Effecs of Dexamethasone and Epidermal Growth Factor on Activity of Viral Promoter p97 and Expression of HPV-16 E6/E7 in Human Oral Keratinocytes Transformed with HPV-16 and Benzo(a)pyrene

Kook, Joong-Ki,Lee, Gene,Woo, Kyung Mi,Min, Byung-Moo The Official Publication of Korean Academy of Oral 1995 International Journal of Oral Biology Vol.19 No.2

Primary human oral keratinocytes were previously transformed by transfection with cloned human papillomavirus type 16(HPV-16)DNA and subsequent exposure to benzo(a)pyrene, and an oral cancer cell line, CTHOK-16B-BaP, was established. To determine the effects of dexamethasone and epidermal growth factor (EGF) on cell proliferation, the expression of HPV-16 E6/E7 and several proto-oncogenes, and activity of HPV-16 E6/E7 promoter, p97, the CTHOK-16B-BaP cells were exposed to either dexamethasone and EGF alone or together. After incubation for 3 days, the degrees of both cell proliferation and the expression of HPV-16 E6/E7, EGF receptor (EGFR), c-myc, and c-fos genes was determined. Dexamethasone and EGF, when added alone or together in the culture media, increased cell proliferation. Although dexamethasone did not affect the transcriptional levels of HPV-16 E6/E7, EGFR, or c-myc in the cells, it down-regulated c-fos mRNA expression. On the other hand, EGF, alone or in conjuction with dexamenthasone, down-regulated the transcriptional levels of HPV-16 E6/E7, c-myc, and c-fos genes, but it had little effect on the level of EGFR transcription. These results suggest that the increased proliferation of CTHOK-16B-BaP cells in the presence of dexamethasone and/or EGF may not depend on the extent of expression of such genes as HPV-16 E6/E7, EGFR, c-myc, and c-fos. In addition, the HPV-16 E6/E7 mRNA level was not changed and reduced by treatment with dexamethasone and EGF, respectively. Unexpectedly, however, dexamethasone and EGF enhanced the activity of the viral promoter p97 in CTHOK-16B-BaP line as analyzed by transient expression assays using the chloramphenicol acetyltransferase gene as a reporter. It appears that dominant regulatory mechanisms presumably depending on the chromosomal integration site are able to override the response of the viral promoter to dexamethasone and EGF. Another EGF-responsive element (7454-7643 nt) which acts as an enhancer may be located within the HPV-16 LCR portion.

Isolation and Characterizaton of Staphylococci in Acute Oral Infection

Kim, Kang-Ju,Kim, Won-Sin,Kim, Ji-Hyun,Yoo, Soo-Kyung,Im, Mi-Kyoung,Chung, Chong-Pyoung The Official Publication of Korean Academy of Oral 1994 International Journal of Oral Biology Vol.18 No.1

To ascertain the distribution and genetic patterns of microorganisms associated with acute oral infections, patients with acute infection such as oral abscess and osteomyelitis were sampled and staphylococci were isolated, identified and characterized. Plasmid and HindⅢ-digested bacterial genomic DNA of staphyllococci was electrophoresed and dot blotting was performed to study the genomic patterns. Most of staphylococci from patients with acute oral infection were Staphylococcus lugdunensis and Staphylococcus aureus, and showed the resistance to ampicillin and penicillin. Four strains of S. lugdunensis produced δ-like hemolysin. In the analysis of plasmid, there was a clear band about 6.5 kb in the S. lugdunensis isolated from patients with infection. In the analysis of genomic pattern, four strains of S. lugdunensis with δ-like hemolysin showed the similar pattern with HindⅢ enzyme digests. These results showed that S. lugdunensis might play a major role in acute oral infection.

δ-Hermolysin like Gene of Staphylococcus lugdunensis in Acute Oral Infection Have Partial Homology with δ-Hemolysin Gene of Staphylococcus aureus

Kim, Kang-Ju,You, Yong-Ouk,Kim, Eun-Sook,Whang, Tae-Suk,Han, Sung-Hi,Kim, Sung-Su,Ryu, Dae-Hyon,Min, Byung-Moo The Official Publication of Korean Academy of Oral 1995 International Journal of Oral Biology Vol.19 No.1

To investigate the distribution and hemolytic activity of staphylococci in acute oral infection, staphylococci were isolated from the patients with acute oral infection and healthy persons, hemolytic activity was measured on sheep blood agar plates, and DNA-DNA hybridization was preformed with δ-hemolysin gene probe of S. aureus under low stringent condition or high stringent condition. The isolation ratio of S.lugdunensis in patients was higher than that of healthy persons. Four strains of S. lugdunensis had δ-like hemolytic activity, but two strains did not. In dot blot analysis, S. lugdunensis was hybridized with δ-hemolysin probe of S. aureus under low stringent condition, but weakly hybridized with δ-hemolysin probe under high stringent condition. There results suggest that S. lugdunensis is an important pathogen in acute oral infection and δ-hemolysin gene of S. lugdunensis have partial homology with δ-hemolysin gene of S. aureus.

Modulation of EGFR and c-Ha-ras Expression During Calcium-induced Diffrentiation in HPV-immortalized Human Oral Keratinocytes

Min, Byung Moo The Official Publication of Korean Academy of Oral 1993 International Journal of Oral Biology Vol.17 No.2

Calcium-induced differentiation in cultures of human oral keratinocytes provides an excellent in vitro model to study differentiation. To understand the genetic events that underlie keratinocyte differentiation, we cultured the human papillomavirus type 16(HPV-16)-immortalized human oral keratinocytes in medium containing 0.15, 0.3, 0.6, or 1.2 mM of calcium for 3 days and determined the degree of cell differentiation and expression of c-fos, c-myc, c-Ha-ras, and epidermal growth factor receptor(EGFR) genes from the cells. Calcium, in a concentration-dependent manner, increased the cell differentiation. Calcium had no effect on the levels of c-myc and c-fos expression. The transcription of c-Ha-ras and EGFR messages, however, was increased by high concentration of calcium(1.2 mM), suggesting transcriptional regulation of these genes during differentiation. These results indicate that expression of c-myc and c-fos does not have an obligate role in the keratinocyte differentiation process, but enhanced levels of c-Ha-ras and EGFR gene messages may be, in part, responsible for the cell differentiation. These findings expand our knowledge of the differentiation process in human oral keratinocytes.

Calcium Modulation of Growth and Differentiation of Immortal Human Oral Keratinocytes

Min, Byung Moo,Kim, Kyung Ae The Official Publication of Korean Academy of Oral 1993 International Journal of Oral Biology Vol.17 No.1

Modification of the medium calcium concentration markedly modulates the pattern of proliferation and differentiation of cultured epidermal cells in human and murine. Correlation of the differences in the molecular weights of some of the keratins with those of NBT reduction abilities of human oral keratinocytes was studied in relation to its differentiation. Immortal human oral keratinocytes were grown in a serumfree keratinocyte growth medium containing 0.15, 0.3, 0.6 or 1.2 mM calcium for 3 days and assayed for the DNA synthesis, NBT reduction, and keratin profiles. The addition of calcium in culture medium produced altered morphology. enhanced percentage of NBT-positive cells and inhibited de novo DNA synthesis showing that extracellular calcium induced the differentiation of immortal oral keratinocytes. In low-calcium-grown cells the low molecular weight keratins were observed. however, the addition of calcium to low-calcium-grown cells resulted in induction of the high molecular weight keratins. These results indicate that the increase of the molecular weights of some of the keratins correlates with the degree of the differentiation of oral keratinocytes. We suggest a possibility that the change of keratin profiles may be used as a molecular marker of calcium-induced keratinocyte differentiation.

Induction of Endocarditis by Strains of Strptococcus sanguis which are Resistant to Bactericidal Activity of Polymorphonuclear leukocytes (PMNs) in a Rat Endocarditis Model

Lee, Si Young The Official Publication of Korean Academy of Oral 1994 International Journal of Oral Biology Vol.18 No.1

The oral cavity is the most common source for the bateria that induce infective endocarditis and the oral viridans streptococci are the most frequent pathogens associated with this fatal disease. Recently, a study showed the correlation between the induction of bacterial endocarditis by streptococcus gordonii, an oral viridans streptococcus, and its resistance to polymorphonuclear leukocytes(PMNs) dependent destruction. That has suggested the major determinant of virulence for endocarditis by S. gordonii strains appeared to be more related to resistance to lectin-mediated killing by PMNs rather than platelet aggregation or binding to attachment proteins or fibrinogen. The purpose of this study is to investigate the correlation between induction of bacterial endocarditis by S. sanguis, anoher type of oral viridans streptococci, and its resistance to destruction by PMNs. Of the 7 strains of S. sanguis, 5 strains were totally resistnat to the killing by PMNs and 2 strains were partially killed (40-65%). The killing was mediated by sialic acid reactive lectin verified by the inhibition of killing by sialidase treatment of PMNs. In rat endocarditis model in which the aortic valve was damaged by insertion of catheter through the carotid artery, both not killed and partially killed strains had the ability to induce severe endocarditis characterized by the multiple vegetations and the high recovered number of bacteria from heart tissue. Thus resistance of S. sanguis to the killing by PMNs appears to be an important virulence determinant in infective endocarditis as also demonstrated by S. gordonii strains in previous studies.

Effects of Cell Cycle Inhibitors on Cell Death of Human Cancer Cell Lines

Whang, Kyung-Tae,Kim, Myung-Soo,Kim, Gwan-Shik The Official Publication of Korean Academy of Oral 1995 International Journal of Oral Biology Vol.19 No.1

Genotoxic agents are known to cause cell death mostly by apoptosis in susceptible cells and this action plays an important role in tumor regression after chemotherapy. It has been thought that the apoptotic process may be associated with cell cycle. In the present study, the effect of aphidicolin and nocodazole, reversible cell cycle inhibitors, on the apoptosis or cell death induced by actinomycin D was studied in 3 cell lines. HeLa, SiHa and NIH/3T3 cells were treated with actinomycin D(100 nM)alone or in combination with cell cycle inhibitors for 20 hours; or presynchronized cells were treated with actinomycin D for 4 hours and further incubated for 20 hours in fresh medium. The synergistic effect of IBMX(5 or 10 μM)on the apoptosis or cell death induced by actinomycin D was also studied. The results were as follows; Majority of HeLa cells showed apoptotic changes after actinomycin D treatment. Aphidicolin or nocodazole blocked neither initiation nor progression of actinomycin D-induced apoptosis in HeLa cell. Aphidicolin pretreatment enhanced the actinomycin D-induced apoptosis of HeLa cells and cell death of SiHa cells and nocodazole pretreatment enhanced cell death of SiHa cells. IBMX moderately enhanced actinomycin D-induced cell death of NIH/3T3 and to less extent that of SiHa cells. These results show that neither initiation nor progression of actinomycin D-induced apoptosis or cell death can be blocked by cell cycle arrest, and that cAMP is partly responsible for actinomycin D-induced apoptosis in certain types of cells.

Effects of Protein Kinase C Modulators on the Osteoclast-like Cell Generation Induced by Osteotropic Hormones

Ko, Seong-Hee,Lee, In-Seok,Kim, Gwan-Shik The Official Publication of Korean Academy of Oral 1995 International Journal of Oral Biology Vol.19 No.1

Protein kinase C (PKC) has been known to play an important role in the process of cell differentiation. The present study was undertaken to investigate the effect of modulators of PKC in the generation of osteoclast-like cells from mouse bone marrow cells. The marrow cells were collected from the femurs and tibiae of 4- to 6-week-old mice and cultured for 8 days. In order to examine the role of PKC in the generation of osteoclast-like cells induced by hormones, a PKC modulator, PMA or H7 was supplemented in the presence of PGE_2, 1,25-dihydroxyvitamin D_3, or PTH. After culture, the cells were stained for tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclast and the TRACP-positive multinucleated cells (MNCs) which have 3 or more nuclei were counted. The present study showed that TRACP-positive MNCs were not formed in the presence of PMA and H7 alone. However, the generation of TRACP-positive MNCs induced by PGE_2 and 1,25-dihydroxyvitamin D_3 was stimulated by PMA at 10^-9 - 10^-7 M. This augmentation of TRACP-positive MNC generation was inhibited by the presence of 10^-5 M H7. The generation of TRACP-positive MNCs induced by PTH was suppressed by 10^-9 - 10^-7 M PMA. This suppression of TRACP-positive MNC generation by 10^-7 M PMA was completely reversed by 10^-5 M H7. H7 at 10^-5 M inhibited PGE_2 induced TRACP-positive MNC generation. These results suggest that PKC may play an important role in the generation of osteoclasts and may modulate the action of osteotropic hormones in the bone resorption.

Isolation and Antibiotic Susceptibility of Staphylococci in Dental Clinic

Moon, Sang-Eun,Kim, Eun-Sook,Hwang, Tae-Suk,Kim, Kang-Ju The Official Publication of Korean Academy of Oral 1995 International Journal of Oral Biology Vol.19 No.1

It was reported that staphylococci present in clinic and personnel are transmissible to patients. The purpose of this study was to investigate nosocomial infection. Staphylococci were isolated from the anterior nares of dental personnel and the environment in dental clinic and characterized by antibiotic sensitivity. Staphylococcus aureus (S. aureus) was identified by Gram stain, catalase test, mannitol fermentation test, DNase test and coagulase test. The antibiotic susceptibility tests were performed according to the National Committee of Clinical Laboratory Standards(NCCLS). The nineteen percent of staphylococci isolated from dental clinic were S. aureus. All the isolates of S. aureus in this study showed the resistance to ampicillin and penicillin, and the susceptibility to vancomycin and chloramphenicol. These results seem to mean that S. aureus might be related to the nosocomial infection in dental clinic.

Effects of Conditioned Media from the Periodontal Ligament Cells and Gingival Fibroblast on the Proliferation and Differentiated in Osteoblastic Clone MC3T3-E1 Cells

Moon, Ki-Ho,Lee, In-Seok,Kim, Gwan-Shik The Official Publication of Korean Academy of Oral 1995 International Journal of Oral Biology Vol.19 No.1

To dotermine whether the human periodontal ligament cell-conditioned medium (PDL-CM) and the human gingival fibroblast-conditioned medium (GF-CM) affect the proliferation and differentiation of osteoblastic clone MC3T3-E1 cells, the effects of both conditioned media on [^3H]thymidine incorporation into DNA and alkaline phosphatase (ALP) activity in clone MC3T3-E1 cells were examined. Both PDL-CM and GF-CM increased [^3H]thymidine incorporation into DNA whereas decreased ALP activity in dose-dependent manner. Especially, their inhibitory effect on ALP activity was proportional to the seeding density of MC3T3-E1 cells and concentration of CM. These findings indicated that PDL and GF produce and release certain soluble factor(s) that stimulate cell proliferation and inhibit differentiation in MC3T3-E1 cells and suggest that PDL and GF may play a role in the regulation of bone cell function in vivo as well.