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Biomedical subjects

T Kiyono

Publications and source records attributed to T Kiyono.

At least 19 recordsLinked to original sources
PubMed2006

HPV16 E6-mediated stabilization of ErbB2 in neoplastic transformation of human cervical keratinocytes.

Whether ErbB2 receptor tyrosine kinase contributes to cervical cancer is controversial. We have examined the effects of E6 and E7 genes of human papillomaviruses type 16 (HPV-16) on ErbB2 expression in primary human cervical keratinocytes (HCK) immortalized with hTERT (HCK1T). In E6-positive cells (HCK1T-E6 and HCK1T-E6E7), ErbB2 expression levels increased with the cell density. HCK1T-E6E7 showed impaired contact inhibition and anchorage-independent growth in soft agar which were abrogated with introduction of ErbB2-specific short hairpin RNA (shRNA) or an ErbB2 specific inhibitor AG825. Furthermore, increased ErbB2 expression was also observed in HPV16 positive cervical cancer cell lines and this was diminished by introduction of HPV16E6- or E6AP-shRNA. At post-confluence cell densities, ErbB2 protein was stabilized in the presence of E6 whereas increased ErbB2 expression was not obvious with E6 mutants incapable of degrading p53. Furthermore, introduction of p53-shRNA to HCK1T resulted in increased ErbB2 protein stability, indicating possible ErbB2 regulation through p53. Finally, we showed that tumor formation of ErbB2-shRNA introduced SiHa cells were almost abolished. Taken together, these data indicate an important role of ErbB2 regulation by HPV16 E6 in oncogenic transformation of human cervical keratinocytes.

Cell Line, Transformed↗
PubMed2006

Establishment of immortalized dental follicle cells for generating periodontal ligament in vivo.

The dental follicle is a mesenchymal tissue that surrounds the developing tooth germ. During tooth root formation, periodontal components, viz., cementum, periodontal ligament (PDL), and alveolar bone, are created by dental follicle progenitors. Here, we report the presence of PDL progenitors in mouse dental follicle (MDF) cells. MDF cells were obtained from mouse incisor tooth germs and immortalized by the expression of a mutant human papilloma virus type 16 E6 gene lacking the PDZ-domain-binding motif. MDF cells expressing the mutant E6 gene (MDF( E6-EGFP ) cells) had an extended life span, beyond 150 population doublings (PD). In contrast, normal MDF cells failed to proliferate beyond 10 PD. MDF( E6-EGFP ) cells expressed tendon/ligament phenotype-related genes such as Scleraxis (Scx), growth and differentiation factor-5, EphA4, Six-1, and type I collagen. In addition, the expression of periostin was observed. To elucidate the differentiation capacity of MDF( E6-EGFP ) cells in vivo, the cells were transplanted into severe combined immunodeficiency mice. At 4 weeks, MDF( E6-EGFP ) cell transplants had the capacity to generate a PDL-like tissue that expressed periostin, Scx, and type XII collagen and the fibrillar assembly of type I collagen. Our findings suggest that MDF( E6-EGFP ) cells can act as PDL progenitors, and that these cells may be a useful research tool for studying PDL formation and for developing regeneration therapies.

Alkaline Phosphatase↗
PubMed2006

Creation of tumorigenic human endometrial epithelial cells with intact chromosomes by introducing defined genetic elements.

Several genetic mutations have been identified in human endometrial cancers, but the specific combinations of mutations required to form endometrial cancer cells remain unknown. In the present study, we established an in vitro model of endometrial carcinogenesis, in which defined genetic elements were introduced into endometrial epithelial cells to create transformed endometrial cells at different stages. Introduction of the human papillomavirus type 16 E6/E7 gene and the human telomerase reverse transcriptase (hTERT) gene into human primary endometrial epithelial cells was sufficient to generate immortalized cells. Introduction of hTERT in early passages stabilized telomeres and created immortalized cells with normal karyotype, whereas introduction of hTERT in later passages generated immortalized cells but with widespread chromosome abnormalities. However, neither of those two immortalized cell lines exhibited tumorigenic phenotypes. Tumorigenic endometrial epithelial cells with invasive capacity were created by introducing a mutant K-ras allele into immortalized cells, keeping their chromosomes intact. Inhibiting the PTEN gene and activating Akt pathways did not create tumorigenic phenotypes, although the latter conferred anchorage-independent growth capacity. These findings suggest that neoplastic transformation of human endometrial cells can occur in the absence of widespread chromosomal abnormality, and that the combination of Rb inactivation, telomerase activation and altered K-ras signaling is sufficient for in vitro neoplastic transformation. The present experimental model can help clarify the genetic requirements for endometrial carcinogenesis, and it is useful for testing and developing specific inhibitors of specific oncogenic pathways.

Animals↗
PubMed2005

Establishment of an immortalised human ovarian surface epithelial cell line without chromosomal instability.

Epithelial ovarian carcinoma is thought to derive from ovarian surface epithelium (OSE). The black box of the early molecular changes in ovarian carcinogenesis is being interpreted by the development of experimental systems employing immortalised human OSE cells. However, the existing cell lines of the OSE cells have limited utility due to chromosomal instability. Our goal was to establish new immortalised human OSE cells that retain the original characteristics of the primary cells without chromosomal alterations. Using primary human OSE cells obtained from a postmenopausal patient with endometrial cancer, five cell lines ('HOSE1' lines) were newly established by infection with retroviral expression vectors containing type 16 human papillomavirus (HPV-16) E6, E7, a variant E6 (E6delta151), and Bmi1 polycomb gene, in combination with telomerase reverse transcriptase (hTERT). Consequently, five HOSE1s cell lines, HOSE1s-E6/hTERT, -E7/hTERT, -E6/E7/hTERT, -E6delta151/E7/hTERT, and -E6delta151/Bmi1/hTERT, grew beyond the population doubling number of 200. These cell lines, except for HOSE1-E6/hTERT, essentially showed the original features of the primary human OSE cells. Of them, HOSE1-E7/hTERT preserved diploidy in a kariotype analysis, and did not show transformed phenotypes in anchorage-independent growth and tumour formation. Thus, HOSE1-E7/hTERT may provide a novel model system with which to investigate the mechanisms of early molecular changes.

Animals↗
PubMed2004

p53-Independent ceramide formation in human glioma cells during gamma-radiation-induced apoptosis.

Although the p53 tumor-suppressor gene product plays a critical role in apoptotic cell death induced by DNA-damaging chemotherapeutic agents, human glioma cells with functional p53 were more resistant to gamma-radiation than those with mutant p53. U-87 MG cells with wild-type p53 were resistant to gamma-radiation. U87-W E6 cells that lost functional p53, by the expression of type 16 human papillomavirus E6 oncoprotein, became susceptible to radiation-induced apoptosis. The formation of ceramide by acid sphingomyelinase (A-SMase), but not by neutral sphingomyelinase, was associated with p53-independent apoptosis. SR33557 (2-isopropyl-1-(4-[3-N-methyl-N-(3,4-dimethoxybphenethyl)amino]propyloxy)benzene-sulfonyl) indolizine, an inhibitor of A-SMase, suppressed radiation-induced apoptotic cell death. In contrast, radiation-induced A-SMase activation was blocked in glioma cells with endogenous functional p53. The expression of acid ceramidase was induced by gamma-radiation, and was more evident in cells with functional p53. N-oleoylethanolamine, which is known to inhibit ceramidase activity, unexpectedly downregulated acid ceramidase and accelerated radiation-induced apoptosis in U87-W E6 cells. Moreover, cells with functional p53 could be sensitized to gamma-radiation by N-oleoylethanolamine, which suppressed radiation-induced acid ceramidase expression and then enhanced ceramide formation. Sensitization to gamma-radiation was also observed in U87-MG cells depleted of functional p53 by retroviral expression of small interfering RNA. These results indicate that ceramide may function as a mediator of p53-independent apoptosis in human glioma cells in response to gamma-radiation, and suggest that p53-dependent expression of acid ceramidase and blockage of A-SMase activation play pivotal roles in protection from gamma-radiation of cells with endogenous functional p53.

Apoptosis↗
PubMed2004

Molecular mechanisms of TNF-alpha-induced ceramide formation in human glioma cells: P53-mediated oxidant stress-dependent and -independent pathways.

The present study was designed to examine the roles of p53, reactive oxygen species (ROS), and ceramide, and to determine their mutual relationships during tumor necrosis factor (TNF)-alpha-induced apoptosis of human glioma cells. In cells possessing wild-type p53, TNF-alpha stimulated ceramide formation via the activation of both neutral and acid sphingomyelinases (SMases), accompanied by superoxide anion (O2-*) production, and induced mitochondrial depolarization and cytochrome c release, whereas p53-deficient cells were partially resistant to TNF-alpha and lacked O2-* generation and neutral SMase activation. Restoration of functional p53 sensitized glioma cells expressing mutant p53 to TNF-alpha by accumulation of O2-*. z-IETD-fmk (benzyloxycarbonyl-Ile-Glu-Thr-Asp fluoromethyl ketone), but not z-DEVD-fmk (benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethyl ketone), blocked TNF-alpha-induced ceramide formation through both SMases as well as O2-* generation. Caspase-8 was processed by TNF-alpha regardless of p53 status of cells or the presence of antioxidants. Two separate signaling cascades, p53-mediated ROS-dependent and -independent pathways, both of which are initiated by caspase-8 activation, thus contribute to ceramide formation in TNF-alpha-induced apoptosis of human glioma cells.

Apoptosis↗
PubMed2002

Cementum matrix formation in vivo by cultured dental follicle cells.

Dental follicle is the fibrous tissue that surrounds the developing tooth germ, and it is believed to contain progenitors for cementoblasts, periodontal ligament cells, and osteoblasts. In this study, we report the presence of cementoblast progenitors in cultures of bovine dental follicle cells and demonstrate their differentiation capacity. Bovine dental follicle cells (BDFC) obtained from tooth germs by collagenase digestion were compared with bovine alveolar bone osteoblasts (BAOB) and bovine periodontal ligament cells (BPDL) in vitro and in vivo. In culture, BDFC exhibited low levels of alkaline phosphatase activity and expressed mRNA for osteopontin (OP) and type I collagen (COLI), as well as low levels of osteocalcin (OC) mRNA. In contrast, cultured BAOB exhibited high alkaline phosphatase activity levels and expressed mRNA for OC, OP, COLI, and bone sialoprotein (BSP). To elucidate the differentiation capacity of BDFC in vivo, cells were transplanted into severe combined immunodeficiency (SCID) mice and analyzed after 4 weeks. Transplanted BDFC formed fibrous tissue and cementum-like matrix, which stained positive for anti-cementum attachment protein (CAP) monoclonal antibody (3G9), and expressed mRNA for OC, OP, COLI, and BSP. On the other hand, transplanted BAOB formed bone-like matrix, but were negative for anti-CAP monoclonal antibody. The BPDL transplants formed fibrous tissue that contained a few cells expressing CAP. These results indicate that cementoblast progenitors are present in BDFC, which can provide a useful model for investigating the molecular mechanisms of cementogenesis.

Animals↗
PubMed2001

Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD.

Keratin 8 and 18 (K8/18) are the major components of intermediate filament (IF) proteins of simple or single-layered epithelia. Recent data show that normal and malignant epithelial cells deficient in K8/18 are nearly 100 times more sensitive to tumor necrosis factor (TNF)-induced cell death. We have now identified human TNF receptor type 1 (TNFR1)-associated death domain protein (TRADD) to be the K18-interacting protein. Among IF proteins tested in two-hybrid systems, TRADD specifically bound K18 and K14, type I (acidic) keratins. The COOH-terminal region of TRADD interacted with the coil Ia of the rod domain of K18. Endogenous TRADD coimmunoprecipitated with K18, and colocalized with K8/18 filaments in human mammary epithelial cells. Overexpression of the NH2 terminus (amino acids 1-270) of K18 containing the TRADD-binding domain as well as overexpression of K8/18 in SW13 cells, which are devoid of keratins, rendered the cells more resistant to killing by TNF. We also showed that overexpressed NH2 termini of K18 and K8/18 were associated with endogenous TRADD in SW13 cells, resulting in the inhibition of caspase-8 activation. These results indicate that K18 may sequester TRADD to attenuate interactions between TRADD and activated TNFR1 and moderate TNF-induced apoptosis in simple epithelial cells.

Aclarubicin↗
PubMed2001

p53 regulates ceramide formation by neutral sphingomyelinase through reactive oxygen species in human glioma cells.

The present study was designed to elucidate the relationship between p53 and ceramide, both of which are involved in apoptotic signaling. Treatment of human glioma cells with etoposide caused apoptosis only in cells expressing functional p53. p53 activation was followed by the formation of reactive oxygen species (ROS), superoxide anion (O2-*) measured by hydroethidium oxidation into ethidium and hydrogen peroxide (H2O2) measured by oxidation of 2',7'-dichlorofluorescin (DCFH) into 2',7'-dichlorofluorescein (DCF), which was accompanied with ceramide generation through the activation of neutral, but not acid, sphingomyelinase. Superoxide dismutase (SOD), a selective antioxidant for O2-*, had no effects on p53 expression but inhibited ceramide generation and apoptotic cell death caused by etoposide. However, catalase, a specific antioxidant for H2O2, only weakly inhibited and sodium formate, a hydroxyl radical (* OH) scavenger, unaffected etoposide-induced apoptosis. Like etoposide-induced cell death, treatment of glioma cells with the O2-*-releasing agent, pyrogallol, induced typical apoptosis and ceramide generation even in the presence of catalase. In contrast, human glioma cells lacking functional p53, either due to mutation or the expression of E6 protein of human papillomavirus, were highly resistant to etoposide and exhibited no significant change in the ceramide level. Moreover, expression of functional p53 protein in glioma cells expressing mutant p53 using a temperature-sensitive human p53(Val138) induced ceramide accumulation by the activation of neutral sphingomyelinase which was dependent on the generation of O2-*. Taken together, these results suggest that p53 may modulate ceramide generation by activation of neutral sphingomyelinase through the formation of O2-*, but not its downstream compounds H2O2 or * OH.

Apoptosis↗
PubMed2001

Persistent increase in chromosome instability in lung cancer: possible indirect involvement of p53 inactivation.

Karyotype and fluorescence in situ hybridization analyses have demonstrated the frequent presence of an altered static state of the number of chromosomes (ie, aneuploidy) in lung cancer, but it has not been directly established whether aneuploidy is in fact associated with a persistent increase in the rate of chromosomal losses and gains (ie, chromosome instability, or CIN). The study presented here used a panel of 10 lung cancer cell lines to provide for the first time direct evidence that CIN is a common feature in lung cancer cell lines in association with the presence of significant aneuploidy. In addition, we found that the CIN phenotype correlates well with the presence of p53 mutations. However, human papilloma virus 16-E6-directed inactivation of p53 in a representative non-CIN lung cancer cell line did not result in the induction of CIN, at least up to the 25th generation, suggesting that inactivation of p53 itself is unlikely to directly induce CIN in lung cancer cells. Interestingly, however, significant CIN could be induced in conjunction with the generation of aneuploid populations when the mitotic spindle formation was transiently abrogated in p53-inactivated cells. These results suggest that inactivation of p53 may allow lung cancer cells to go through an inappropriate second division cycle under certain forms of mitotic stresses, which would result in the induction of the CIN phenotype in conjunction with the generation of aneuploidy.

Aneuploidy↗
PubMed2000

Co-expression of human chaperone Hsp70 and Hsdj or Hsp40 co-factor increases solubility of overexpressed target proteins in insect cells.

The insect-baculovirus expression system has proved particularly useful for producing recombinant proteins that are biologically active. Overexpression of foreign proteins using the recombinant baculovirus system is often accompanied by aggregation of the overexpressed protein, which is thought to be due to a limitation of the translated protein folding in the infected cells. Co-infection of a recombinant baculovirus capable of expressing the human chaperone Hsp70 slightly increased the solubility of the overexpressed Epstein-Barr virus replication protein, BZLF1. Co-expression of Hsp70 and its co-factor, Hsdj or Hsp40, was here found to improve the solubility of the target protein several fold. Thus, a baculovirus expression system producing these molecular chaperones may find application for improved production of target foreign gene products in insect cells.

Animals↗
PubMed2000

The Epstein-Barr virus pol catalytic subunit physically interacts with the BBLF4-BSLF1-BBLF2/3 complex.

The Epstein-Barr virus (EBV)-encoded replication proteins that account for the basic reactions at the replication fork are thought to be the EBV Pol holoenzyme, consisting of the BALF5 Pol catalytic and the BMRF1 Pol accessory subunits, the putative helicase-primase complex, comprising the BBLF4, BSLF1, and BBLF2/3 proteins, and the BALF2 single-stranded DNA-binding protein. Immunoprecipitation analyses using anti-BSLF1 or anti-BBLF2/3 protein-specific antibody with clarified lysates of B95-8 cells in a viral productive cycle suggested that the EBV Pol holoenzyme physically interacts with the BBLF4-BSLF1-BBLF2/3 complex to form a large complex. Although the complex was stable in 500 mM NaCl and 1% NP-40, the BALF5 protein became dissociated in the presence of 0.1% sodium dodecyl sulfate. Experiments using lysates from insect cells superinfected with combinations of recombinant baculoviruses capable of expressing each of viral replication proteins showed that not the BMRF1 Pol accessory subunit but rather the BALF5 Pol catalytic subunit directly interacts with the BBLF4-BSLF1-BBLF2/3 complex. Furthermore, double infection with pairs of recombinant viruses revealed that each component of the BBLF4-BSLF1-BBLF2/3 complex makes contact with the BALF5 Pol catalytic subunit. The interactions of the EBV DNA polymerase with the EBV putative helicase-primase complex warrant particular attention because they are thought to coordinate leading- and lagging-strand DNA synthesis at the replication fork.

Animals↗
PubMed1999

Rapid determination of Epstein-Barr virus-specific CD8(+) T-cell frequencies by flow cytometry.

We have developed an efficient and rapid method for detection of Epstein-Barr virus (EBV)-specific CD8(+) T-cell frequencies both in freshly isolated peripheral blood mononuclear cells (PBMCs) and in vitro established cytotoxic T lymphocyte (CTL) lines. Responder cells are thereby stimulated with an autologous lymphoblastoid cell line for 5 hours and intracellular accumulation of interferon gamma (IFNgamma) is detected by multiparameter flow cytometric analysis. EBV-specific CD8(+) T-cell frequencies ranged between 0.63% and 1. 29% in PBMCs of 5 healthy long-term EBV carriers. Using EBV-specific T-cell lines, it was shown that flow cytometric analysis is more sensitive than limiting dilution analysis for CTL precursors and enzyme-linked immunospot assay detecting IFNgamma-producing T cells. The class I restriction of IFNgamma production was confirmed using an anti-class I monoclonal antibody (MoAb). Information on other cytokine production of EBV-specific CTLs could be obtained using combinations of anti-cytokine MoAbs. The sensitive and rapid nature of the flow cytometric assay for EBV-specific CD8(+) T-cell frequency has significant advantages for evaluation of EBV-specific CD8(+) T-cell responses in PBMCs of patients with EBV-related diseases.

Antigens, Viral↗
PubMed1999

The G(2) checkpoint is maintained by redundant pathways.

While p53 activity is critical for a DNA damage-induced G(1) checkpoint, its role in the G(2) checkpoint has not been compelling because cells lacking p53 retain the ability to arrest in G(2) following DNA damage. Comparison between normal human foreskin fibroblasts (HFFs) and HFFs in which p53 was eliminated by transduction with human papillomavirus type 16 E6 showed that treatment with adriamycin initiated arrest in G(2) with active cyclin B/CDC2 kinase, regardless of p53 status. Both E6-transduced HFFs and control (LXSN)-transduced cells maintained a prolonged arrest in G(2); however cells with functional p53 extinguished cyclin B-associated kinase activity. Down regulation was mediated by p53-dependent transcriptional repression of the CDC2 and cyclin B promoters. In contrast, cells lacking p53 showed a prolonged G(2) arrest despite high levels of cyclin B/CDC2 kinase activity, at least some of which translocated into the nucleus. Furthermore, the G(2) checkpoint became attenuated as p53-deficient cells aged in culture. Thus, at late passage, E6-transduced HFFs entered mitosis following DNA damage, whereas the age-matched parental HFFs sustained a G(2) arrest. These results indicate that normal cells have p53-independent pathways to maintain DNA damage-induced G(2) arrest, which may be augmented by p53-dependent functions, and that cells lacking p53 are at greater risk of losing the pathway that protects against aneuploidy.

Animals↗
PubMed1999

Increased frequency of antigen-specific CD8(+) cytotoxic T lymphocytes infiltrating an Epstein-Barr virus-associated gastric carcinoma.

Gastric adenocarcinomas carrying Epstein-Barr virus (EBV) are known to be accompanied by massive lymphocyte infiltration. To characterize the tumor-infiltrating lymphocytes (TILs), we isolated and cultured such cells from a surgically resected EBV-associated gastric carcinoma. They were found to be positive for CD3, CD8, T-cell receptor beta chain, and cytotoxic molecules. The isolated TILs consisted of human leukocyte antigen (HLA) class I-restricted CD8(+) cytotoxic T lymphocytes (CTLs), which killed autologous EBV-transformed cells (but not phytohemagglutinin blast cells) and recognized HLA-A24 as restriction molecules. However, the TILs did not recognize known EBV antigenic peptides presented by HLA-A24 molecules, nor HLA-A24(+) fibroblasts infected with vaccinia recombinant virus expressing each of the EBV latent proteins. EBV(+) gastric carcinomas do not express conventional target proteins of EBV-specific CTLs, and the data suggest that some cellular proteins may be involved in the strong T-cell response to EBV-associated gastric carcinoma. In addition, our data suggest that class I-restricted, antigen-specific CD8(+) CTLs are specifically expanded within EBV(+) gastric carcinoma tissue.

HLA-A Antigens↗
PubMed1999

[Successful treatment of pleuritis carcinomatosa using combination therapy of 5'-DFUR, MPA and CPA as maintenance therapy].

A 44-year-old female patient with inoperable, local advanced left breast cancer was treated with 3 cycles of high dose CAF therapy followed by combination therapy of 5'-DFUR, MPA and CPA. The patient was discharged after receiving 3 cycles of high-dose CAF therapy and continued to receive daily oral doses of 5'-DFUR (800 mg), MPA (800 mg), and CPA (100 mg) for 15 months. After 3 cycles of high-dose CAF therapy, tumor marker (CEA, CA 15-3) levels were reduced. Six months later, after 3 cycles of high-dose CAF therapy, the tumor marker levels were within the normal range. No serious side effects were observed during chemotherapy. The patient enjoyed a good quality of life. We thus confirmed that this combination regimen was effective as a maintenance therapy for local advanced breast cancer.

Adenocarcinoma↗