Biomedical subjects

Shin-ichi Kumakura

Publications and source records attributed to Shin-ichi Kumakura.

5 recordsLinked to original sources

PubMed2006

Effects of minocycline and doxycycline on cell survival and gene expression in human gingival and periodontal ligament cells.

OBJECTIVES AND BACKGROUND: Periodontitis is an infectious disease in the gingival crevice caused by periodontopathic bacteria, including Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomycetemcomitans, and Tennerella forsythensis, and antibacterial agents are directly administered to the site of infection to treat it. To maximize the therapeutic effects while reducing the adverse effects, the antibacterial agents should be administered at concentrations greater than their MIC(90) doses required to inhibit the growth of 90% of periodontopathic bacteria and the administration should not damage the periodontal tissue. One approach for estimating cellular damage in the periodontal tissue caused by the administration is to assay cytological damages following exposures of cultured human cells derived from periodontal tissues to antibacterial agents. In the present study, we investigated the cytotoxic effect of minocycline (MINO) and doxycycline (DOX) by using a human gingival fibroblast cell line, a human gingival epithelial cell line, and a human periodontal ligament fibroblast cell line. We also used these cell lines to study the effect of MINO or DOX on the mRNA and protein expressions of genes associated with the differentiation of fibroblasts and the proliferation, differentiation, or cellular adhesion important to the epithelial regeneration of the periodontal attachment. METHODS: The cytotoxic effect of MINO or DOX was measured as a decrease in cell survivals. The effects of these antibiotics on the mRNA and protein expressions in the cell lines were studied by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analyses, respectively. RESULTS: The maximum concentration of MINO or DOX that has little effect on the cell survivals and the mRNA and protein expressions of genes for alkaline phosphatase, type I procollagen, keratinocyte growth factor receptor, keratin 18 or 8/18, integrin beta1, integrin beta4, and laminin 5gamma2 was 10 or 30 microm, respectively, which are greater than their MIC(90) doses against periodontopathic bacteria described above. CONCLUSIONS: These findings suggest that little, if any, cellular damage would be expected with topical administration of MINO or DOX to the periodontal pocket at a dose equivalent to the MIC(90). It is important to note, however, that the extrapolation of these findings to in vivo conditions has yet to be undertaken.

Administration, Topical↗

PubMed2005

Reversible conversion of immortal human cells from telomerase-positive to telomerase-negative cells.

Immortal cell lines and tumors maintain their telomeres via the telomerase pathway or via a telomerase-independent pathway, referred to as alternative lengthening of telomeres (ALT). Here, we show the reversible conversion of the human papillomavirus type 16 E6-induced immortal human fibroblasts E6 Cl 6 from telomerase-positive (Tel(+)) to telomerase-negative (Tel(-)) cells. Tel(+) cells converted spontaneously to Tel(-) cells that reverted to Tel(+) cells following treatment with trichostatin A (TSA) and/or 5-aza-2'-deoxycytidine (5-AZC), which induced the reversion from complete to partial methylation of the CpG islands of the human telomerase reverse transcriptase (hTERT) promoter in Tel(-) E6 Cl 6 cells. Tel(-) E6 Cl 6 cells lacked the phenotypes characteristic of ALT cell lines such as very long and heterogenous telomeres and ALT-associated promyelocytic leukemia nuclear bodies (APB) but grew for >240 population doublings (PD) after they became telomerase negative. The ratios of histone H3 (H3) lysine (K) 9 methylation to each of H3-K4 methylation, H3-K9 acetylation, and H3-K14 acetylation of the chromatin containing the hTERT promoter in Tel(-) E6 Cl 6 cells and ALT cell lines were greater than those in Tel(+) cells and decreased following treatment with TSA and/or 5-AZC, inversely corresponding to telomerase activity. Our findings suggest the possibility that human tumors may be able to reversibly interconvert their telomere maintenance phenotypes by chromatin structure-mediated regulation of hTERT expression.

Acetylation↗

PubMed2003

Immortalization of normal human embryonic fibroblasts by introduction of either the human papillomavirus type 16 E6 or E7 gene alone.

The ability of the human papillomavirus type 16 (HPV-16) E6 or E7 gene to induce immortalization of normal human embryonic fibroblast WHE-7 cells was examined. WHE-7 cells at 9 population doublings (PD) were infected with retrovirus vectors encoding either HPV-16 E6 or E7 alone or both E6 and E7 (E6/E7). One of 4 isolated clones carrying E6 alone became immortal and is currently at >445 PD. Four of 4 isolated clones carrying E7 alone escaped from crisis and are currently at >330 PD. Three of 5 isolated clones carrying E6/E7 were also immortalized and are currently at >268 PD. The immortal clone carrying E6 only and 2 of the 3 immortal clones carrying E6/E7 expressed a high level of E6 protein, and all the immortal clones carrying E7 alone and the other immortal clone carrying E6/E7 expressed a high level of E7 protein when compared to their mortal or precrisis clones. The immortal clones expressing a high level of E6 or E7 protein were positive for telomerase activity or an alternative mechanism of telomere maintenance, respectively, known as ALT (alternative lengthening of telomeres). All the mortal or precrisis clones were negative for both phenotypes. All the immortal clones exhibited abrogation of G1 arrest after DNA damage by X-ray irradiation. The expression of INK4a protein (p16(INK4a)) was undetectable in the E6-infected mortal and immortal clones, whereas Rb protein (pRb) was hyperphosphorylated only in the immortal clone. The p16(INK4a) protein was overexpressed in all the E7-infected immortal clones and their clones in the pre-crisis period as well as all the E6/E7-infected mortal and immortal clones, but the pRb expression was downregulated in all of these clones. These results demonstrate for the first time to our knowledge that HPV-16 E6 or E7 alone can induce immortalization of normal human embryonic fibroblasts. Inactivation of p16(INK4a)/pRb pathways in combination with activation of a telomere maintenance mechanism is suggested to be necessary for immortalization of normal human embryonic fibroblasts by these viral oncogenes. The susceptibility of human cells to immortalization may be related to the state of differentiation of the cells.

Blotting, Western↗

PubMed2002

Enhancer elements in the mouse Cyp1a2 gene for constitutive expression.

CYP1A2 is one of the major hepatic cytochrome P450s that is involved in the metabolism of many drugs, as well as in the activation of chemical carcinogens. To elucidate the transcriptional regulation of the constitutive expression of the mouse Cypla2 gene, the 4.8-kbp 5(')-flanking region of the gene was analyzed for transcriptional activity using a primary cultured mouse hepatocyte system. With 5(')- and 3(')-deletion analysis, two enhancer elements, i.e., a 20-bp DNA fragment (E1) from -4401 to -4382 and a 9-bp (E2) from -4300 to -4292, were identified. E1 and E2 contain a phorbol 12-O-tetradecanoate-13-acetate (TPA)-responsive element (TRE) and TRE-like element, respectively. Electrophoretic mobility shift assay confirmed specific binding between these two enhancer elements and nuclear proteins. Site-directed mutagenesis assay suggested that the TRE element in E1 is essential for constitutive expression of the mouse Cypla2 gene.

Animals↗

PubMed2002

Telomere length and telomerase activity in the process of human fibroblast immortalization.

To examine the telomere maintenance mechanism in the process of human fibroblast immortalization, we studied telomere length, telomerase activity, chromosome instability, and minisatellite alterations in human fibroblasts following the introduction of the human papillomavirus type 16 E6 gene or E7 gene, or both. One cell line immortalized by E6 alone maintained short telomeres, and its telomerase activity was positive. Fairly long and heterogeneous telomeres were maintained in all four E7-immortalized cell lines lacking telomerase activity. Of the three clones immortalized by both E6 and E7, two cell lines with telomerase activity maintained short telomeres, and the other cell line, which lacked telomerase activity, maintained long and heterogeneous telomeres. Although all immortal cell lines expressed mRNAs for human telomerase RNA component and telomerase-associated protein, expression of mRNA for human telomerase reverse transcriptase was detected only in the telomerase-positive cell lines. All immortal cell lines showed both chromosomal abnormalities, including structural and numerical changes, and minisatellite alterations detected by DNA fingerprinting. These findings indicate the existence of different telomere maintenance mechanisms in telomerase-positive and -negative fibroblast cell lines immortalized by E6, E7, or E6/ E7, and the possible involvement of chromosome instability and minisatellite alterations in the activation of the telomere maintenance mechanisms.

Journal Article↗