf1/Cip1) promoter activity was also increased in p53-null Saos2 osteosarcoma cells, but was markedly reduced when the ets-binding sites were deleted. These results indicate that E1AF positively regulates transcription from the p21(Waf1/Cip1) promoter in response to genotoxic stresses.

Adenovirus E1A Proteins↗
PubMed1997

Cloning and characterization of rat p27Kip1, a cyclin-dependent kinase inhibitor.

Cyclin-dependent kinase (Cdk) inhibitors play significant roles in the cell cycle control of various biological phenomena. To characterize the role of Cdk inhibitors in rat cells, we isolated a cDNA encoding rat p27Kip1, a 27-kDa Cdk inhibitor. The 1.04-kb cDNA of rat p27 contained an open reading frame of 197 amino acids that shared high homology with mammalian p27 and significant homology with mammalian p21Cip1 and p57Kip2. p27 mRNA was detected in most rat tissues and cell lines. The levels of p27 protein expression were similar in rat cell lines transformed by E1A and in normal cells. Rat p27 was able to interact with Cdk 2/4 and cyclin A/D in rat cells, but the amounts of rat p27 in Cdk2 complexes were different between transformed cells and normal cells. Thus, the formation of stable complexes of rat p27 may be modulated by E1A. Rat p27 protein could inhibit the increased Cdk2-associated kinase activity in transformed rat cells.

Adenovirus E1A Proteins↗
PubMed1997

A clearer distinction between HIV-1 paired isolates from peripheral blood mononuclear cells of asymptomatic carriers with and without CD8+ T-cells at nef rather than env V3 loci.

In asymptomatic carriers, the vast majority of human immunodeficiency virus type 1 (HIV-1) infection is non-productive whilst the clinical stage of disease is associated with significant virus expression. Virus-specific CD8+ T-cell functions are believed to play a major role in the generation of heterogeneous virus populations and in subsequent disease progression. Here, we prepared two types of HIV-1 isolate by culturing whole and CD8+ T cell-depleted peripheral blood mononuclear cells (PBMCs) from five asymptomatic carriers. The former is expected to be escape variant populations, whereas the latter would be mixed populations including the former viruses. The analyses of Nef and Env V3 sequence variations of viruses in a total of 77 and 44 DNA clones, respectively, allowed a direct comparison to be made of the differences between the paired isolates. Comparison of Nef sequences between the paired isolates showed them to be more distinct in two carriers with a relatively stable CD4/CD8 ratio (Nos 68 and 69), than in two other carriers with similar CD4/CD8 ratios (Nos 53 and 57), or in carrier No. 67, which had an extremely lower CD4/CD8 ratio. By contrast, a distinction between the paired isolates by use of the Env V3 sequences was only apparent in the latter three carriers. These results indicate that the predominant populations of HIV-1 in Nos 68 and 69 were sensitive to selective pressure from Nef-specific CD8+ T-cells, while those in Nos 53, 57, and 67 were sensitive to pressure from V3-specific CD8+ T-cells. It is noteworthy that Nos 53 and 57 progressed to an AIDS-related complex shortly and several years after this examination. These data suggest that HIV-1-induced pathogenesis is more strongly associated with the generation of variant nef alleles than with env V3 variants, and that these arise by CD8+ T-cell pressure.

Amino Acid Sequence↗
PubMed1997

Two transcription factors, E1AF and N-myc, correlate with the invasiveness of neuroblastoma cell lines.

The ets transcription factor E1AF can activate several matrix-degrading metalloproteinase (MMP) genes and is implicated in enhancement of tumor cell invasion. Here we compared the invasive activity of five human neuroblastoma cell lines (TGW, GOTO, SK-N-BE, SK-N-SH and SK-N-AS), which exhibit distinct levels of N-myc amplification, together with the expression of E1AF. Extracellular matrix-degrading proteases and their inhibitor proteins, which play an important role in local invasion, were also analyzed. The activity to invade through reconstituted basement membrane was high in cells (TGW, GOTO, and SK-N-BE) with N-myc amplification, and these cells produced relatively large amounts of E1AF mRNA, correlating with the invasive activities. Of several matrix metalloproteinases (MMPs) and a tissue inhibitor of MMPs (TIMP), only membrane-bound type 1 MMP (MT1-MMP) was specifically detected in N-myc-amplified cells, suggesting a role of MT1-MMP in neuroblastoma cell invasion. MMP-2 (72 kD type IV collagenase), TIMP-1 and TIMP-2 were expressed in all five cell lines. Urokinase-type plasminogen activator was undetectable. These findings indicate that the transcription factors E1AF and N-myc are related to malignant phenotypes of neuroblastoma.

Adenovirus E1A Proteins↗
PubMed1997

Decoy approach using RNA-DNA chimera oligonucleotides to inhibit the regulatory function of human immunodeficiency virus type 1 Rev protein.

Human immunodeficiency virus type 1 (HIV-1) encodes two regulatory proteins, Tat and Rev, that bind to target RNA sequences. These are the trans-activation responsive (TAR) RNA and the Rev-responsive element (RRE), respectively. The Rev protein shifts RNA synthesis to viral transcripts by binding to the RRE within the env gene. In the present study we prepared a RNA-DNA chimera consisting of 29 or 31 nucleotides to inhibit the Rev regulatory function by means of the decoy approach. The chimera oligonucleotides (anti-Rev oligonucleotides [AROs]) contained an RNA "bubble" structure (13 oligonucleotides; the Rev-binding element in RRE) that bound Rev with a high affinity in an in vitro assay. The controls were RNA-DNA chimera oligonucleotides (negative control oligonucleotides [NCOs]) similar to ARO, but without the bubble structure, that bound with considerably less affinity to Rev. When the inhibitory effects of these decoys on HIV-1 replication were examined, we found that AROs, but no NCOs, reduced more than 90% of the HIV-1 production generated by productively infected human T-cell lines. The production of primary HIV-1 isolates in healthy donor-derived peripheral blood mononuclear cells was also similarly inhibited by AROs. In addition, the induction of viral mRNAs and antigens in latently HIV-1-infected ACH-2 cells by tumor necrosis factor alpha was specifically inhibited by AROs, but not by NCOs. No apparent cytotoxicity was caused by either decoy. Thus, the use of a Rev-binding element-based decoy, the RNA-DNA chimera oligonucleotide, may represent a safer approach to gene therapy for reducing the virus load in HIV-1-infected individuals.

Cell Line↗
PubMed1997

Protease-defective, gp120-containing human immunodeficiency virus type 1 particles induce apoptosis more efficiently than does wild-type virus or recombinant gp120 protein in healthy donor-derived peripheral blood T cells.

Apoptosis and syncytium formation are two mechanisms by which human immunodeficiency virus type 1 (HIV-1) impairs uninfected CD4+ T-cell function and are mainly involved in the progression of the disease to AIDS. Previously, we showed that gp120-containing, protease-deficient HIV-1 (L-2) particles generated syncytia by particle-mediated fusion with uninfected cultured CD4+ T cells. Here, we present evidence that such L-2 particles can induce apoptosis in 40 to 50% of T cells which were enriched from HIV-1-negative healthy donor-derived peripheral blood mononuclear cells (PBMC-Ts). Activation of PBMC-Ts with phytohemagglutinin, concanavalin A, or ionomycin after incubation with L-2 particles resulted in the loss of proliferative capacity and gradual induction of apoptosis over 3 days. Wild-type strain LAI particles or recombinant gp120 were markedly less efficient (< or = 15%) at inducing such apoptosis. Western blot (immunoblot) analysis revealed that L-2 particles contained a larger amount of Env gp120 than LAI particles. Either preincubation of PBMC-Ts with a Fas antagonist or preincubation of L-2 particles with soluble CD4 blocked most of the apoptosis. This suggests that L-2-like particles can play a major role in HIV-1-induced apoptosis of uninfected bystander cells.

Apoptosis↗
PubMed1997

Identification of human papilloma virus DNA sequence in the hyperplastic epithelium of an oral denture fibroma.

The human papilloma virus (HPV) associated with hyperplastic epithelium in an oral denture fibroma was examined by southern blot hybridization. Extracted DNA was hybridized with full length linear HPV type 2a, 6b, 11, 16, 18, 31 and 33 DNAs as a mixed probe only under low stringent conditions. The hybridized bands digested with Bam HI and Eco RI were approximately 8.8 kbp and 15 kbp, respectively. Thus the lesional HPV DNA was different from HPV types used as probes and was probably integrated into host cell chromosomal DNA judging by the off-size high molecular weight bands. Considering the contaminating mesenchymal region and uninfected epithelial cells as well as the evidently limited homology with probe HPV DNAs, the virus copy number in infected cells was poorly defined. In situ antigen staining signals were widely detected in the hyperplastic epithelial layer.

Blotting, Southern↗
PubMed1997

Antisense E1AF transfection restrains oral cancer invasion by reducing matrix metalloproteinase activities.

E1AF is a newly identified human ets-family transcription factor. We have reported that E1AF can up-regulate transcription of matrix metalloproteinase (MMP) genes and confers invasive phenotype on human cancer cells. HSC3 is an oral squamous-cell-carcinoma-derived cell line, and it manifests high levels of E1AF and MMP-1 and -9 gene expression that are associated with invasive potential. We reconstructed an E1AF antisense expression vector, transfected HSC3 cells with the vector, and obtained HSC3AS cells that express E1AF antisense RNA. HSC3AS showed decreasing mRNA and protein levels of MMP-1, -3, and -9. Moreover, HSC3AS showed lower invasive potential in vitro three-dimensional raft culture and in vivo implantation into nude mice. These results imply that transfection of antisense E1AF inhibits tumor invasion by down-regulating MMP genes.

Adenovirus E1A Proteins↗
PubMed1997

Inhibition of HIV-1 replication by targeting the Rev protein.

Human immunodeficiency virus type 1 (HIV-1) encodes two regulatory proteins, Tat and Rev. The Rev protein facilitates the transport of unspliced and singly-spliced RNA to the cytoplasm in infected host cells by binding to target RNA (Rev response element: RRE). A variety of approaches targeting Rev function, including gene therapy, have been developed that inhibit HIV-1 replication in cells cultured in vitro. This minireview summarizes the recent developments as well as our application of the Rev-binding element-based decoy approach using RNA-DNA chimera oligonucleotide modeling.

Anti-HIV Agents↗
PubMed1996

Growth dominance of a revertant virus generated during in vitro serial passage of nef frameshift mutant of HIV-1.

We prepared a series of nef mutant HIV-1 with a frameshift mutation at the Xho I site by up to 50 serial transfers into MT-4 cells. Here, we studied revertants. Immunofluorescence using an anti-Nef monoclonal antibody revealed that cells first became Nef antigen-positive at the 23rd passage. The percentage of Nef antigen-positive cells gradually increased and reached almost 100% by the 27th passage. The sequence of the provirus in the cells supported the generation of a revertant. This revertant mutated at the site immediately after the initially introduced frameshift mutation. This resulted in the substitution of only three amino acids and the insertion of two, which restored the proline-rich domain, a conserved region believed essential to viral replication, at the middle of Nef. Thus, the growth dominance of the revertant virus, compared with the original nef mutant, was directly demonstrated in vitro using serial passages consisting of mixed HIV-1 populations in a single cell.

Amino Acid Sequence↗
PubMed1996

High-risk HPV-positive human cancer cell lines show different sensitivity to cisplatin-induced apoptosis correlated with the p21Waf1/Cip1 level.

We investigated the sensitivity and cell-cycle inhibitory gene expression of human papillomavirus (HPV) 16- and 18-positive human cancer cell lines after DNA damage induced by treatment with the anti-cancer drug cisplatin. Four HPV-positive cell lines (Caski, SiHa, HeLa and KB) were treated with cisplatin at various concentrations. Apoptotic cell death was observed in a dose-dependent manner in all cell lines treated with cisplatin; however, colony assay for chemosensitivity revealed that HeLa and KB cells (HPV 18-positive cell lines) were more sensitive than SiHa and Caski cells (HPV 16-positive cell lines). Northern blot analyses showed that p53 and p21Waf1/Cip1 mRNA were detectable in all untreated cells, and increasing amounts of these transcripts were identified in all cell lines treated with cisplatin. However, signals were more prominent in HeLa and KB, HPV 18-positive-cells. Immunohistochemical detection of p21Waf1/Cip1 protein showed that the p21-positive cells with apoptotic features were more distinct in KB and HeLa cells (HPV 18-positive) than in SiHa and Caski cells (HPV 16-positive). Our results show that there were differences in sensitivity to cisplatin among four types of high risk HPV-positive cells, possibly due to different levels of p21Waf1/Cip1 up-regulation by functional p53.

Apoptosis↗
PubMed1996

The 273rd codon mutants of p53 show growth modulation activities not correlated with p53-specific transactivation activity.

Cancer-related mutations of the p53 tumor suppressor gene are clustered in the four so-called 'hot spots', codons 175, 248, 273 and 281/282. By using recombination PCR in vitro mutagenesis, we introduced point mutations into the codon 273 of wild-type (wt) p53 (pC53-SN3) from Arg to His (pC53-273H [273H]), Asp (273D), Pro (273P), Lys (273K), Leu (273L) or Thr (273T), and compared their biological and biochemical activities with wt p53 and cancer-derived 175H, 248W and 273H/309S. Among them, 273H/309S, 273H and 273D as well as wt p53 transactivated the chloramphenicol acetyltransferase (CAT) gene placed downstream of the p53 binding consensus, while none of the other mutants including 273L did. Transcriptions from human c-fos and rat PCNA promoters were suppressed by wt p53 and 273D, while they were enhanced variously by all other mutants in Saos-2 and/or NIH3T3 cells. On the other hand, growth of human squamous carcinoma cell lines measured by the plating efficiency of G418-resistant colonies was enhanced by transfection of 175H, 248W, 273H/309S and 273P, while suppressed by not only wt p53, 273D and 273H but also 273L. Thus, 273H/309S enhanced cell growth in spite of its p53-specific transactivation activity, while 273L suppressed cell growth in spite of its complete loss of the p53-specific transactivation. We concluded that the sequence-specific transactivation of p53 is not always correlated with its growth inhibitory activity.

3T3 Cells↗
PubMed1996

Mutations in the p53 gene and human papillomavirus infection as significant prognostic factors in squamous cell carcinomas of the oral cavity.

The p53 gene has been indicated to be a tumour suppressor gene that is found in mutated form in common human cancers. Human papillomavirus (HPV) has oncogenic activity in cervical and oral squamous cell carcinomas (SCCs). The E6 protein of HPV is known to bind with p53 protein and inactive the tumor suppressor activity by promoting p53 degradation. Because of this background, we examined 38 primary, resected specimens of oral SCCs for detection of p53 mutations and HPV DNAs. Exons 5 through 8 of the p53 Mutations were observed in nine cases (24%). HPV-DNA detection and typing were performed using PCR with ¿high risk group' HPV-specified primers. HPV DNA sequences were detected in eight cases (21%). The AvaII digestion pattern of PCR-amplified HPV DNA showed that HPV-16 was present in all eight cases. Seven cases were p53 mutation-positive/HPV-negative, six cases were p53 mutation-negative/HPV-positive, and two intraosseus SCC cases were p53 mutation-positive/ HPV-positive. Thus, 15/38 (40%) cases had inactivation of the p53 protein. Interestingly, p53 mutation-negative/ HPV-negative cases had a poorer prognosis than p53 mutation positive or HPV-positive cases (P < 0.01). We conclude that (1) mutation in the p53 gene and/or HPV infection are frequent (40%) in oral SCC; (2) inactivation of p53 function by mutation and HPV infection are important genetic events in the development of 40% integral of oral SCCs; (3) p53 mutation and HPV infection are not mutually exclusive events and (4) other oncogenes or tumor suppressor genes may be crucial in the development of oral SCC if the prognosis is poor.

Aged↗
PubMed1996

A single ets-related transcription factor, E1AF, confers invasive phenotype on human cancer cells.

Invasion of cancer cells is the first step of metastasis. The invasive activity is thought to be dependent on the production of matrix metalloproteinases (MMPs). The transcription regulatory regions of MMP genes often contain binding sites for Ets and AP-1 transcription factors and they mediate oncogene- and growth factor-induced transcription of the genes. We recently isolated the cDNA encoding human E1AF, a new member of ets oncogene family. E1AF highly stimulated transcription from three different subclasses of MMP genes in transient expression assays. Here we show that transfection of the non-invasive human breast cancer cell line MCF-7 with the E1AF expression plasmid results in induction of invasive and motile activities, accompanied by an increase of 92 kD type IV collagenase (MMP-9) gene expression. Tumors derived from the E1AF transfectant were highly invasive and produced MMP-9. Expression of E1AF and MMP-9 genes was elevated in several invasive tumor cell lines. These results provide evidence for an important role of ets-related E1AF in tumor cell invasion.

Collagenases↗
PubMed1996

Fusion of an ETS-family gene, EIAF, to EWS by t(17;22)(q12;q12) chromosome translocation in an undifferentiated sarcoma of infancy.

EIAF is a newly isolated ETS-family gene that is located on 17q21 and codes for the adenovirus EIA enhancer-binding protein. In our chromosome analysis of 18 of the Ewing family of tumors and undifferentiated sarcomas, we found t(17;22)(q12;q12) in an MIC2 antigen-positive undifferentiated sarcoma of infancy. On Southern blot analysis, EWS and EIAF cDNA probes hybridized to the same rearranged band, indicating that an EWS-EIAF fusion gene was formed in the tumor. Further Southern blot analysis using four EIAF cDNA probes of different sizes showed that the breakpoint lies in the region upstream to the ETS domain of the EIAF gene. EIAF may be the fourth ETS-family gene to be identified forming a fusion gene with EWS. We assume that the RNA binding domain of EWS may have been replaced by the DNA binding domain of EIAF in the EWS-EIAF fusion protein as in other fusion proteins previously characterized in Ewing sarcoma and other types of sarcomas.

Adenovirus E1A Proteins↗
PubMed1996

High susceptibility of U937-derived subclones to human immunodeficiency virus type 1 infection correlates with accumulation of unintegrated circular viral DNA.

Our previous report showed that U937-derived subclones were differentiated into at least three types (high, middle, and low types), even in the subclones expressing similar levels of surface CD4, in terms of the kinetics of the appearance of viral antigens and virus production after infection with human immunodeficiency virus type 1 (HIV-1). Here we showed the evidence that high susceptibility to HIV-1 infection, which was confirmed by the profound expression of viral messages and antigens, was exclusively associated with a high number of the unintegrated extrachromosomal form of viral DNA, but not with the amounts of adsorbed virus RNA nor those of integrated DNA form. The difference in the amounts of extrachromosomal form of viral DNA was also observed in the culture with 3'-azido-3'-deoxythymidine (AZT), indicating that the susceptibility is essentially unrelated to multiple infection events. Thus, the susceptibility of U937-derived subclones to HIV-1 infection seems to be affected by the occurrence of specific events involved in the accumulation of unintegrated viral DNA after viral adsorption.

DNA, Circular↗