The p16 gene encodes an inhibitor of the cyclin-dependent kinase, which inactivates cyclin-dependent kinase and contro1s the cell cycle progression, The 10ss of p16 expression or overexpression has been reported in many kinds of tumors, Both p16 and PCNA regu1ates cell cycle progression at the Gl/8 checkpoint, Although many researches about the p16 expression in ora1 cancer have been carried out, there are few studies about the corre1ation between p16 ex pression and pro1iferation of ora1 cancer cells The object of this study was to eva1uate the avai1ability of p16 as ear1y diagnostic factor and prognostic factor through corre1atión ana1ysis of p16 expression in ora1 squamous cell carcinoma and its re1ation to PCNA index and clinicopatho1ogic factors 80 we investigated p16 immunohistochemica1 expression of 83 ora1 squmaous cell carcinomas, and obtained the resu1ts as followed, 18 out of the 83 cases(21, 69%) showed p16 positive and 65 samp1es(78,31%) showed p16 negative, Whi1e the mean va1ue of PCNA indices of p16 positive cases was 65,94 ::t 18,32, that of PCNA indices at p16 negati ve ones 54,79 ::t 18, 39, This difference between them showed statistica1 sígnificance, (P=O, 030) p16 positive group was 12/60(20, 0%) of well differentiated tumors and p16 negative group was 6/23(16, 1%) of moderate1y or poor1y differentiated tumors, This difference did not show statistica1 significance. (P=O. 372) From the resu1ts above, it was suggested p16 expression is re1ated to PCNA index in ora1 squamous cell carcinomas.
Cyclooxygenase- 2 (COX-2) is an inducible enzyme that is not found in normal conditions,. but is induced by a varie ty of pathophysiologic conditions of tissues by growth factors. inflammatory stimuli. oncogenes and tumor promoters, COX-2 is upregulated in a number of epithelial cancers. including in oral premalignant and malignant lesions, The mode of action of COX-2 in carcinogenesis may include mutiple mechanisms that may be acting at different stages of malignant disease, In this study. the expression of COX- 2 protein was assessed quantitatively 없d qualitatively by immunohistochemistry during DMBA-induced hamster buccal pouch carcinogenesis, The immunoreactivity for COX-2 protein increased as the tissue passed from hyperplasia to dysplasia and SCC, The highest mean expression was SCC at 14 week, The differences between COX-2 expression in the normal and that the dysplastic and carcinomatous lesions was statistically significant, In addition. the mean values of COX -2 expression in the stromal cells increased gradually during malignant progression, The results suggest that increased COX-2 expression may be associated with the chemically induced carcinogenic progression of hamster buccal pouch model, The gradual increasing COX-2 expression de tected during the progressive manner toward more malignant lesions shows that the COX-2 protein can have an important role in both the early and the later stages of multistep oral carcinogenesis
We conducted a series of in vitro experiments to evaluate the efficiency of photodynamic therapy on head and neck cancer cell using hydroxybacteriochlorine from photosynthetic bacteria. We tested the cytotoxicity of the hydroxybacteriochlorine by MTI assay and observed the cell death pattern(apoptosis or necrosis) after PDT by hoechst 33342 and propidium iodide staining methods IC50 value of the hydroxybacteriochlorine was 0.22μg/rrúi. At higher doses of hydroxybacteriochlorine () 0.6μg/rrúi) , cancer cells died exclusively by necrosis after PDT. By contrast, at IC50 value, hydroxybacteriochlorine induced cancer cell to undergo apoptotic cell death. The induction begins approximately 6 hours after PDT. We investigates intracellular localization of hydroxybacteriochlorine by head & neck cancer cell via confocal laser scanning microscopy. Head & neck cancer cells dual-stained with hydroxybacteriochlorine and a panel of organelle- specific fluorescence probes (Mitotracker, Lysotracker, ER-Tracker) revealed an intracellular fluorescence distribution restricted to cytoplasmic compartments with no detectable fluorescence in the nucleus Confocal images of cells containing hydroxybacteriochlorine were never overlap in subcellular organelle fluorescence when digitally over layed with the organelle-specific fluorescence probe images of the same cells. These results demonstrated that the hydroxybacteriochlorine may have a function as a photosensitizer.
This study was conducted to test the anticancer effect of photodynamic therapy using chlorophyll derivative (9-HpbD-a) and 632nm diode laser. Human SNU 1041 cells were seeded into 96 well plate of 104cells/well and cultured for 24 hours. Cells were washed with media containing various concentration of 9-HpbD-a ranging from Oug/ml to 3.75ug/ml. Then 932 nm diode laser was given at various lasering time setting, and at various starting time after ini tial 24 hours of culture. The treated cells were incubated 48 hours and tetrazolium-based colorimetric(M'IT) assay was done to measure the viability of cells For in vivo study, SNU- 1041 cells were xenografted into the back of nude mouse. When the xenografted tumors grew up to 400-600 mm3, the animals were randomly placed into 4 groups: Group 1 (n=20) , PDT group, interstitial injection of 9-HpbD- a (47 ug/kg) followed by irradiation with 3.2 J/c야 of light 6 hours after then i띠 ection; Group II (n=lO) , irradiation with 3.2 J/crrf of light using diode laser; Group III (n=lO), in terstitial injection of 9-HpbD- a only(47 ug/kg); Group IV (n=lO), normal control group. The viability of cells was de creased with increasing lasering time No significant difference of cell viability was noted by variously delayed starting time of lasering. PDT effects were observed in the xenografted nude mouse model Group IV (no 9-HpbD-a, no laser irradiation) was a control group which showed a continuous tumor growth. Group III (9-HpbD-a i띠 ection only) showed no response, Group II (laser irradiation only) sho￦ed 1 complete remission out of 10 (10%) , Group 1 (9-HpbD-a and laser irradiation) showed 13 cpmplete remission out of 20 (65%) , Group 1 showed significant remission rate, comparing to other groups (p<0.05). This study demonstrated anticancer effect of photodynamic therapy using 9-HpbD-a and 632nm diode laser on human squamous cell carcinoma cell line.
Nuclear factor 1 (NFI) was discovered as a protein required for adenovirus DNA replication in vitro, but it is now clear that NFI protein plays an important role in the expression of many cellular genes. NFI-C null mice demonstrated aberrant odontoblast differentiation, abnormal dentin formation, and thus molar lacking roots while other tissues/or gans in the body, including ameloblasts appear to be unaffected and normal. However, little is known about the mechanism of NFI -C function in odontoblast differentiation and dentin formation. In this study, in order to elucidate the molecular mechanisms of odntoblast differentiation, we examined morphological characteristics of the aberrant odontoblast in NFI-C null mice. we also evaluate the expression of dentin sialophosphoprotein (DSPP) and bone sialoprotein (BSP) mRNAs in the MDPC-23 cells by northern analysis after over-expression and inactiγation of NFI -C into mouse MDPC-23 cells Odontoblasts of the NFI-C null mouse were round in shape, lost their polarity, organized as a sheet of cells, and trapped in osteodentin-like mineralized tissue. Abnormal odontoblasts of NFI-C null mouse revealed the absence of an intercellular junctional complex known as the t erminal webs. MDPC-23 cells started to express DSPP mRNA beginning from the postnatal day of 14 and showed a steady increase as differentiating into odontoblasts. Over-expression of NFI -C increased the expression of DSPP mRNA. Inactivation of NFI - C induced BSP mRNA expression. These results suggest that NFI-C plays an important role in odontoblast differentiation in a cell-type specific manner and thus in dentin formation
Meckel’s cartilage is one of the earliest structure to appear in a mandible derived from the first branchial arch and serves as the primordium for the formation of mandible, malleus, incus, and sphenomandibular ligament. However, its direct role and the mechanism in mandibular development are not well elucidated. To address this issue, we observed morphological and histological changes and gene expression patterns in the Meckel's cartilage of a developng mouse, from E13.5 to E18.5 embryos, using skeletal preparation samples and routinely prepared slide sections for light rnicro scopic observation in various sectional planes. The following methods were performed: H&E staining for general histo logical observation, Von Kossa staining for detection of rnineralization, TRAP actiγity staining for localization of osteoclastic cells, immunohistochernistry for MMP- 1 and - 9 for evaluation of enzymatic activity of osteoclastic cells, and in situ hybridization for detection of collagen type 1. II, and X mRNA expression, respectively. At E13.5 Meckel's cartilage appeared as a V- shaped rod fused at the rnidline and thin rnineralized mandibular buccal plate was formed lateral to, and at some distance from, Meckel's cartilage in an intramembranous ossification mode With the progression of tooth development, the Meckel’s in cartilage adjacent incisors revealed hypertrophic chondrocyte differentiation with rnineralization of the chondroid matrix The Meckel’s cartilage was replaced with bone by - osteoclasts, showing strong immunoreactivity for MMP-1 and - 9 from E16.5. With time, this bony replacement of Meckel’s cartilage in an endochondral ossification mode was extended up to the rnid- portion of the molar sockets till E18 5 The bony replacement of rnineralized hypertrophic chondrocyte zone expressing X collagen mRNA contributed to the formation of thick mandibular lingual plate. These findings suggest that mandibular formation and development is closely related with not only Meckel's cartilage, but also with the developing tooth, and that certain influence from the developing tooth may play a role in deterrnining the fate of Meckel’s cartilage during mandibular development.
In order to obtain novel genes related to the human craniofacial development, molecular cloning and sequencing, and in situ hybridization using craniofacial tissue sections were performed and followed by protein structure simulation. Totally 231 clones were obtained from the subtracted craniofacial tissue cDNA library of human embryo. Random cloning using the non-redundant clones from the craniofacial tissue of human embryo was done and obtained 398 clones from the premade human chondrocyte cDNA library. Their partial sequence data showed that 214 clones of subtracted cDNA library of craniofacial tissue were still non-redundant in Genebank search. And 20 clones among 498 clones of premade chondrocyte cDNA library were known to be undefined genes. Through in situ hybridization screening in the craniofacial tissue sections of 10 weeks old human embryo 36 clones were found to be positive in specific tissues. Depending on the cell types of sirnilar developmental origin, the positive reactions could be divided into five groups. Among the 20 clones of undefined genes from human chondrocyte cDNA library, 7 clones showed characteristic positive reaction in human cartilage tissue by in situ hybridization. From the simulated protein structure, motif analysis and in situ hybridization studies for the 7 undefined clones, Ch89, Ch96, Ch129, Ch285 clones may function in the outer space of the cell constituting a part of matrix protein complex, and Ch276 as a transmembrane protein which might partic ipate in matrix calcification around chondrocytes. Ch153 is a kind of antirnicrobial protein also acting as an inflammation mediator, and Ch334 clone is a zinc finger protein, of which expression increases in human adult tissues We presume these novel genes from human chondrocytes may provide a new path of chondrocyte development and functions of human craniofacial tissues
The study of cornified cell envelope has been used to investigate the differentiation factors and to advance oral carcinogenesis, CE of human oral keratinocytes are in wet condition as saliva containing many proteases, growth factors, and many kinds of bacteria, The analysis of CE in Immortalized human oral keratinocyte(IHOK) derived from normal human oral keratinocyte(NHOK) will be used to study the pathogenesis of oral squamous cell carcinoma, The purpose of this study was to analyze the amino acid component derived from CE of cultured NHOK and IHOK, It will be helpful to study the role of transfected E6/E7 gene in forming CE, and to examine the pathogenesis of oral squamous cell carcinoma, After primry culture of NHOK, IHOK were cultured in KBM bullet kit at 370C under 95% C02 incubator, Growth curve according to calcium concentration, cornified cell envelope measurement(CEM), and protein chemistry for amino acid component of CE were done(Mena :f::SD) , respectively. The obtained results were as follows, lHOK showed small areas of stratification, more compact, with irregular border and tightly apposed cells in 1,2 mM Ca++, Cornified cell envelope exhibited an aggregated group of empty space surrounded by the remained cell membrane, During the terminal differentiation in cultured NHOK and IHOK, insoluble cornified cell envelope formation was increased, CEM of NHOK was about 4 folds than that of lHOK under high calcium, Amino acid component of both groups showed Pro/Glu(SPR) , Gln/Glu(lnvolucrin) , and Gly(Loricrin) in descending order, From the aboving results, ít was suggested that when the terminal dífferentiation in cultured NHOK and IHOK, major amino acid component of CE in cultured lHOK was the same to that of cultured NHOK, It was thought that E6 and E7 gene should be involved in preventing the differentiation and proliferation of IHOK from making CE,
It is very little known that the molecular mechanisms control growth, cell differentiation, and invasion of ameloblastoma into bone. Tissue culture methods have also been used extensively for studies of the cell biology of ameloblastoma. The purpose of this study were to examined the ultrastructural features of ameloblastoma, and to apply these results to examine the pathogenesis of ameloblastoma in the future. Amelobalstoma was primarily cultured under 0.1, 0.15 and 1.2mM Ca++ of KBM bullet kit at 370C and 5% C02. For transmission electronmicroscopy(TEM), cultured ameloblastoma cells were immediately fixed in 2.0% glutaraldehyde in O.lM cacodylate buffer(pH 7.4) at 40C for 1h The ultrathin sections were stained with uranyl acetate and lead citrate, and examined by TEM. The obtained results were as follows. Primay culture ameloblastoma grown in 0.1 mM Ca++ showed interlacing papillary projections without desmosome within early passage(3-4). Primary culture amelobalstoma under high calcium showed prominent desmosomes or tight junctions within early passage. There was evidence of cellular degeneration, as exemplified by nuclear pyknosis, the margination and clumping of the chromatin, and vaculolation under high calcium. The sparse ribosomes, the cytoplasmic space filled with vacuoles, and the condensed mitochondria were seen under high calcium. From the aboving results, under high calcium primary culture ameloblastoma showed rapid cellular degeneration within early passage, indicating that the cells were gradually losing metabolíc actívitíes, leading to enventual cell death. It was thought that it would be necessary to establish cultured immortalized amelobalstoma cell line for studying the pathogenesis of odontogenic tumors.
Abnormal number of deciduous teeth causes esthetic and dental problems on infants or children, which has effect on articulation disorder and emotional development as well as their physical growth. Therefore, it is important to detect dental problems early and to provide comparable indications. The purose of this study was to find out the prevalence and pattern in abnormal number of deciduous teeth. The clinical and radiographic examination was undertaken for 200 at age from 1 to 10 years and statistical analysis was done. The result were as follows. Among the examined patients, congenital missing teeth 8.6% and supernumerary teeth 36.4% were seen(p<0.05). And abnormal number of deciduous teeth was prevalent in male. Most supernumerary teeth located on middle area(88.6%) was seen(p<0.05). The most frequently missing teeth was the mandibular primary lateral incisor and the mandibular 2nd. premolar. And these teeth were mainly located in maxilla and right portion. It suggested that this study would play an important role for the basis of the demographic research of abnormal teeth in oral pathologic field