Primary Normal Human Oral Keratinocytes Research Articles

Granulocyte macrophage-colony stimulating factor and keratinocyte growth factor control of early stages of differentiation of oral epithelium.

Oral epithelial differentiation is known to be directed by underlying fibroblasts, but the responsible factor(s) have not been identified. We aimed here to identify fibroblast‐derived factors responsible for oral epithelial differentiation. Primary normal human oral keratinocytes and fibroblasts were isolated from healthy volunteers after informed consent (n = 5) and 3D‐organotypic (3D‐OT) cultures were constructed. Various growth factors were added at a range of 0.1‐100 ng/ml. 3D‐OTs were harvested after ten days and assessed histologically, by immunohistochemistry and the TUNEL method. Epithelium developed in 3D‐OT without fibroblasts showed an undifferentiated phenotype. Addition of granulocyte macrophage‐colony stimulating factor (GM‐CSF) induced expression of cytokeratin 13 in suprabasal cell layers. Admixture of GM‐CSF and keratinocyte growth factor (KGF) induced, in addition, polarization of epidermal growth factor (EGF) receptor and β1‐integrin to basal cell layer and collagen IV deposition. Terminal differentiation with polarization of TUNEL‐positive cells to superficial layers occurred only in the presence of fibroblasts in collagen gels either in direct contact or at distance from normal oral keratinocytes. Taken together, these results show that major aspects of oral epithelial differentiation are regulated by the synergic combination of GM‐CSF and KGF. However, the terminal stage seems to be controlled by other yet unidentified fibroblast‐derived diffusible factor(s).

Epithelial cells release IL-36α in extracellular vesicles following mechanical damage

The epithelium is an integral part of barrier tissues, and plays a critical role in the initiation of the innate immune responses. The pro-inflammatory cytokine IL-36α has been previously reported to be strongly expressed during oral mucosal wound healing, but regulation of IL-36α expression and secretion in the oral mucosa are not well known. The objective of this study was to determine the types of stimuli that lead to expression and secretion of IL-36α in epithelial cells. Maxillary tissues from C57BL/6J mice during wound healing were utilized to identify endogenous expression of IL-36α, β, and γ in oral epithelial tissue. Immortalized HaCaT cells and primary normal human oral keratinocytes were subjected to Escherichia coli derived lipopolysaccharide (LPS), Poly(I:C), heat killed Candida albicans (HKCa), and mechanical damage. IL-36α and IL-1β levels in supernatant were assessed by sandwich ELISA, and expression of pro-inflammatory cytokines and IL-36 family genes were assessed by quantitative real-time PCR in HaCaT cells. Migration ability of keratinocytes was assessed with or without functional IL-36 signaling. IL-36α but not IL-36β or γ levels in the oral epithelium were elevated during wound healing. Treatment of epithelial cells with LPS, Poly(I:C), HKCa and mechanical damage revealed little to no soluble IL-36α in the media supernatant. However, sonication of the supernatant to disrupt the membranes of extracellular vesicles revealed a dose-dependent increase in IL-36α for each of the tested conditions. IL-1 superfamily genes were upregulated following mechanical damage in keratinocytes. Abrogation of IL-36 signaling led to severe inhibition of migration. Our data show for the first time that IL-36α is released primarily in extracellular vesicles by oral keratinocytes. Additionally, we show that IL-36α – but not IL-36β or γ – is upregulated in keratinocytes following mechanical damage, and that IL-36 signaling is important for keratinocyte migration.

Rebamipide suppresses 5-fluorouracil-induced cell death via the activation of Akt/mTOR pathway and regulates the expression of Bcl-2 family proteins

Oral mucositis is a common adverse effect of chemotherapy that limits the required dose of chemotherapeutic agents. Numerous attempts to mitigate chemotherapy-induced oral mucositis have failed to identify an appropriate treatment. Recently, it has been indicated that rebamipide prevents chemoradiotherapy-induced oral mucositis in patients. However, the details of the underlying mechanism involved in the cytoprotective effect of rebamipide remain obscure. In the present study, we investigated the mechanism behind rebamipide cytoprotective effect in the oral mucosa using primary normal human oral keratinocytes (NHOK cells). We found that rebamipide prevented 5-fluorouracil (5-FU)-induced cell death in NHOK cells. In addition, rebamipide increased the levels of phosphorylated Akt and mTOR, enhanced the Bcl-2 and Bcl-xL expressions, and suppressed the expression of Bax and Bim. This is in contrast to 5-FU-induced suppression of Akt and mTOR activation, Bcl-2 and Bcl-xL expressions, and the enhanced expression of Bax and Bim. These findings suggest that rebamipide can potentially be used for the protection of oral mucosa from chemotherapy-induced mucositis. This is the first study that elucidates the specific molecular pathway for the cytoprotective effect of rebamipide.

Nano-TiO2 penetration of oral mucosa: in vitro analysis using 3D organotypic human buccal mucosa models.

BackgroundOral cavity is a doorway for a variety of products containing titanium dioxide (TiO2) nanoparticles (NPs) (nano‐TiO2) such as food additives, oral healthcare products and dental materials. Their potential to penetrate and affect normal human oral mucosa is not yet determined.ObjectivesTo evaluate the ability of nano‐TiO2 to penetrate the in vitro reconstructed normal human buccal mucosa (RNHBM).Methods RNHBM was generated from primary normal human oral keratinocytes and fibroblasts isolated from buccal oral mucosa of healthy patients (n = 6). The reconstructed tissues were exposed after 10 days to clinically relevant concentrations of spherical or spindle rutile nano‐TiO2 in suspension for short (20 min) and longer time (24 h). Ultrahigh‐resolution imaging (URI) microscopy (CytoViva™, Auburn, AL, USA) was used to assess the depth of penetration into reconstructed tissues.ResultsUltrahigh‐resolution imaging microscopy demonstrated the presence of nano‐TiO2 mostly in the epithelium of RNHBM at both 20 min and 24‐h exposure, and this was shape and doze dependent at 24 h of exposure. The depth of penetration diminished in time at higher concentrations. The exposed epithelium showed increased desquamation but preserved thickness.ConclusionNano‐TiO2 is able to penetrate RNHBM and to activate its barrier function in a doze‐ and time‐dependent manner.

Gene expression signatures affected by ethanol and/or nicotine in normal human normal oral keratinocytes (NHOKs).

It has been reported that nicotine/alcohol alters epigenetic control and leads to abrogated DNA methylation and histone modifications, which could subsequently perturb transcriptional regulation critically important in cellular transformation. The aim of this study is to determine the molecular mechanisms of nicotine/alcohol-induced epigenetic alterations and their mechanistic roles in transcriptional regulation in human adult stem cells. We hypothesized that nicotine/alcohol induces deregulation of epigenetic machinery and leads to epigenetic alterations, which subsequently affect transcriptional regulation in oral epithelial stem cells. As an initiating step we have profiled transcriptomic alterations induced by the combinatory administration of EtOH and nicotine in primary normal human oral keratinocytes. Here we provide detailed experimental methods, analysis and information associated with our data deposited into Gene Expression Omnibus (GEO) under GSE57634. Our data provide comprehensive transcriptomic map describing molecular changes induced by EtOH and nicotine on normal human oral keratinocytes.

Licochalcone A induces apoptosis in KB human oral cancer cells via a caspase-dependent FasL signaling pathway

Licochalcone A (Lico-A) is a natural phenol licorice compound with multiple bioactivities, including anti-inflammatory, anti-microbial, anti-fungal and osteogenesis-inducing properties. In the present study, we investigated the Lico-A-induced apoptotic effects and examined the associated apoptosis pathway in KB human oral cancer cells. Lico-A decreased the number of viable KB oral cancer cells. However, Lico-A did not have an effect on primary normal human oral keratinocytes. In addition, the IC50 value of Lico-A was determined to be ~50 μM following dose-dependent stimulation. KB oral cancer cells stimulated with Lico-A for 24 h showed chromatin condensation by DAPI staining, genomic DNA fragmentation by agarose gel electrophoresis and a gradually increased apoptotic cell population by FACS analysis. These data suggest that Lico-A induces apoptosis in KB oral cancer cells. Additionally, Lico-A-induced apoptosis in KB oral cancer cells was mediated by the expression of factor associated suicide ligand (FasL) and activated caspase-8 and −3 and poly(ADP-ribose) polymerase (PARP). Furthermore, in the KB oral cancer cells co-stimulation with a caspase inhibitor (Z-VAD-fmk) and Lico-A significantly abolished the apoptotic phenomena. Our findings demonstrated that Lico-A-induced apoptosis in KB oral cancer cells involves the extrinsic apoptotic signaling pathway, which involves a caspase-dependent FasL-mediated death receptor pathway. Our data suggest that Lico-A be developed as a chemotherapeutic agent for the management of oral cancer.

FOXM1 Induces a Global Methylation Signature That Mimics the Cancer Epigenome in Head and Neck Squamous Cell Carcinoma

The oncogene FOXM1 has been implicated in all major types of human cancer. We recently showed that aberrant FOXM1 expression causes stem cell compartment expansion resulting in the initiation of hyperplasia. We have previously shown that FOXM1 regulates HELLS, a SNF2/helicase involved in DNA methylation, implicating FOXM1 in epigenetic regulation. Here, we have demonstrated using primary normal human oral keratinocytes (NOK) that upregulation of FOXM1 suppressed the tumour suppressor gene p16INK4A (CDKN2A) through promoter hypermethylation. Knockdown of HELLS using siRNA re-activated the mRNA expression of p16INK4A and concomitant downregulation of two DNA methyltransferases DNMT1 and DNMT3B. The dose-dependent upregulation of endogenous FOXM1 (isoform B) expression during tumour progression across a panel of normal primary NOK strains (n = 8), dysplasias (n = 5) and head and neck squamous cell carcinoma (HNSCC) cell lines (n = 11) correlated positively with endogenous expressions of HELLS, BMI1, DNMT1 and DNMT3B and negatively with p16INK4A and involucrin. Bisulfite modification and methylation-specific promoter analysis using absolute quantitative PCR (MS-qPCR) showed that upregulation of FOXM1 significantly induced p16INK4A promoter hypermethylation (10-fold, P<0.05) in primary NOK cells. Using a non-bias genome-wide promoter methylation microarray profiling method, we revealed that aberrant FOXM1 expression in primary NOK induced a global hypomethylation pattern similar to that found in an HNSCC (SCC15) cell line. Following validation experiments using absolute qPCR, we have identified a set of differentially methylated genes, found to be inversely correlated with in vivo mRNA expression levels of clinical HNSCC tumour biopsy samples. This study provided the first evidence, using primary normal human cells and tumour tissues, that aberrant upregulation of FOXM1 orchestrated a DNA methylation signature that mimics the cancer methylome landscape, from which we have identified a unique FOXM1-induced epigenetic signature which may have clinical translational potentials as biomarkers for early cancer screening, diagnostic and/or therapeutic interventions.

Downstream targets of FOXM1: CEP55 and HELLS are cancer progression markers of head and neck squamous cell carcinoma

We recently showed that upregulation of a key oncogene FOXM1 precedes head and neck squamous cell carcinoma (HNSCC) malignancy. Furthermore, we also identified a centrosomal protein CEP55 and a DNA helicase/putative stem cell marker HELLS, which are both downstream targets of FOXM1. In this study, we have investigated the expression profiles of CEP55 and HELLS using immunohistochemistry and quantified by digital densitometry in a tissue panel (20 samples) consisting of normal oral mucosa, dysplasias, HNSCC and lymph node metastasis (LnMet) samples. Furthermore, we corroborated our findings using absolute real-time PCR (qPCR) on a panel of 12 primary normal human oral keratinocytes, five dysplasia and 10 HNSCC cell lines. Finally, we validated our study using bioinformatics microarray analysis on an independent HNSCC patient cohort (four normal and 16 tumours). In normal oral mucosa, CEP55 protein was detected at very low level within the upper differentiated layers. In contrast, CEP55 was highly expressed in oral dysplasia whereas only moderate expression was detected in HNSCC and LnMet. Low level of HELLS expression was detected in the basal cell layer of the normal oral mucosa, moderate level was seen in dysplasia and high levels in both HNSCC and LnMet. These expression patterns were consistent with both qPCR data from the cell line panel and microarray data analysis of TNM-stage defined HNSCC samples confirming the progressive expression pattern of CEP55 and HELLS. To our knowledge, this is the first pilot study demonstrating that both CEP55 and HELLS mRNA and protein expression positively correlate with pre-malignancy and HNSCC progression. This study provides strong evidence that CEP55 and HELLS may be used in conjunction with FOXM1 as a biomarker set for early cancer detection and indicators of malignant conversion and progression.

Adverse effects of Sudanesetoombakvs. Swedish snuff on human oral cells

The high incidence of oral cancer in Sudan has been associated with the use of toombak, the local type of smokeless tobacco. However, its specific effects on human oral cells are not known. We aimed to investigate the effects of toombak on primary normal human oral keratinocytes, fibroblasts, and a dysplastic oral keratinocytic cell line, and to compare them with the effects induced by Swedish snuff. Aqueous extracts were prepared from moist toombak and Swedish snuff and added in serial dilutions on in vitro monolayer cultured cells. Cell viability, morphology and growth, DNA double-strand breaks (gammaH2AX staining), expression of phosphatidylserine (Annexin V staining), and cell cycle were assessed after various exposure time periods. Significant decrease in cell number, occurrence of DNA double-strain breaks, morphological and biochemical signs of programmed cell death were detected in all oral cell types exposed to clinically relevant dilutions of toombak extract, although to a lesser extent in normal oral fibroblasts and dysplastic keratinocytes. G2/M-block was also detected in normal oral keratinocytes and fibroblasts exposed to clinically relevant dilutions of toombak extract. Swedish snuff extract had less adverse effects on oral cells, mainly at non-clinically relevant dilutions. This study indicates a potential for toombak, higher than for Swedish snuff, to damage human oral epithelium. Dysplastic oral keratinocytes were less sensitive than their normal counterparts, suggesting that they might have acquired a partially resistant phenotype to toombak-induced cytotoxic effects while still being prone to DNA damage that could lead to further malignant progression.

Cancer progression is associated with increased expression of basement membrane proteins in three-dimensional in vitro models of human oral cancer

Background Although basement membrane was traditionally considered an inert barrier that tumour cells had to cross before invasion into the surrounding stroma, recent studies suggest that basement membrane components are not only degraded during tumour progression, but also newly synthesised at the invasive front. Objective This study aimed at evaluating (1) the expression of basement membrane proteins in human oral carcinogenesis and (2) the role that epithelial–mesenchymal interactions play on it, by using an in vitro oral cancer progression model. Material and methods In vitro three-dimensional (3D) organotypic cultures of normal, early neoplastic and neoplastic human oral mucosa were developed by growing primary normal human oral keratinocytes, dysplastic human oral keratinocytes (DOK cell line), and neoplastic human oral keratinocytes (PE/CA-PJ15 cell line) on type I collagen biomatrices, with or without primary fibroblasts isolated from normal human oral mucosa. The cultured tissues were immunohistochemically assessed for the expression of the major basement membrane proteins laminin-332, type IV collagen, and fibronectin. Results Expression of laminin-332, type IV collagen, and fibronectin was gradually more pronounced in neoplastic models when compared to normal mucosa models, and, with the exception of laminin-332, it was further enhanced by presence of fibroblasts. Deposition of type IV collagen at the epithelium–biomatrix interface occurred only in presence of fibroblasts, as well as the extracellular matrix deposition of fibronectin. Conclusions These findings, obtained in a 3D in vitro model that closely mirrors the in vivo human oral cancer progression, show an enhanced basement membrane protein expression during human oral cancer progression that is dependent on the epithelial–mesenchymal environment, respectively the existence of fibroblasts.

Early loss of mitochondrial inner transmembrane potential in khat-induced cell death of primary normal human oral cells

Previous studies suggest the use of khat, a psychostimulant plant used by millions of people in Middle East and Africa, as risk factor for oral cancer. We previously reported that khat is able to induce adverse affects, as cell cycle arrest and apoptosis, in normal human oral cells cultured in vitro. This study further investigates the more specific role played by mitochondria in khat-induced cell death and the kinetics of the events involved in this process. Exposure of primary normal human oral keratinocytes and fibroblasts to khat extract resulted in a swift and sustained decrease of the mitochondrial inner transmembrane potential occurring within 0.5–1 h. Loss of mitochondrial membrane potential preceded all other biochemical and morphologic changes, and was associated with a significant decrease in cell survival. Subsequently, apoptosis-inducing factor was released from mitochondria into cytosol and relocated to nucleus. Cyclosporine A and bongkrekic acid delayed both the loss of mitochondrial inner transmembrane potential and the onset of cell death. This study describes a novel mechanism of khat-induced cell death in primary normal oral keratinocytes and fibroblasts involving an early pivotal effect on mitochondrial function and integrity.

Khat (Catha edulis) Induces Reactive Oxygen Species and Apoptosis in Normal Human Oral Keratinocytes and Fibroblasts

Khat chewing is widely practiced in Eastern Africa and the Middle East. Khat is genotoxic to cells within the oral mucosa, and several studies have suggested an association between khat use and oral lesions like hyperkeratosis and oral cancer. This study investigated the mechanism of khat-induced cytotoxicity using primary normal human oral keratinocytes (NOK) and fibroblasts (NOF). Khat induced rounding up of cells, plasma membrane blebbing, and condensation of nuclear chromatin within 3-6 h of exposure. The cells also showed externalization of phosphatidylserine and fragmentation of DNA. Morphological and biochemical features were compatible with cell death by apoptosis. Khat also induced an increase in cytosolic reactive oxygen species (ROS) and a depletion of intracellular glutathione (GSH) within 1 h of exposure. Antioxidants reduced ROS generation, GSH depletion and delayed the onset of cytotoxicity in both cell types. Generally, NOF cells were more sensitive to khat-induced cytotoxicity than NOK cells. These effects were elicited at concentrations of khat expected to occur in the oral cavity during khat chewing. In summary, khat induced apoptotic cell death in primary normal oral keratinocytes and fibroblasts by an early effect on mechanisms that regulate oxidative stress.

Khat induces G1‐phase arrest and increased expression of stress‐sensitive p53 and p16 proteins in normal human oral keratinocytes and fibroblasts

Khat is a psychostimulant plant used by over 10 million people daily, mainly in eastern Africa and the Middle East. Previous studies have suggested an association between khat use and oral lesions such as hyperkeratosis and oral cancer. This study investigated the effects of an extract of khat on primary normal human oral keratinocytes (NOK) and normal human oral fibroblasts (NOF). Low (sublethal) concentrations of khat inhibited the proliferation of both cell types in a dose-dependent and time-dependent manner. Both NOK and NOF treated with khat accumulated in the G1-phase of the cell cycle and showed increased expression of the stress-sensitive p53 protein after 24 h. Normal human oral keratinocytes showed a profound increase in p16(INK4A) (p16) after 24 h and showed morphological changes suggesting cell differentiation. Normal human oral fibroblasts showed growth inhibition and increased expression of p21(WAF1/CIP1) (p21) within 24 h. The concentrations of khat tested in this study were within the range of those found in the oral cavity of khat chewers. The results show that stress induced by khat modulates the cell cycle in oral keratinocytes and fibroblasts. It is further speculated whether khat could have similar effects in vivo, especially in keratinocytes.

Dual effects of sodium lauryl sulphate on human oral epithelial structure

Sodium lauryl sulphate (SLS) is a common detergent known to cause irritation and inflammatory reactions in skin. SLS is also the most commonly used toothpaste detergent and has been related to intraoral adverse effects. However, its specific biological effects on the oral mucosa (OM) have not yet been identified. The objective of this study was to investigate the putative effects of SLS on human oral epithelium using a novel in vitro reconstructed three-dimensional cell culture model. Reconstructed human OM, generated from primary normal human oral keratinocytes and fibroblasts, was exposed to clinically relevant concentrations of SLS (range 0.015-1.5%). The cultured tissues were evaluated by histomorphometry, immunohistochemistry (Ki-67, epithelial (E)-cadherin, alpha6-, beta1-integrins, cleaved caspase-3) and the TUNEL method. Increased epithelial thickness, enhanced proliferation (Ki-67), a more pronounced expression of E-cadherin throughout all epithelial cell layers and single TUNEL-positive cells in the middle spinous cell layers were observed in cultures exposed to low concentrations (0.015%) of SLS. At exposure to higher SLS concentrations (>or=0.15%), epithelial thickness, cell proliferation and E-cadherin expression gradually decreased and in the central areas of exposed regions, cells detached from each other and underwent cell death. In conclusion, clinically relevant concentrations of SLS have dual effects on reconstituted human OM; although occasional cell death within the epithelium was also observed, the increased epithelial thickness, proliferation and E-cadherin expression induced at lower concentrations might be associated with a protective mucosal response, whereas at higher concentrations a more destructive type of reaction predominated.

Fibroblast control on epithelial differentiation is gradually lost during in vitro tumor progression

This study aimed to investigate the role of underlying fibroblasts on morphogenesis of in vitro epithelium reconstituted with normal and neoplastic human oral keratinocytes at various stages of malignant transformation. Primary normal human oral keratinocytes (NOKs), early neoplastic/dysplastic human oral keratinocytes (DOK cell line), and neoplastic human oral keratinocytes (PE/CA-PJ 15 cell line) were organotypically grown on top of a collagen type I matrix with or without primary normal human oral fibroblasts. Morphogenesis of the reconstituted epithelia was assessed by histomorphometry, immunohistochemistry (Ki-67, cyclin D1, cytokeratin 13 (CK13), collagen IV, E-cadherin, p53, CD40), and the terminal deoxynucleotidyl transferase-mediated dUTP in situ nick end-labelling method. Reproducible in vitro models of multistage oral carcinogenesis were established. Presence of fibroblasts in the collagen matrix significantly increased cell proliferation in all three models (p<0.05), and induced an invasive pattern of growth in the neoplastic cell lines (p<0.05). In normal, but not in neoplastic oral keratinocytes fibroblasts induced the expression of CD40, and polarized the expression of E-cadherin and p53 to the basal cell layer. In both normal and early neoplastic keratinocytes (DOK cell line), fibroblasts induced the expression of CK13 and collagen IV. In the neoplastic oral keratinocytes (PE/CA-PJ 15 cell line), the presence of underlying fibroblasts did not change the expression of any of the protein markers assessed. This study showed that (1) major steps of oral carcinogenesis can be reproduced in vitro, and (2) the tight control exerted by fibroblasts on epithelial morphogenesis of in vitro reconstituted normal human oral mucosa is gradually lost during neoplastic progression.

Common genes responsible for differentiation and senescence of human mucosal and epidermal keratinocytes

Serial subcultures of normal human oral keratinocytes (NHOKs) and normal human epidermal keratinocytes (NHEKs) to the postmitotic stage result in terminal differentiation and replicative senescence. In order to investigate the common mode of differentiation and/or senescence between mucosal and epidermal keratinocytes, gene expression profiling on both NHOKs and NHEKs was performed by a cDNA microarray analysis. Primary NHOKs and NHEKs were serially subcultured, and the expression level of 3,063 genes was compared between the exponentially growing and senescent cultures. The senescent NHOKs and NHEKs highly expressed 55 and 37 genes, respectively. Among these genes, 16 genes were common in both NHOKs and NHEKs while the other genes were upregulated either in the NHOKs or in the NHEKs. Furthermore, the expression levels of the common genes did not change in the human diploid fibroblasts during the subcultures. These results suggest that subculture-induced differentiation and/or replicative senescence in NHOKs and NHEKs has similar characteristics, but that the pathways leading to these processes are distinct and keratinocyte specific.

Senescence-associated genes in normal human oral keratinocytes

The current study was undertaken to identify senescence-associated (SA) genes in cultured normal human oral keratinocytes (NHOK). Primary NHOK were serially subcultured in vitro as dispersed cells in low (0.15 mM) Ca 2+ medium until senescence. The SA genes of NHOK were identified by comparing the expression levels of 3195 human genes between exponentially replicating and senescing cultures. Approximately 5% of the screened genes were upregulated in senescing NHOK by a factor greater than 3 compared with rapidly dividing NHOK culture. Among them, we identified discrete gene groups, i.e., cyclin-dependent kinase inhibitors, G-protein-coupled receptors, apolipoproteins, matrix metalloproteinases, and mitochondrial proteins. To validate the microarray results, we confirmed the enhanced expression of a few selected SA genes, i.e., gpr1, apo-D, apo-E, apo-L, mmp-1, mmp-3, cyb561, cyp1b1, and cyp4b1, by reverse transcription-PCR. These SA genes were upregulated in three independent cultures of NHOK at high population doubling (PD) levels compared with those of low PDs. The enhanced expression of these SA genes was also found in senescing NHOK maintained in 3T3 feeder cell system, as well as in the chemically defined medium containing low Ca 2+. These results indicate that the onset of senescence in NHOK is associated with altered expression of the SA genes, which represent discrete gene groups, independently of the donor variation or culture conditions.

Phospholipase C-γ1 is required for subculture-induced terminal differentiation of normal human oral keratinocytes

Serial subculture of primary normal human oral keratinocytes (NHOKs) to the post-mitotic stage induces terminal differentiation, which is in part linked to elevated levels of phospholipase C (PLC)-gamma1. Therefore, PLC-gamma1 may be involved in the signal transduction system that leads to the calcium regulation of subculture-induced keratinocyte differentiation. To test this hypothesis, the expression of PLC-gamma1 in primary NHOKs was blocked by transfecting cells with the antisense PLC-gamma1 cDNA construct. These cells demonstrated dramatic reductions in PLC-gamma1 protein and in the differentiation markers involucrin and transglutaminase following calcium exposure and an increase (15-20%) in in vitro life span versus empty vector-transfected cells. In addition, we established the ability of antisense PLC-gamma1 to block the serial subculture-induced rise in intracellular calcium. Similar observations were made following treatment with the specific PLC inhibitor U73122. These results indicate that the terminal differentiation of NHOKs by serial subculture is associated with PLC-gamma1, which mediates calcium regulation by mobilizing intracellular calcium.

Analysis of herpes simplex virus entering into cells of oral origin

The entry of herpes simplex virus (HSV) into an oral epithelial cell line, primary normal human oral keratinocytes (NHOK) and gingival fibroblasts (GF) was examined. Infection of these cells by HSV-1 and HSV-2 was blocked by heparin. Further examination indicated that heparin reduced viral attachment but not penetration. Moreover, neomycin inhibited HSV-1 infection more effectively than HSV-2 infection in GF, but not in NHOK. In conclusion, our results elucidated some aspects of the HSV entry process into oral cells and revealed some differences in HSV entering into NHOK and GF.

Decreased expression of alpha3 and beta1 integrin subunits is responsible for differentiation-associated changes in cells behavior in terminally differentiated human oral keratinocytes.

Primary normal human oral keratinocytes (NHOKs) terminally differentiate in serial subculture. To investigate whether this subculture-induced differentiation of NHOKs affects integrin expression and cell-matrix interaction, we studied the expression levels of integrin subunits and cellular response to the extracellular matrix (ECM) proteins in NHOKs at different population doublings. The phosphorylation statuses of focal adhesion kinase (FAK), extracellular signal regulated kinase (ERK), p38, and c-Jun amino-terminal kinase (JNK) were also determined in NHOK cells cultured on ECM proteins, to evaluate the functions of integrins with respect to cellular responses to ECM proteins. The expression levels of alpha3 and beta1 integrin subunits progressively decreased in NHOKs undergoing terminal differentiation. The ability of NHOKs to spread upon laminin and type I collagen significantly decreased in terminally differentiated oral keratinocytes. Keratinocyte migration was significantly increased on type I collagen for terminally differentiated NHOKs. Similar results were seen following preincubation of rapidly proliferating NHOKs with function-blocking antibodies to alpha3 or beta1 integrin subunit. In contrast, fibronectin had no effect on cellular responses in NHOKs, which were almost negligible in the expression levels of alpha5 integrin subunits. The extent of FAK phosphorylation in terminally differentiated NHOKs was notably lower than that of rapidly proliferating cells, but was enhanced in terminally differentiated cells that were cultured on type I collagen. Our results indicate that decreased expression of alpha3 and beta1 integrin subunits is responsible for differentiation-associated changes in cells behavior in terminally differentiated oral keratinocytes. Our data also show that the abrogation of the alpha5beta1 integrin function caused by omitting alpha5 subunit is linked to the loss of a cell-fibronectin interaction in human oral keratinocytes.