Human β- defensin-2 (hBD-2) is an antimicrobial peptide which is produced by epithelial cells after stimulation with microorganisms or inflammatory mediators. However, little is known as to whether the LPS and nicotine induces the expression of hBD-2 in periodontal l igament (PDL) cells. T he p urpose o f this s tudy was t o investigate t he r oles o f MAPKs pathway o f nicotine a nd L PS-induced hBD-2 expression in PDL cells. The maximal expression of hBD-2 was observed in nicotine 5 mM and LPS 1 μg/ml treated PDL cells. Nicotine and LPS induced the phosphorylation of p38, c-Jun N-terminal kinase 1 and 2 (JNK1/2), and extracellular signal-regulated kinases 1 and 2 (ERK1/2) MAPK. ERK1/2 inhibitor SB203580, p38 inhibitor PD98059, and JNK inhibitor SP600125, blocked the effects of nicotine and LPS on hBD-2 upregulation in PDL cells. These results collectively suggest that hBD-2 is up-regulated in nicotine and LPS-treated PDL cells through activation of p38, ERK and JNK MAPKs pathway. Our data regarding the up-regulation of hBD-2 may help us to understand the antimicrobial mechanism in periodontal disease

The present study aimed to verify the effects of DFO on PDL cells, with particular emphasis on focusing on osteoblastic differentiation. Its mechanisms related to heme oxygenase-1 (HO-1) pathway were also analyzed. DFO increased the expression of HO-1 and early osteoblastic differentiation markers, such as alkaline phosphatase (ALP) and bone sialoprotein (BSP). DFO upregulated heme oxygenase-1. Treatment with HO-1 siRNA blocked the DFO-stimulated osteoblastic differentiation and HO-1 expression. The NF-kB inhibitor pyrrolidine dithiocarbamate, phosphatidylinositol 3-kinase inhibitor Wortmannin, and p38 MAPK inhibitor U0126 blocked the effects of DFO on HO-1 expression and osteoblastic differentiation in PDL cells. Collectively, these data suggest that DFO promotes osteoblastic differentiation and induces the expression of defense protein HO-1 probably via PI3K, p38 MAPK, and NF-kB signalling pathways in PDL cells.

Human papillomavirus (HPV) has been classified as one of the causing factors of head and neck squamous cell carcinoma (HNSCC). However, little is known about HPV-related carcinogenesis in HNSCC. The purpose o f this s tudy i s to characterize immortalized human oral keratinocyte (IHOK) transfected by HPV16 E6/E7, IHOK/hcdk4 (IHOK transfected by pLXRN-hcdk4) and IHOK/hcdk4/hTERT (IHOK transfected by pLPC-hTERT-hcdk4) to reconstitute HNSCC in vitro. Conclusively, we established a new immortalized cell lines, IHOK/hcdk4 and IHOK/hcdk4/hTERT, to understand multistep carcinogenic process of oncogenic HPV16 E6/E7 in HNSCC.

Eukaryotic translation initiation factor 5A (eIF-5A) is essential for proliferation of eukaryotic cells, andwas identified as diagnotic marker in cervical intraepithelial neoplasia, cervical and endometrial cancer, but relatively little is known about thein vivo and in vitro expression patterns of eIF-5A in oral premalignant and malignant lesions mirror the expression levels observed in vitro with cells derived from normal oral mucosa, immortalized oral keratinocytes (IHOK) and primary and metastatic oral squamous cell carcinoma (OSCC). We used an oral squamous cell carcinoma (OSCC) progression model composed of cell lines and tissue specimens to characterize expression patterns by Western blotting and immunohistochemistry. eIF-5A and PCNA levels are elevated in IHOKand primary and metastatic OSCC cella as compatred to normal human oral keraitinocytes. eIF5A and PCNA expression was l imited to basal cells of normal oral mucosa. eIF-5A and PCNA expression is increased in dysplastic epithelium spreading to more superficial layers, and its expression levels correlated significantly with the degree of dysplasia. Well and moderately differentiated OSCC showed strong expression of eIF-5A and PCNA. These results suggest that upregulated expression of eIF-5A seems to be an important epigenetic alteration that accompanies oral carcinogenic progression, and eIF-5A could be used as an biomarker for oral premalignat lesion or squamous cell carcinoma

Considering the great potential of iron chelators at inhibiting the proliferation of tumor cells, in order to determine the molecular and biological basis for the effects of iron chelator in oral cancer, we investigated the effects of iron chelator, desferrioxamine (DFO), on the gene profiling analysis of immortalized human oral keratinocytes (IHOK), and oral cancer cells (HN12), using the cDNA microarray. We identified 46 clones cDNA exhibiting more than 2 fold overexpression in DFO treated IHOK and HN12 cells, and 94 cDNA reveal more than 2 fold down-regulated expression. Examination of gene expression that differs between DFO treated vs. control IHOK and HN12 cells apprear to be related to : cell cycle regulator, cell growth and apoptosis, signal transduction and stress. p21 for cell cell cycle factor was upregualted, and cyclin-cdk gene was decreased expression, so we observed cell cycle arrest in DFO treated IHOK and HN12 cells. In tumor growth, we have identified downregulation of hemidesmosomal protein (bullous pemphigoid antigen 1) and epiregulin expression in DFO treated IHOK and oral cancer cells. Signal transducers including mitogen-activated protein kinase-activated protein kinase 5, serine/thereonine kinase 6 were downregulated with DFO treated cells, suggesting the DFO regulates the p38 MAP kianse pathway in immortalized and maignant oral keratincytes. In conclusion, we have demonstrated the high-throughput utility of cDNA array hybridization in parallel to the gene expression analysis to identify genes that are expressed differentially in DFO treated with immortalized and malignant oral keratinocytes. The differentially expressed genes identified here should be informative in DFO-induced anti-cancer effects.

The purpose of this study was to examine the effects of vi tamin D3 and 1'etinoic acid(RA) on the human mesenchymal stem ce!ls(MSC) g1'owth and osteogenic differentiations. Cell proliferation, mineralization, cell cycle, expression of cell cycle regu l atOJγ proteins and markers fo1' osteogenic differenatiaiton were determined by MTI assay, mineralization assay, flow cytomet1'Y‘ and Western blot analysis, respectively. Cell viability was dec1'ease by each vitamin D3 and RA added to MSC. it was more decrease by vitamin D3 and RA. Mineralized nodule formation revealed similar expression pattern with positive cont rol group at vitamin D3 and RA mixed add to MSC. At vitamin D3 and RA mixed add to MSC after 7 days of incubation was increase G1 s tage. after 21 days of incubation was inhibit cell cycle prog1'ess by inc1'ease of sub-G1 Treatment vitamin D3 to MSC inhibits p53 and p21, but inc1'ease pRb. RA inhibit p53, but increase p21 and pRb, vitamin D3 plus RA group was same as added RA group. so two vitamin was effect to inhibited cell growth each different mechanism. Expression of BMP-2 protein was prominent in osteogonic supplement treated g1'oup of MSC at 2 weeks cultivation days, but vi tamin D3 treatment decreased BMP-2 expression rather than in (+) control group. BSP protein was notably increased in the OS compa red to positive controls at 2 weeks cultivation, but similar to that of vitamin D3 group t1'eatment group and was least expressed in plus RA mixed group, at 3 weeks, BSP expression was similar to 1'esult of 2 weeks Collectively, these results shows that vitamin D3 and RA have diffe1'ential effects on the MSCs g1'owth and differ entia tion 211

We have examined the effect of NO donor, S-nitl‘ oso-N-acetyl-DL-penicillamine(SNAP) on heme oxygenase-1 (HQ-l) ex pression in human oral immortalized & malignant keratinocytes, and investigated in the control of keratinocyte proliferation evidence tha t HO-1 cou ld be involved in a low dose of NO, NO inhibitor, HOinducer, and HO inhibitor medi ated cytoprotect ion against cytotoxi city induced by a high dose of NO Oral keratinocyte growth inhibitory or anti-proliferative effects were exerted by with SNAP and hemin in a dose- and cul tivation time dependent manner The level of HQ-1 protein was increased in all cell types after exposure hernin dose, and the hemin induced HQ-1 protein achieved at higher maximum level by 12 hrs in all kind of cells , The pretreatment of cells with 0, 2 μ M SNAP reduced 1 mM SNAP-induced death in IHOK and HN4 cells , These cytoprotective effects on high dose of NO induced HQ-1 expresion and cell ular toxicity were blocked by low dose of SNAP, HCB, and ZnPP IX supporting the involvement of HQ-1 in high dose NO induced growth arrest or cell death, But these cytoprotection pattern is different from immortalized and malignant keratinocytes , These results indirectly demonstrate that HQ-1 could be involved in cytoprotection by NO priming against high dose NO induced cytotoxicity in immortalized and maigla nt oral keratinocytes, Thus, HQ-1 might be an important cellular target of NO donor, with clinical implications for the pre vention of inJlammatory di seases and anti-tumor immunity

In order to perform the protein analysis using the paraffin sections previous fixed with formalin, we applied the ImmunoMemBlot (IMB) method1) to detect the epitopes of target proteins with specific antibodies. In this study the protein extracts were obtained from the paraffin sections of each representative case of ameloblastoma, adenomatoid odontogenic tumor (AOT), and normal gingiva, and more a protein extract from fresh tissue of ameloblastoma was also compared to evaluate the IMB results used with 24 different antibodies. First of all, in the comparison between the paraffin section extract and fresh tissue extract of ameloblastoma, the latter consistently showed more positive IMB reaction than the former. Meanwhile, the paraffin section extract of ameloblastoma was more comparable with that of normal gingival, disclosing that most of proliferating genes, oncogenes, and apoptosis related genes, i.e., PCNA, CDK4, c-erbB2, CEA, p53, Bax, Bad, FLIP, FAS, Bcl-2, p21, N-ras, MMP-2, MMP-9, caspase-3, -8, -9, were highly expressed in ameloblastoma, but EGFR, HGF, and VEGF were similarly expressed both in the ameloblastoma and in normal human gingiva. On the other hand, the comparison between ameloblastoma and AOT both in the immunohistochemistry and IMB using their paraffin section extracts clearly demonstrated that the ameloblastoma showed more expression of proliferating genes and oncogenes while the AOT showed more expression of apoptosis related genes, i.e., Bax, Bad, FLIP, and caspase-9. Taken together, these data suggest that the IMB can be used for the primary screening of quantitative protein analysis using the paraffin section extract, and that the IMB results could be evaluated in conjunction with the immunohistochemical observation.

구강암의 발생원인 중 하나인 인간유두종바이 러스(HPV)로 불멸화시 킨 구깅 각화세 포(1 HOK) 외 정상 인긴 구강각화세 포 (NHOK) 의 표지자를 비 교 연구하는 것은 정상과 그 전암냉소의 생화학적 및 세 포회 학적 띤호l을 평가히는 적젤힌 모탤이지띤 떤 구된바 많지 않았다 본 연구는 구강 각화 세 포의 형 질전환을 조절히는 분자적 상태를 연구하기 위 해 익 6000711 의 유전자가 pri nt된 cONA mì croarray를 이용하여 인간 정상 구강각화세 포(NHOK) 와 HPV16으로 불띨회한 각화세 포(J HO K) 간에 유전지 발현을 비교하였다， NHOK외 IHOK세포는 96%의 유전자가 유사한 발현을 보였으며 2배이 상의 발현을 보이 는 경우-는 NHOK가 IHOK에 비해 85개 유전지가‘ IHOK가 쩌OK에 비해 1477H 의 유전자 발현이 u p-regul at i on되 어 총 4%미 만이 발현차이를 보 였고 반복된 hybridì zation 으로부터 얻은 선택된 spot의 Pearson 싱관계수는 074 에서 091로 냐티 났다 NHOK외 IHOK간의 유전자 발현을 기능별로 분석한 결과 몇 가지 주요 발현 유전자 그룹을 확인하였는데 세포부착 및 인식 세포주기 초칠인지 세 포자떨사， 전사인지- 성장인자 및 수용기 ， 세포골격 및 세 포외기질 단백 . 신호진달 조절자 및 기 타 그룹으로 분류할 수 있었다 IHOK에서 는 세 포인식 인자 중 endothelin 1, collagen IV, fibronectin , SPR1 이 발현증가 되었고 CK7, POG1"2, F'G1"1"R2가 세 포골격 및 성장인지중에서 upregulation 되었지만 신호전달 인지중 발현증가된 것은 없었고 동일힌 유전자를 나타내 는 cJ ustered gene map을 그릴 수 있었다 따라서 이러힌 Illicroan‘ay를 이 용한 분자학적 표지자 얀구가 구강 임 빌임과정 에서 유전지발현 을 확인히는데 큰 도웅을 줄 수 있을 뿐 아니라 유전자 조절에 의힌 진단 에 후. 치 료의 정획성을 개선시 킬 수 있으리리 여겨진다

We studi ed the difTerential elTect of vitamins A, C, U. and E on normal human 이 al keratinocyte(NHOK) , HPV-16 E6E7 immor talized human oral keratinocyte(1HOK) , Oncogene transfected HPV-16 immortalized ce1ls(OTOK) , and two ol'al sq ua mous cell line(HNSCC30‘ HNSCC31) according to carcinogenesis stage. The vitamin effect was evaluated by morphology. ce ll viabi lity. a nd orgnaotypic culture Vitamin A has a greater negative effect on growth for all NHOK IHOK HNSCC. es pec ially N-Ras t rans fected IHOK, Vitamin D & E revealed no significant cell activity on NHOK lHOK, ad OTOK Vitamin C was found increased cell viability to IHOK and OTOK 1n primary oral squmaous cell ca rcino ma (HN30 ). vitam in 0 and C showing increased cell growth , but vitamin E showing no effect 1n metastatic oral squamous cell ca rcinoma(HN31), vitamin C has prol iferative effect , but vitamin 0 & E has anti-proliferative effect Vitamin A t reated normal a nd ma lignant ce1ls by organotypic cu lt ure. showed reduction of epithelial layer and in vasion to connective tissue. , especia lly in 1HOK & oncogene-transfected 1HOK, 1n conclusion. three-dimensional culture sys tem may be useful as a model to acess the efficiency of agents such as a1l trans retinoic acid can preventing progression of these premaligant lesion to maligant oral carcinoma(ch emopreventive agent) .

Studies to evaluate distribution of markers in normal keratinocyte and their immortalized keratinocyte are appropriate to evaluate the normal and preneoplastic lesion of oral cancers as biochemical and cytochemical changes associate with tumorigenesis being not completely understood. Complementary DNA microarray containing 6000 sequence -verified cDNA elements was used to systematically characterize the variation in gene expression patterns of NHOK cells vs. immortalized keratinocyte by HPV16 E6-E7(IHOK). Examination of gene expression that is 85 clones cDNAs exhibits greater than 2 fold overexpression in NHOK probes relative to IHOK probe, 147 cDNAs reveal greater 2 fold overexpression in IHOK relative to NHOK probe.The high similarity in gene expression (96.5%) between IHOK and NHOK cells suggests that only an additional 232/6720 (3.5%) of the genome is differentially gene activated during HPV16 immoratlized keratinocyte growth and differentiation. Examination of gene expression that differs between NHOK and IHOK cellsapprear to be related to : cell adhesion & recognition, cell cycle regulator, apoptosis, transciption factors, growth factors and therir receptors, cytoskeletal and extracellular matrix proteins, signal transduction modulators and effectors, and miscellaneous. The gene expression of cell recognition factor such as endothelin 1, collagen IV, fibronectin, and SPR1 in IHOK were upregulated. Distinct or duplicated cDNA clones representing the same gene were typically clustered in adjacent rows in the clustered gene map. Therefore the differentially expressed and identified genes should be informative in studying oral epithelial cell carcinogenesis and such studies should foster the research of molecular markers allowing to assess the phenotypeof malignant epithelial tumor.

Nitric oxide (NO) has been known to inf1uence cell fate through apoptotic or necrotic cell death. Here, we investigated the role of nitric oxide on the growth and viability of immortalized human salivary gland (HSG) cells 띠 vitro. Treatrnent of HSG with a NO donor, S-nitroso-N-acetyl-DL-penκi1lamine (SNAP), significantly diminished the growth rate of HSG in a concentration dependent manner. However, this retardation of cell비ar growth rate was not corresponded to the apoptotic cell death of HSG cells, because there were no characteristic apopto디c features such as condensation of nuclear chromatin, nuclear fragmentation, and the apoptotic peak of propidium iodide (PI)-stained nuclei by flow cytome띠. 까ùs implies that HSG cells are resistant to NO-mediated 다π。to:잉city. 1n SNAP treated HSG cells, cell cycle analysis revealed that the number of G2/M phase increased markedly, according to while the percentage of cells in GO/Gl and S phases was not significantly affected. Otherwise, high concentrations of SNAP increased both P1 and annexin V positive cells. 1nterestingly, preincubation of HSG cells with iron chelator, deferoxamine (DFO), significantly diminished NO cytotoxicity more than when HSG cells are only incubated with SNAP which su잃.ests the role of iron homeostasis in NO-mediated cell death of HSG cells. 1n addi디。n ， treatrnent of HSG cells with SNAP specifically cleaved iron regulatory protein-2 (IRP2) while not affecting 1RP1. Collectively, the mπent results s멍gest that NO has a potential to control HSG cell growth through cell cycle arresting at G2/M phase. 1n addi디on ， intracellular iron homeostasis nùght play an important role in regulating cell survival of HSG cells

Epithelial-mesenchymal interaction is well known to have an importance during the organ development as well as cell growth and differentiation. However, in vitro experimental model is not well developed to reproduce in vivo cellular micro-environment which provide a epithelial-mesenchymal interaction. The aims of this study were to develop and evaluate the in vitro experimental model that maintains epithelial-mesenchymal interaction by organotypic raft culture, and to characterize biologic properties of three-dimensionally reconstituted human normal oral kertinocyte(NHOK) and immortalized human oral keratinocytes(IHOK) by histological and immunohistochemical analysis. The results were as follows; 1. Best condition of three dimensionally reconstituted IHOK & HaCaT cells are 14 days air-exposure cultivation, 3 days of submerged state, and dermal equivalent consisting type I collagen and IGF cells. 2. In comparison to IHOK, there was better preservation of the overall epidermal strucutures in oral cracioma cells (HN30) & HaCaT cells by organotypic cultures. But orgnaotypic co-culture of the normal keratinocyte showed the thinnest epithelial layer formation. 3. PCNA was detected primarily in the basal layer of normal mucosa and NHOK, whereas was shown throught the epithellium except surface layer of IHOK cells, and it's expression was similar to that of CIS of biopsied patient's tissue 4. Involucrin is expressed in the upper layer of oral mucosa and NHOK raft, but staining for involucrin was induced in the IHOK rafts indicating differentiation is incomplete, and the staining pattern in the IHOK raft was not uniform. 5. Normal oral keratinocyte raft showed weak immunostaining for p53, and p53 expression of IHOK raft increased rahter than in NHOK. In organotypic cultures of normal cells and IHOK, p53 expression was restricted to the proliferative part of epithelium. This is consistent with expression pattern in biopsy specimens of the normal and CIS tissue. 6. In artifically reconstructed NHOK, the pattern of keratin staining showed both similarity and differences from that of intact normal mucosa. An obvious difference was increased expression of CK10 & CK19, and decreased expression of CK6 in a reconstructed NHOK rather than in normal mucosa, and similar expression was in CK4 and CK16. 7. CK19 & CK16 were strongly positive in HPV immortlaized keratinocyte rafts rahter than in NHOK, an indicator of premalignant or malignant changes, while CK10 & CK6 were decreased, and organotypic cultures of IHOK express was similar keratin expression as epithelial dysplasia or CIS tissue. These results suggest that three-dimensional organotypic co-culture of normal oral & immortalized keratinocytes with dermal equivalent consisting type I collagen and fibroblasts results in similar morphologic and immunohistochemical characteristics to in vivo patient specimens. Thus this organotypic system can be used for studing mechanism of epitehlial-mesenchymal interaction in particular regulating epidermal diffenentiation and morphogenes

We used three-dimensional Matrigel culture system to examine the morphognesis of normal and malignant salivary glands cell in vitro including acinar cells(AC), myoepithelial cell(MC), salivary gland adenocarcinoma cells(SGT), mucoepidermoid carcinoma cells(MEC), and immortalized human salivary gland cells(HSG). For this purpose, normal and salivary gland tumor cells cultured in 3-D Matrigel, and characterized histologically and immunohistochemically, compared with same cells grown on monolayer culture and patient tissue from biopsy. 1. In three-dimensional Matrigel culture, HSG cells form acinar structure, SGT cells shows duct like structure, and other AC, MC and MEC cells dont' form any structure , and their morphology was different from that of monolayer cells. 2. Matrigel involved cell proliferation at a similar pattern to cells on plastic monolayer cell cultures, and monolayer cell revealed higher cell viability than that of Matrigel cultured cells. 3. All salivary glands cells on Matrigel or monolayer showed strong PCNA expression, and there is no expression difference in these cells. But some cells including myoepithelial cells in normal and salivary gland tumor tissue showing PCNA lavelling, so there is PCNA expression difference among normal and tumor tissue cells. 4. Actin expression was noted in AC cells on Matrigel, were rare expressed in the other cells except in MEC cells, and was present in myoepithelial cell and ductal cells of normal gland tissue. There is actin expression difference between tissue and cultured cells . 5. S-100 immunoreaction was moderateively positive in MC cells of monolayer culture, myoepithelial cells of normal tissue and pleomorphic adenoma, all cancer cells of mucoepidermoid carcinoma tissue, but significantly decreased in all salivary cells on Matrigel. 6. TGase 2 expression was prominent in MC cells of monolayer and Matrigel cultured, in myoepithelial cells of normal gland and pleomorphic adenoma, epidermoid cells of mucoepidermopid carcioma, and strong reaction in MEC and AC cells of monolayer and Matrigel cultured. 7. Expression of CK in monolayer culture showed strong reaction to CK6 in all sailvary gland cells, and mild reaction to CK10 and CK16 for all salivary cells, CK16 and CK19 expression in monolayer culture was similar to that of Matrigel culture. 8. CK6 and CK10 expression was strongest in AC and MC cells on Matrigel, and CK 4 was negative reaction in AC, SGT, MEC cells, strong reaction in MC cells but mild in SGT cells on Matrigel. Expression of CK was rare in HSG cells compared with other salivary gland cells, CK16 was prominent in SGT cells, CK10 and CK16 showed strongest expression in MEC cells of Matrigel. 9. Monolayer culture of HSG cell shwoing strong reaction to CK6, moderate to CK19 and mild to the others CK, but 3D cultured HSG cells reveal mild expression to CK16, and rare to others CK, intercallated duct in normal gland tissue showing strong to CK19, and mild to the others Ck, so there are CK expression difference in tissue, monolayer and 3-D cultured cells. 10. Monolayer culture of MEC cells represent strong reaction to CK6, mild to other CK, 3-D cells showing increased CK expression including CK6, epidermoid cells and intermediate cells in mucoepidermoid carcinoma tissue reveal positive to CK6 and CK16, mucous cell positive to CK10 and CK19, so Matrigel showed similar CK pattern compared to mucoepidermoid carcinoma tissue rather than monolThese data indicate that the interaction of salivary gland cells with basement membrane is an important factor in salivary gland development and cytodifferentiation, so this model system will be useful to study acinar or ductal differentiation in vitro.ayer cultred.