Human Oral Keratinocyte Cell Line Research Articles

Role of oxidative stress and MAPK signaling in reference moist smokeless tobacco-induced HOK-16B cell death

The use of smokeless tobacco products is often associated with an oral injury at the site of repeated use. To further our understanding of this injury process, the effect of reference moist smokeless tobacco extract (STE) on cell death, oxidative stress, and MAPK signaling in a human oral keratinocyte cell line, HOK-16B, was investigated. STE caused dose-dependent cell death and reactive oxygen species (ROS) production within 30 min to 3 h of exposure. This same insult enhanced the activity of ERK1/2, JNK1/2, p38 MAPK and ASK1, an upstream activator of JNK1/2 and p38 MAPK. Inhibition of JNK1/2 and to a lesser extent p38 MAPK, but not ERK1/2, suppressed STE-induced cell death. Pretreatment with antioxidants and an iron chelator, deferoxamine suppressed ROS production, ASK1, JNK1/2 and p38 MAPK activation, and reduced cell death after STE exposure. Interestingly, extracellular free iron levels in STE (29.4 ± 0.5 μM) were significantly elevated as compared with cell culture medium (4.9 ± 0.6 μM) and the addition of extracellular free iron (14, 30 or 70 μM) to HOK-16B cultures (without STE) caused dose-dependent cell death after 3 h. Thus, acute exposure to STE leads to HOK-16B cell death in part through oxidative stress via activation of ASK1 and the JNK1/2 and p38 MAPK pathways.

Reference moist smokeless tobacco‐induced oxidative stress in human oral keratinocyte cell lines leads to cell death via JNK and p38 MAPK signaling

The use of moist smokeless tobacco products is associated with acute oral injuries such as leukoplakia. Unfortunately, there is a limited understanding of the mechanisms and signaling pathways associated with these injuries. The present studies investigate the effects of a Reference 1S3 moist smokeless tobacco extract (STE) on oral injury, oxidative stress and MAPK signaling in multiple human oral keratinocyte cell lines utilizing cytospin slide analysis to observe alterations in cell morphology, DCF‐DA fluorescence, and western blotting techniques. STE caused dose‐dependent keratinocyte cell death within 30 minutes to 3 h of exposure. STE enhanced the activity of ERK1/2, JNK1/2, p38 MAPK and ASK1, an upstream activator of JNK1/2 and p38 MAPK. Inhibition of JNK1/2 or p38 MAPK, but not ERK1/2, suppressed STE‐induced cell death. STE promoted the generation of reactive oxygen species (ROS). The anti‐oxidants, Trolox and L‐ascorbic acid, suppressed ROS production, ASK1, JNK1/2 and p38 MAPK activation, and reduced cell death after STE exposure. Thus, reference STE leads to cell death in human oral keratinocyte cell lines in part through oxidative stress via activation of ASK1 and the JNK1/2 and p38 MAPK pathways.Research supported by Altria Client Services, 601 East Jackson St. Richmond, VA 23219 U.S.A.

Mucotoxicity of dental composite resins on a tissue-engineered human oral mucosal model

Objective Tissue-engineered human oral mucosal models have been developed for biocompatibility assessment of biomaterials. The aim of this study was to evaluate the biological effects of three different composite resin systems on a three-dimensional human oral mucosal model. Methods Full-thickness oral mucosal models were engineered by air/liquid interface culture of a human oral keratinocyte cell line on a lamina propria composed of oral fibroblasts seeded into a porous scaffold. The surface of the tissue models was exposed to three types of experimental composite resins: a TEGDMA-based, a UDMA-based, and a BisGMA/TEGDMA (80:20)-based composite resin for 24 h. The response of the engineered oral mucosa to the test materials was assessed using routine histology, the Alamar Blue tissue viability assay and IL-1β release measured by ELISA. Results Compared to the other materials tested, the TEGDMA-based composite resin caused significant damage to the oral mucosal model. Statistical analysis by one-way ANOVA followed by Tukey's analysis showed that there was a significant decrease in the viability of tissue models after 24 h exposure to TEGDMA-based composite resin. Also exposure to TEGDMA-based composite resin significantly increased the amount of IL-1β released from the oral mucosal model. Conclusion The 3D human oral mucosal model has the potential to be a more relevant and more informative model than monolayer cell culture systems for biocompatibility testing of dental materials. The results obtained from multiple-endpoint analysis of the oral mucosal model indicate significant mucotoxicity of high TEGDMA-containing composite resins.

MICA and MICB overexpression in oral squamous cell carcinoma

The concentration of MICA in serum seems be a good candidate marker in cancer. Previous studies from our laboratory have shown that the polymorphic MIC gene may confer a risk for oral squamous cell carcinoma (OSCC). The study investigated the expression levels of MICA and MICB of OSCC patients and cancer cell lines. We used RT-PCR to analyze the mRNA expression of MICA and MICB in four oral cancer cell lines compared with three normal human oral keratinocyte (NHOK) cell lines and in tissues from 36 patients with OSCC comparing tumor tissue with non-cancerous matched tissue (NCMT). Endogenous MICB mRNA expression in OSCC cell lines was significantly higher than that in NHOK (1.40 +/- 0.27 vs. 0.40 +/- 0.16; P = 0.04). In 20 of 36 sets of tissue from patients with OSCC, MICB mRNA expression was higher in the cancerous tissue than in the NCMT. The mean MICB mRNA expression in OSCC tissues was significantly higher than in NCMT (0.39 +/- 0.08 vs. 0.14 +/- 0.03, P = 0.009, paired t-test). A significantly lower MICA mRNA was found in patients who chewed areca nut compared with those who did not use areca (P = 0.001) and in patients with well-differentiated tumors compared with those with less well-differentiated tumors (P = 0.02). MICA and MICB mRNA expression may be increased in OSCC but there appears to be individual variation.

Development of a highly reproducible three-dimensional organotypic model of the oral mucosa

In this report we describe the development of a standardized three-dimensional (3D) system of the human oral mucosa based on an immortalized human oral keratinocyte cell line (OKF6/TERT-2). The procedure takes approximately 2-3 weeks to complete and includes three main stages: preparation of collagen-embedded fibroblasts, addition of the mucosal component and airlifting of cultures to ensure adequate differentiation/stratification. This procedure results in a multilayer epithelial structure in which layers are organized similarly to the cells in native oral mucosa. Specifically, this model system consists of a stratum basale, having one layer of columnar to round cells, a relatively flattened stratum spinosum and stratum granulosum, and a non-keratinizing stratum corneum. This 3D system resembles the commercially available system based on the cell line TR146 (SkinEthic), with the exception that our model system does not contain dyskeratotic changes and has a submucosal component, and thus better represents the normal human mucosa and submucosa.

The effect of cyclosporin on cell division and apoptosis in human oral keratinocytes

Gingival overgrowth (GO) is a side-effect of cyclosporin A (CsA) therapy and is characterised by enlargement of the gingiva with epithelial thickening and overproduction of extracellular matrix components. The pathogenesis of the epithelial thickening in GO remains obscure. The objective of the present study was to investigate the effects of CsA on the growth of oral epithelial cells in vitro and to test the hypothesis that CsA influences apoptosis in these cells. Cyclosporin was cocultured with an immortalized normal human oral keratinocyte cell line (HOK-16B), an epitheloid cervical carcinoma cell line (HeLa) and primary oral keratinocytes. Cell division was quantified using a CyQUANT kit. Apoptosis was induced using tumour necrosis factor-alpha (TNF-alpha) and assayed by analysis of caspase-3 activity. Expression of the anti-apoptotic protein, Bcl-2, was measured by western blotting. CsA exhibited a dose- and time-dependent inhibition of cell division in all three keratinocyte cell cultures. Significantly, HOK-16B cells treated with high doses of CsA (10 alphag/ml) did not recover their proliferative capacity 3 d after withdrawal of CsA, indicating that CsA-induced inhibition of growth is not temporary. Concentrations of CsA that inhibited cell division (1 microg/ml) did not have any effect on constitutive or TNF-alpha -induced apoptosis or Bcl-2 expression in HOK-16B cells. CsA inhibits oral epithelial cell division and this effect is not associated with changes in apoptosis in these cells. The action of CsA on oral epithelial cells may be associated with a long-lasting stress signal, which might account for some of the pathological effects of this drug.

A human oral keratinocyte cell line responds to human heat shock protein 60 through activation of ERK1/2 MAP kinases and up- regulation of IL-1β

Heat shock proteins (HSP) are released by cells in response to stress signals. It is hypothesized that pathogenic bacteria stimulate the cells in the periodontium to up-regulate the expression of HSP60, which would stimulate macrophages, and possibly other cells, to produce proinflammatory cytokines. We sought to determine whether oral keratinocytes responded to recombinant human HSP60 and to identify the signalling pathways involved. In addition, whether oral keratinocytes are a source of endogenous HSP60 was also investigated. RT-PCR revealed that rhHSP60 induced expression of the IL-1beta gene in the Human Oral Keratinocyte (HOK-16B) cell line and it was highest at the lowest concentration used (0.1 microg/ml). These responses were mediated via activation of p44/42 MAP-kinases and to a lesser extend the MAP-kinase SAP/JNK. Similar data was obtained from analysis of intracellular signalling pathways in HOK-16B cells by rhHSP70 and LPS (from both E. coli and the oral pathogen Porphyromonas gingivalis). However, there was little activation of p38 by rhHSP60. Blocking of the p44/42 pathway decreased HSP60-induced IL-1beta gene expression and protein secretion. In addition, we discovered that self-HSP60 proteins were constitutively secreted by HOK-16B cells. Secretion of self-HSP60 was up-regulated in cells treated with LPS from P. gingivalis, but down-regulated with LPS from E. coli. To summarize, oral keratinocytes respond to exogenous HSP60 by triggering expression of the inflammatory cytokine IL-1beta through activation of p44/42 MAP kinase. Oral keratinocytes are also a source for self-HSP60 and the secretion of this protein may be differentially modified by LPS from different bacterial species. These results highlight the importance of oral keratinocytes and HSPs in the development of an immune response against bacterial infection.

Presentation of ICAM-1 Protein at the Cell Surface of Oral Keratinocytes in the Presence of Adrenomedullin and Corticotrophin

Increasing evidence suggests that adrenomedullin (AM) and corticotrophin (ACTH) are immunomodulatory. Intercellular adhesion molecule-1 (ICAM-1) plays an important role in the recruitment of leukocytes not only from peripheral blood into inflamed tissues but also into epithelia. We have investigated the effects of AM and ACTH on the expression of ICAM-1 by human oral keratinocytes. The human oral keratinocyte cell line H357 was incubated with either AM or ACTH for up to 8 hrs and ICAM-1 expression was measured by cell surface ELISA. ICAM-1 was up regulated by both peptides and this was attenuated by the adenylyl cyclase inhibitor SQ22,536 and the NF-κB inhibitor SN-50. H357 cells constitutively express ICAM-1 mRNA and expression of this gene was significantly modulated by AM and ACTH. Furthermore AM caused translocation of NF-κB from the cytoplasm to the nucleus. This is the first report describing up regulation of ICAM-1 in oral keratinocytes by AM and ACTH and the results suggest both cAMP and NF-κB may play a role. These results further suggest both peptides may have an immunostimulatory role in oral muocsa and skin.

Global gene expression profiles of human head and neck squamous carcinoma cell lines.

For genomewide monitoring and identification of biomarkers of head and neck squamous cell carcinoma (HNSCC), we have conducted a systematic characterization of gene expression profiles, using human cDNA microarrays containing 9K clones, in 25 HNSCC cell lines and 1 immortalized human oral keratinocyte cell line. We used normal human oral keratinocytes (NHOKs) as a reference. Our study showed that genes primarily involved in cell cycle regulation, oncogenesis, cell proliferation, differentiation, apoptosis and cell adhesion were widely altered in the 26 cell lines. Upregulated genes included known oncogenes, protein kinases, DNA-binding proteins and cell cycle regulators, while those commonly downregulated included differentiation markers, cell adhesion proteins, extracellular matrix proteins, structural proteins (keratins) and protease inhibitor proteins. Compared to NHOK, we observed a striking reduction in the expression of genes involved in terminal differentiation, suggesting that a loss in this process is an important signature of HNSCC. In addition, hierarchical clustering analysis as well as principal component analysis revealed 2 distinctive subtypes of gene expression patterns among the 26 cell lines, reflecting a degree of heterogeneity in HNSCC. By applying significance analysis of microarrays, 128 genes were selected for being distinctively expressed between the 2 groups. Genes differentially expressed in the 2 subgroups include cell proliferation-related genes, IGFBP6, EGFR and VEGFC; tumor suppression and apoptosis-related genes such as Tp53, Tp63; as well as cell cycle regulators such as CCND1 and CCND2 (cyclins D1 and D2), suggesting that the 2 subgroups might have undergone different pathways of carcinogenesis.

Metastatic dissemination of human malignant oral keratinocyte cell lines following orthotopic transplantation reflects response to TGF-beta 1.

This study examined the behaviour of nine human malignant oral keratinocyte cell lines following orthotopic transplantation to the floor of the mouth of athymic mice. Tumourigenesis, local spread, and metastatic dissemination were correlated with known cellular responses to transforming growth factor-beta 1 (TGF-beta 1). Six of nine cell lines were tumourigenic; four of these cell lines showed local spread which was characterized by vascular and bone invasion. Metastatic spread was uncommon, with only 9% of animals with primary tumours developing metastases and these were almost exclusively found in the regional lymph nodes; there was one pulmonary metastasis and no liver deposits. Tumour cell behaviour did not reflect the clinical stage of the original tumours. Cell lines that were resistant to TGF-beta 1-induced growth inhibition were more likely to form primary tumours, exhibit local spread, and metastasize than cells that were growth-inhibited by the ligand. The data demonstrate that tumourigenicity and tumour behaviour in this orthotopic mouse model varied between cell lines and that the pattern of local invasion and metastasis was similar to that seen in human oral cancer. Furthermore, cell lines that were refractory to the growth inhibitory effects of TGF-beta 1 behaved more aggressively than cells that underwent ligand-induced cell-cycle arrest.

Functional significance of MMP-2 and MMP-9 expression by human malignant oral keratinocyte cell lines

This study examined the expression of MMP-2 and MMP-9 in normal and human malignant oral keratinocytes. The expression of pro-MMP-2 and pro-MMP-9 was heterogeneous in the malignant cell lines. Normal oral keratinocytes expressed less pro-MMP-2 and more pro-MMP-9 than their malignant counterparts. Cells that expressed high levels of both MMP-2 and MMP-9 showed the greatest degree of invasion through Matrigel in vitro compared to cells with either low or variable levels of these enzymes; normal keratinocytes were non-invasive in these conditions. The degree to which the cells invaded through Matrigel was similar to their motility in the absence of Matrigel and was not influenced by the activation of the pro-enzymes or the inhibition of enzyme activity using a chemical inhibitor of gelatinases. Cells were transplanted orthotopically to athymic mice and demonstrated a variable capacity not only to form tumours at the site of inoculation but, also, to metastasise; normal oral keratinocytes were non-tumorigenic. There was no correlation between the expression of either MMP-2 or MMP-9 and the tumorigenic/metastatic phenotype. The results emphasise the limitations of correlating in vitro and in vivo assays of tumour cell behaviour and suggest that invasion/motility in vitro may be a distinct phenotype from tumorigenicity/metastasis in vivo.

Loss of heterozygosity on chromosome 7q in in vitro-immortalized human oral keratinocyte cell lines

Loss of heterozygosity in two in vitro-immortalized human oral keratinocyte cell lines was analysed by polymerase chain reaction using 42 polymorphic microsatellite markers on chromosomes 4, 6, 7 and 15. These chromosomes are regarded as candidates for harbouring genes involved in the immortalization of human cells or tumour-suppressor genes in several tumours, including oral cancers, and karyotypic analysis has revealed that both cell lines have non-random alterations in these chromosomes. No allele losses were detected at any informative loci on chromosomes 4 and 6 in the cell lines, including genomic regions adjacent to putative human tumour-suppressor genes and putative senescence genes. When analysed for loss of heterozygosity on chromosomes 7 and 15, allele losses common to both cell lines were detected in the regions at 7q11.2, 7q21.1–21.3 and 7q31.1. High frequencies of loss of heterozygosity on chromosome 7q in at least two distinct regions, particularly centred around 7q31, are observed in a variety of tumours, including oral squamous-cell carcinoma, suggesting that multiple genes involved in immortalization of these cell lines might be present on chromosome 7q.

TGF-beta1 acts as a tumor suppressor of human malignant keratinocytes independently of Smad 4 expression and ligand-induced G(1) arrest.

This study examined the role of TGF-beta1 in human keratinocyte malignancy. Two carcinoma-derived human oral keratinocyte cell lines, BICR 31 and H314, were selected on the basis of their known resistance to TGF-beta1-induced G(1) arrest, the presence of wild type TGF-beta cell surface receptors and normal Ras. Smad 4 protein was undetectable in both cell lines, but Smad 2 and Smad 3 were expressed at levels comparable with a fully TGF-beta responsive cell line, and treatment of the cells with TGF-beta1 resulted in the phosphorylation of Smad 2. Treatment with exogenous TGF-beta1 resulted in a failure to induce transcription from an artificial Smad-dependent promoter and a failure to down-regulate c-myc, but resulted in an up-regulation of AP-1 associated genes (Fra-1, JunB and fibronectin). Transient transfection of Smad 4 into BICR 31 restored TGF-beta1-induced growth inhibition and Smad-dependent transcriptional activation. Protracted treatment of cells with exogenous TGF-beta1 resulted in the attenuation of cell growth in vitro. To over-express TGF-beta1, both cell lines were transfected with latent TGF-beta1 cDNA; neutralization studies of conditioned media demonstrated that whilst the majority of the peptide was in the latent form, a small proportion was present as the active peptide. Cells that over-expressed endogenous TGF-beta1 grew more slowly in vitro compared to both the vector-only controls and cells that did not over-express the peptide. Orthotopic transplantation of cells that over-expressed endogenous TGF-beta1 to the floor of the mouth in athymic mice resulted in marked inhibition of primary tumor formation compared to controls. Expression of a dominant-negative TGF-beta type II receptor in cells that over-expressed endogenous TGF-beta1 resulted in enhanced cell growth in vitro and diminished the tumor suppressor effect of the ligand in vivo, indicating that the endogenous TGF-beta1 was acting in an autocrine capacity. The results demonstrate that over-expression of endogenous TGF-beta1 in human malignant oral keratinocytes leads to growth inhibition in vivo and tumor suppression in vitro by mechanisms that are independent of Smad 4 expression and TGF-beta1-induced G(1) arrest.

An AP-1 binding site mutation in HPV-16 LCR enhances E6/E7 promoter activity in human oral epithelial cells.

Expression of the human papillomavirus (HPV) E6 and E7 oncoproteins is responsible for the transforming ability of the virus. The HPV long control region (LCR) and E6/E7 promoter regulate transcription of the E6 and E7 viral oncogenes. However, factors involved in the stimulation of E6/E7 promoter activity in carcinogenesis are unclear. We previously identified a point mutation in an HPV-16 immortalized human oral keratinocyte cell line subsequently exposed to a tobacco-specific carcinogen. This mutation was located in the LCR at nucleotide 7633 and contains binding sites for the transcription activator, AP-1, overlapping with putative binding regions for the transcription factor, C/EBP, which represses the E6/E7 promoter. In this study, this mutation was analyzed by both electrophoretic mobility shift analysis and luciferase assays. We found that the point mutation enhanced the binding affinity of AP-1 to the LCR, thus stimulating the E6/E7 promoter activity. Our results suggest that mutations in binding sites for crucial regulators may be the result of exposure to carcinogens and could induce expression of the E6 and E7 oncogenes.

Direct effects of corticotrophin on oral keratinocyte cell proliferation.

Corticotropin is produced by keratinocytes and may have an immunoregulatory role in oral mucosa and skin. We have investigated its effects on a human oral keratinocyte cell line and shown that corticotropin, acting via its specific receptor, stimulates a dose-dependent increase in DNA synthesis and induces cell proliferation. When cells were incubated in the presence of increasing concentrations of corticotropin, there were significant increases in intracellular cAMP levels. Corticotropin-stimulated mitogenesis and cell proliferation were attenuated by the adenylyl cyclase inhibitor SQ22,536, but were unaffected by inhibitors of protein kinase C or tyrosine kinase. These data identify corticotropin as a mitogenic regulatory peptide of keratinocytes acting via cAMP.

Normal human oral keratinocytes are more sensitive to N-methyl-N'-nitro-N-nitrosoguanidine-induced cytotoxicity and apoptosis than HPV-immortalized oral keratinocytes

Exposure of HPV-immortalized, but not normal human oral keratinocytes, to the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) renders the cells tumorigenic. The underlying mechanism of this differential response of normal and immortalized cells was investigated. Normal primary human oral keratinocytes and three HPV-immortalized human oral keratinocyte cell lines exposed to MNNG were evaluated for survival rate, single and double-strand DNA breaks, and the expression of p53 and bcl-2 proteins. MNNG exposure for 2 h induced both greater cytotoxicity and a more rapid kinetic of cell death in normal keratinocytes than in the immortalized cells. Further, normal keratinocytes were more sensitive to lower concentrations of MNNG that were subtoxic for the immortalized cells. Likewise, with lower concentration of MNNG (50 mu M), significant single-strand DNA breaks in normal keratinocytes were induced whereas no such effect was seen in the immortalized cells. Double-strand DNA fragmentation (apoptosis) was observed in normal keratinocytes exposed to 50 mu M MNNG but not in the immortalized cells. Higher concentrations of MNNG (100 mu M) were toxic to both normal and immortalized cells although the normal cells were still more sensitive and with faster kinetics of cell death. MNNG-induced apoptosis was not attributable to down regulation of the anti-apoptotic product bcl-2 in normal cells; however, exposure of normal keratinocytes to MNNG did result in induction of the apoptotic gene p53. No change in p53 level was seen in the immortalized cells. These findings suggest that the selective sensitivity of normal keratinocytes to MNNG-induced apoptosis is in part due to the induction of p53. The HPV-immortalized cells are resistant to MNNG-induced apoptosis and therefore are capable of undergoing mutations affecting cell proliferation and resulting in tumori-genicity.

Chemokine expression in human oral keratinocyte cell lines and keratinized mucosa.

Chemoattractant cytokines regulate the immune response within the tissue by recruiting neutrophils and macrophages. These so-called chemokines include a large family of peptide molecules encoded by distinct genes. Their expression is controlled by a variety of microbial and host factors. Among host factors, interleukin-1 (IL-1) is thought to be a key regulator of tissue destruction and mediator of the local immune response. To study its influence on chemokine expression, we used a highly sensitive, semi-quantitative method to assess gene expression at the level of mRNA. RNA was extracted from human oral keratinocyte cell lines after treatment with recombinant human IL-1. To test the method further and possibly establish a chemokine mRNA expression pattern, we also extracted RNA from healthy oral keratinized mucosa. Purified RNA was reverse-transcribed and subsequently amplified in a polymerase chain reaction (RT-PCR) by means of specific primer pairs. Amplified sequences were analyzed by agarose gel electrophoresis, visualized by ethidium bromide staining, transferred to nylon membranes, and hybridized to biotinylated oligonucleotide probes. Detection was achieved by streptavidin-conjugated alkaline phosphatase, a chemiluminescent substrate, and autoradiography. Autoradiographs were analyzed by densitometric measurements. IL-1 stimulation resulted in an increase of the chemokine mRNAs encoding interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), and GRO gamma. Macrophage inflammatory protein-1 alpha (MIP-1 alpha) mRNA was not detectable in keratinocytes. In healthy oral mucosa, we found considerable variation between the subjects. Detection of chemokine mRNAs by RT-PCR proved to be sensitive, specific, and fast. It allows for the study of not only cell-line-derived RNA, but also of RNA isolated directly from biopsy material. The latter feature makes this method well-suited for diagnostic purposes.

The effect of smokeless tobacco exposure on P53 expression in a human oral keratinocyte cell line

The effect of smokeless tobacco exposure on P53 expression in a human oral keratinocyte cell line