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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.

Drug-induced osteonecrosis of the jaw (ONJ) is a detrimental intraoral lesion that often occurs after dental-related interventions in patients undergoing treatment with bisphosphonates or denosumab, the neutralizing human anti–receptor activator of NF-κB ligand (RANKL) antibody (Ab). The cause of ONJ by these drugs has been speculated to their direct effects on osteoclasts. However, the extent to which osteoclasts contribute to ONJ pathogenesis remains controversial. Herein, by using a tooth-extraction mouse model with i.v. administration of mouse anti-RANKL Ab or the bisphosphonate zoledronate (ZOL), we show that unresorbed bone due to impaired formation or suppressed functions of osteoclasts, respectively, is associated with ONJ development. After tooth extraction, ONJ-like lesions developed 50% in the anti-RANKL Ab-treated mice and 30% in the ZOL-treated mice. Nonviable and unresorbed bone was found more in anti-RANKL Ab-treated mice compared with mice receiving ZOL. All mice receiving anti-RANKL Ab had an undetectable tartrate-resistant acid phosphatase (TRAP) level in the serum and no TRAP-positive osteoclasts at the extracted sockets, whereas ZOL-treated mice had a decreased TRAP level without altering the numbers of TRAP-positive osteoclasts. Interestingly, the absence of newly formed woven bone in the extracted sockets was evident in ONJ-like lesions from both anti-RANKL Ab- and ZOL-treated mice. Our study suggests that the lack of osteoclasts' bone-resorptive functions by these drugs and suppression of woven bone formation after dental trauma may be associated with ONJ development.

Introduction Revascularization of necrotic pulp has been successful in the resolution of periradicular inflammation; yet, several case studies suggest the need for cell-based therapies using mesenchymal stem cells (MSCs) as an alternative for de novo pulp regeneration. Because the availability of MSCs may be limited, especially in an aged population, the current study reports an alternative approach in generating MSCs from epidermal keratinocytes through a process called epithelial-mesenchymal transition (EMT). Methods We induced EMT in primary normal human epidermal keratinocytes (NHEKs) by transient transfection of small interfering RNA targeting the p63 gene. The resulting cells were assayed for their mesenchymal marker expression, proliferation capacities as a monolayer and in a 3-dimensional collagen scaffold, and differentiation capacities. Results Transient transfection of p63 small-interfering RNA successfully abolished the expression of endogenous p63 in NHEKs and induced the expression of mesenchymal markers (eg, vimentin and fibronectin), whereas epithelial markers (eg, E-cadherin and involucrin) were lost. The NHEKs exhibiting the EMT phenotype acquired extended replicative potential and an increased telomere length compared with the control cells. Similar to the established MSCs, the NHEKs with p63 knockdown showed attachment onto the 3-dimensional collagen scaffold and underwent progressive proliferation and differentiation. Upon differentiation, these EMT cells expressed alkaline phosphatase activity, osteocalcin, and osteonectin and readily formed mineralized nodules detected by alizarin S red staining, showing osteo-/odontogenic differentiation. Conclusions The induction of EMT in primary NHEKs by means of transient p63 knockdown allows the generation of induced MSCs from autologous sources. These cells may be used for tissues engineering purposes, including that of dental pulp.

Somatic mutations within a mononucleotide repeat sequence present in the hMSH6 and hMSH3 coding regions have been frequently observed in various human cancer tissues and cell lines showing genomic instability. However, relatively few germline mutations of the repeat sequence have been identified. Two germline mutations in the hMSH6 region have been reported in hereditary nonpolyposis colorectal cancer (HNPCC); however, no germline mutations in the hMSH3 gene have been reported yet. To investigate genetic alterations within an 8 bp polycytosine repeat of the hMSH6 gene and an 8-bp polyadenine repeat of the hMSH3 gene, we amplified the mononucleotide repeat sequences of 35 HNPCC patients, 44 patients suspected of having HNPCC who did not ful-fill the criteria of the International Collaborative Group on HNPCC, and 45 patients with sporadic early-onset colorectal cancer who developed colorectal cancer before the age of 40 years without any family history of colorectal cancer. Genetic alteration of the repeat sequence of the hMSH3 gene was not observed, whereas germline frameshift mutations (one C insertion) in the hMSH6 gene were found in two of the 44 suspected HNPCC patients in whom germline mutations of hMSH2 or hMLH1 had not been detected. An identical frameshift mutation was also observed in another affected member of a suspected HNPCC family. These results suggest that the mutation of hMSH6 is responsible for tumorigenesis in minor groups of suspected HNPCC patients.

Defective hMLH1 function has been increasingly associated with acquired cellular resistance to DNA alkylation damage in human colorectal and endometrial cancer cells. To investigate the relationship between the DNA alkylation tolerance and the hMLH1 status in human gastric carcinoma cells, we determined the cellular response to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), the mutational changes, and the expression of hMLH1 in 11 human gastric carcinoma cell lines. Of 11 cell lines, 4 (SNU-5, -16, -620, and -719) were sensitive, whereas 7 (SNU-1, -216, -484, -520, -601, -638, and -668) were resistant to the cytotoxic effect of MNNG. As determined by Western analysis, it was evident that all the MNNG-resistant cell lines except one (SNU-601) produced very low or undetectable levels of hMLH1 protein compared to the MNNG-sensitive cell lines. A homozygous non-sense mutation that resulted in truncated protein was found in one MNNG-resistant cell line (SNU-1). Therefore, to determine whether the sensitivity of cells to MNNG can be restored by exogenous expression of hMLH1 protein, wild-type hMLH1 cDNA was introduced into the MNNG-resistant cells (SNU-1). The cytotoxicity test showed that expression of exogenous wild-type hMLH1 protein caused an increase in sensitivity to the cytotoxic effect of MNNG. This restoration was confirmed by an increase in the cell population containing less than the G1 amount of DNA (cell death) in the wild-type hMLH1-transfected cells, as determined by flow cytometry analysis. Together our results suggest that (1) the absence or decreased level of wild-type hMLH1 protein may be a frequent event in the human gastric carcinoma cell lines, (2) such alterations in the hMLH1 protein are closely associated with the MNNG tolerance in the human gastric carcinoma cell lines, and (3) the hMLH1 protein participates not only in the repair of DNA mismatches but also in the mechanism of escape from the cytotoxic effects of DNA alkylation damage.

Introduction Cell migration is an important step in pulpal wound healing. Although components in the resin-based dental materials are known to have adverse effects on pulp wound healing including proliferation and mineralization, their effects on cell migration have been scarcely examined. Here, we investigated the effects of 2-hydroxyethyl methacrylate (HEMA) on the migration of dental pulp stem cells (DPSC) in vitro. Methods Cell viability was assessed using the MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay, and cell migration was evaluated using the wound scratch assay and transwell migration assay at noncytotoxic doses. The Western blot was used to examine pathways associated with migration such as focal adhesion kinase, mitogen-activated protein kinase, and glycogen synthase kinase 3. Results There were no drastic changes in the cell viability below 3 mmol/L HEMA. When DPSCs were treated with HEMA at 0.5, 1.0, and 2.5 mmol/L, cell migration was diminished. HEMA-treated DPSCs exhibited the loss of phosphorylated focal adhesion kinase in a dose-dependent manner. The HEMA-mediated inhibition of cell migration was associated with phosphorylation of p38 but not glycogen synthase kinase 3, Extracellular signal-related kinase (ERK), or c-Jun N-terminal kinase (JNK) pathways. When we inhibited the p38 signaling pathway using a p38 inhibitor, the migration of DPSCs was suppressed. Conclusions HEMA inhibits the migration of dental pulp cells in vitro, suggesting that poor pulpal wound healing under resin-based dental materials may be caused, in part, by the inhibition of cell migration by HEMA.

Camphorquinone (CQ) is a photoinitiator that triggers polymerization of light-curing materials such as dental adhesives and composites. CQ does not become a part of the polymer network, suggesting that CQ can be leached out into surrounding environment including dental pulp and exert adversary effects on tissues. In order to understand the mechanisms of CQ-induced side effects, we investigated the effect of CQ on cell viability, cytokine secretion, and odontogenic differentiation of dental pulp stem cells in vitro.Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after CQ exposure. Western blotting was performed for p16(INK4A), p21(WAF1), and p53. Secretory cytokines were evaluated using the membrane-enzyme-linked immunosorbent assay as well as conventional and quantitative reverse-transcription polymerase chain reaction. The effects of CQ on odontogenic differentiation were evaluated using alkaline phosphatase and alizarin red S staining methods.CQ treatment suppressed the proliferation of DPSCs and induced the expression of p16(INK4A), p21(WAF1), and p53. Levels of proinflammatory cytokines (eg, interleukin 6, interleukin 8, and matrix metalloproteinase-3 [MMP3]) were increased by CQ treatment. CQ also inhibited odontogenic differentiation and mineralization capacities of DPSC and MC3T3-E1 cells.Our study showed that CQ may trigger pulpal inflammation by inducing proinflammatory cytokine production from the pulpal cells and may impair odontogenic differentiation of dental pulp cells, resulting in pulpal irritation and inflammation.

We immortalized oral keratinocytes by transfecting them with recombinant human papillomavirus (HPV) type 18 DNA and established three cell lines. These lines were morphologically different from their normal counterpart, contained integrated entire HPV-18 DNA, and expressed the viral E6/E7 genes. The cells contained less p53 protein and more c-myc mRNA than normal cells. However, they proliferated only in keratinocyte growth medium (KGM) containing low calcium and were not tumorigenic in nude mice. To test the hypothesis that tumors result from the combined effect of a "high-risk" HPV and chemical carcinogens in the human oral cavity, we exposed the immortalized cells to the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine. Three chemically transformed cell colonies were isolated. These cells (a) proliferated well in both KGM and Dulbecco's modified minimum essential medium containing physiological levels of calcium; (b) were capable of proliferating in nude mice; (c) contained intact, integrated HPV-18 sequences; (d) transcribed substantially more HPV-18 E6/E7, transforming growth factor-alpha, and c-myc than the immortalized counterpart; and (e) contained, like the immortalized counterpart, less wild-type p53 protein and DCC message. These data indicate that human oral keratinocytes can be transformed by sequential exposure of normal keratinocytes to a "high-risk" HPV and chemical carcinogens.

MicroRNAs (miRNAs) are epigenetic regulators of eukaryotic gene expression and play key roles in many cellular processes. However, the role of miRNAs for replicative senescence of normal human keratinocytes (NHKs) remains unknown. Thus, we examined the expression profiles of 847 miRNAs in exponentially replicating and senescent NHKs and identified 126 senescence-associated miRNAs (SA-miRs). Among SA-miRs, 117 miRNAs (93%) were upregulated and 9 miRNAs (7%) were downregulated in senescent NHKs compared to those of exponentially replicating cells. Among the above miRNAs, we selected two miRNAs, miR-137 and miR-668, for further investigation because they were consistently upregulated with replicative senescence of three independent NHK cultures. Ectopic overexpression of miR-137 or miR-668 induced senescence in rapidly proliferating NHKs; a notable increase in senescence-associated β-galactosidase activity, p16INK4A and p53 was observed, indicating that they are novel senescence-inducing miRNAs. In addition, these senescence-inducing miRNAs were gradually increased during organismal aging of normal human oral epithelia. We also detected downregulation of miR-137 and miR-668 in many tested human head and neck squamous cell carcinoma cell lines. Since senescence would be viewed as a potent tumor suppressive pathway, the newly identified senescence-inducing miRNAs deserve to be further investigated for their therapeutic application in cancer treatment.

We previously reported that wild type (wt) hnRNP G exhibited tumor suppressive activity in human oral squamous cell carcinoma (HOSCC) cell lines lacking hnRNP G. Wt hnRNP G markedly inhibited the proliferation capacity, anchorage independency and in vivo tumorigenicity of HOSCC cells and notably enhanced the DNA repair capabilities of these cells. In the present study, we studied the genetic and expression states of hnRNP G in normal, premalignant and malignant human oral tissues to further understand the relationship between the hnRNP G alterations and the development of human oral cancer. To correlate the cancer development and the level of hnRNP G expression, we performed an immunohistochemistry staining of hnRNP G in normal, premalignant and malignant human oral tissues. Moreover, we examined the entire coding regions of hnRNP G from selected samples to understand the cause of the alterations of the gene expression. The expression of hnRNP G was notably decreased or completely abolished in 80% of premalignant-dysplastic and malignant oral epithelial tissues, whereas 100% of normal and 90% of hyperplastic non-dysplastic epithelium showed high level of hnRNP G in the nucleus of the basal cell layers. Approximately 80% of HOSCC lacking the expression of hnRNP G showed genetic alteration in hnRNP G, i.e., point mutation and exonic deletion. This study suggest that genetic alterations and aberrant expression of hnRNP G occurring during oral carcinogenesis might be useful markers for the early detection of human oral cancer. Published by Elsevier Ltd.

We recently identified Grainyhead-like 2 (GRHL2) as a novel transcription factor that binds to and regulates the activity of the human telomerase reverse transcriptase (hTERT) gene promoter. In this study, we investigated the biological functions of GRHL2 and the molecular mechanism underlying hTERT gene regulation by GRHL2. Retroviral transduction of GRHL2 in normal human keratinocytes (NHK) led to a significant extension of replicative life span, whereas GRHL2 knockdown notably repressed telomerase activity and cell proliferation. Using promoter magnetic precipitation coupled with Western blotting, we confirmed the binding of GRHL2 to the hTERT promoter and mapped the minimal binding region at −53 to −13 of the promoter. Furthermore, mutation analysis revealed the three nucleotides from −21 to −19 to be critical for GRHL2 binding. Because hTERT expression is regulated in part by DNA methylation, we determined the effects of GRHL2 on the methylation status of the hTERT promoter. Senescent NHK exhibited hypermethylation of the CpG island, which occurred with the loss of hTERT expression. On the contrary, the promoter remained hypomethylated in GRHL2-transduced NHK, irrespective of cell proliferation status. Also, knockdown of endogenous GRHL2 led to hypermethylation of the promoter. These results indicate that GRHL2 regulates the hTERT expression through an epigenetic mechanism and controls the cellular life span.

Enhanced expression of human telomerase reverse transcriptase (hTERT) occurs frequently during cellular immortalization. The current study was undertaken to determine the mechanism regulating the hTERT promoter activity during cellular immortalization of human oral keratinocytes. Normal human oral keratinocytes (NHOKs) were immortalized with Bmi-1 and the E6 oncoprotein of human papillomavirus type 16 to establish the telomerase-positive HOK-Bmi-1/E6 cell line. Using DNA-protein-binding assay, we found that heat shock protein 90 (hsp90) physically interacts with the hTERT promoter in vitro. The hsp90 interaction with the promoter was detected more strongly in the telomerase-positive HOK-Bmi-1/E6 cells compared with that in senescing NHOK. Chromatin immunoprecipitation confirmed the in vivo interaction between hsp90 and the hTERT promoter in SCC4 cells, a telomerase-positive oral cancer cell line, but not in the NHOK. To determine the physiological significance of this interaction, SCC4 cells were exposed to geldanamycin (GA), a competitive inhibitor of hsp90. GA exposure led to decrease in telomerase activity, hTERT promoter activity and hTERT messenger RNA expression in SCC4 cells, even in the absence of de novo protein synthesis. Also, it abolished the in vivo interaction of the hTERT promoter region with hsp90 but not with Sp1 or c-Myc. These results indicate that physical interaction between hsp90 and the hTERT promoter occurs in telomerase-positive cells but not in normal human cells and is necessary for the enhanced hTERT expression and telomerase activity in cancer cells.

Introducing highly active antiretroviral therapy (HAART) has significantly decreased the morbidity and mortality in human immunodeficiency virus-positive (HIV+) individuals by decreasing the viral loads and increasing the CD4+ T-cell counts. Subsequently, the occurrence of many HIV-associated diseases has been dramatically declined except human papillomavirus (HPV)-associated lesions. Such notion suggests that immune response is not a major determinant, and that the direct interaction between HIV and HPV may be involved in the HPV-associated pathogenesis. In the current study, we investigated whether HIV plays a direct role in HPV-associated oral carcinogenesis by using HIV-1 transactivator protein (Tat), which is known to have oncogenic properties. We found that HIV-1 Tat not only increased the expression of HPV-16 E6 and E7 oncogenes in human oral keratinocytes harboring the HPV type-16 genome (HOK-16B), but also notably enhanced the proliferative capacity of the cells in vitro. Moreover, HOK-16B cells expressing HIV-1 Tat was capable of inducing cystic nodules in nude mice, while the control HOK-16B cells failed to produce nodules in the mice. Our results indicate that HIV could play a role in the HPV-associated pathogenesis by exerting oncogenic stimulus via Tat protein.

Nitric oxide (NO) is a multifunctional regulator, critical to various biochemical processes, including inflammation, vasodilatation, intra- and intercellular signaling, apoptosis, and carcinogenesis. In particular, recent studies have indicated the association between elevated NO production and neoplastic cell transformation, suggesting procarcinogenic effects of NO. To investigate the mechanism by which NO facilitates oral carcinogenesis, we tested the effects of exogenous NO on the expression of hnRNP G, a novel protein demonstrating tumor suppressive effects against oral squamous cell carcinomas. Oral epithelial cells exposed to NO donor demonstrated significant reduction in the level of hnRNP G protein and mRNA expression. Also, exposure to NO donor led to decreased hnRNP G promoter activity in cells, indicating that NO negatively regulates hnRNP G expression at the level of transcription. Since hnRNP G expression is markedly decreased or completely abolished in precancerous and malignant oral lesions in situ, these results suggest the possibility that NO facilitates the progression of the disease by targeting hnRNP G expression. In this article, we review the role of hnRNP G in tumor suppression and maintenance of genetic integrity, with focus on its potential association with NO in the context of oral carcinogenesis.

Purpose: Grainyhead-like 2 (GRHL2) is one of the three mammalian homologues of Drosophila Grainyhead involved in epithelial morphogenesis. GRHL2 also controls epithelial cell proliferation and differentiation. Our objective was to evaluate the role of GRHL2 in oral carcinogenesis and the underlying mechanism. Experimental Design: GRHL2 expression was evaluated in multiple oral cancer cell lines and tissues of oral squamous cell carcinomas (OSCCs) by qRT-PCR, western blot assay, immunohistochemistry and laser-captured microdissection assay. The effects of shRNA-mediated GRHL2 knockdown were evaluated by tumor spheroid assay, colony formation assay, gene promoter luciferase assay and immunofluorescent staining and chromatin immunoprecipitation assay. In vivo the effect of GRHL2 loss on cancer cell tumorigenicity was evaluated by tumor xenograft assay. Results: GRHL2 expression was elevated in cells and tissues of oral squamous cell carcinomas (OSCCs) compared with normal counterparts. Knockdown of GRHL2 resulted in the loss of in vivo tumorigenicity, cancer stemness, and epithelial phenotype of oral cancer cells. GRHL2 inhibition decreased oral cancer cell proliferation and colony formation. GRHL2 regulated the expression of miR-200 family and Octamer-binding transcription factor 4 (Oct-4) genes through direct promoter DNA binding. Overexpression of miR-200 genes in the oral cancer cells depleted of GRHL2 partially restored the epithelial phenotype, proliferative rate and cancer stemness. Conclusion: GRHL2 plays pivotal role in the maintenance of the transformed phenotype in OSCCs. Downregulation of GRHL2 leads to the decrease of tumorigenicity, cancer stemness of oral cancer cell, in which miR-200 and Oct-4 genes partially mediate the tumorigenic effects of GRHL2 in OSCC. This study was supported in part by the grants R56DE024593 and Jack Weichman Endowed Fund. Citation Format: Wei Chen, Shebli Mehrazarin, Ki-Hyuk S Shin, Yi-Ling Lin, Reuben H. Kim, No-Hee Park, Mo K. Kang. Grainyhead-like 2 (GRHL2) regulates epithelial plasticity and stemness in oral cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2097. doi:10.1158/1538-7445.AM2015-2097

In human cells, DNA double-strand breaks (DSBs) are repaired primarily by the DNA end-joining (EJ) process and thus, abnormal DNA EJ activities lead to an accumulation of mutations and/or aneuploidy, resulting in genetic instability of cells. Since genetic instability is the hallmark of cancer cells, we studied the DNA EJ activities of normal, non-malignant immortalized and malignant human epithelial cells to investigate the association between DNA EJ and carcinogenesis. We found a significant diminution of precise (error-free) DNA EJ activities in non-malignant immortalized human oral keratinocytes (HOK-16B) and human head and neck squamous cell carcinoma (HNSCC) cells compared to that in normal human oral keratinocytes (NHOK). Moreover, abnormal DNA EJ activities were detected exclusively in HOK-16B and HNSCC cells due to microhomology-mediated and non-microhomology-mediated end-joining activities in these cells. These data indicated that aberrant DNA EJ activity may be partly responsible for genetic instability and oncogenic transformation.

In human cells, DNA double-strand breaks (DSBs) are repaired primarily by the DNA end-joining (EJ) process and thus, abnormal DNA EJ activities lead to an accumulation of mutations and/or aneuploidy, resulting in genetic instability of cells. Since genetic instability is the hallmark of cancer cells, we studied the DNA EJ activities of normal, non-malignant immortalized and malignant human epithelial cells to investigate the association between DNA EJ and carcinogenesis. We found a significant diminution of precise (error-free) DNA EJ activities in non-malignant immortalized human oral keratinocytes (HOK-16B) and human head and neck squamous cell carcinoma (HNSCC) cells compared to that in normal human oral keratinocytes (NHOK). Moreover, abnormal DNA EJ activities were detected exclusively in HOK-16B and HNSCC cells due to microhomology-mediated and non-microhomology-mediated end-joining activities in these cells. These data indicated that aberrant DNA EJ activity may be partly responsible for genetic instability and oncogenic transformation.

We previously reported that the E6 oncoprotein of high-risk human papillomavirus (HPV) caused genetic instability and oncogenesis by disrupting cellular DNA repair. Here, to investigate the effect of different domains of E6 on DNA double-strand break (DSB) repair, we infected normal human oral fibroblasts (NHOF) with retroviruses expressing wild-type (wt) or mutant (mt) HPV-16 E6 and examined the cellular DNA end-joining (EJ) activity. The cells expressing E6 showed not only a diminution of error-free DNA EJ but also an increase in erroneous DNA EJ capacity if compared with cells without wt E6. Analysis of DNA EJ activities from the cells expressing mt HPV-16 E6 indicated that binding to p53 and the presence of both intact zinc finger domains of E6 are necessary for inducing the E6-mediated aberrant DNA EJ activity. Also, deletion of the PDZ binding C-terminal region reduced this activity by 50%. These findings suggest that E6 can disrupt the fidelity of DSB repair via both p53-dependent and -independent pathways and the impaired fidelity might contribute to the development of genetic instability found in HPV-associated cancer.

We previously reported that the E6 oncoprotein of high-risk human papillomavirus (HPV) caused genetic instability and oncogenesis by disrupting cellular DNA repair. Here, to investigate the effect of different domains of E6 on DNA double-strand break (DSB) repair, we infected normal human oral fibroblasts (NHOF) with retroviruses expressing wild-type (wt) or mutant (mt) HPV-16 E6 and examined the cellular DNA end-joining (EJ) activity. The cells expressing E6 showed not only a diminution of error-free DNA EJ but also an increase in erroneous DNA EJ capacity if compared with cells without wt E6. Analysis of DNA EJ activities from the cells expressing mt HPV-16 E6 indicated that binding to p53 and the presence of both intact zinc finger domains of E6 are necessary for inducing the E6-mediated aberrant DNA EJ activity. Also, deletion of the PDZ binding C-terminal region reduced this activity by 50%. These findings suggest that E6 can disrupt the fidelity of DSB repair via both p53-dependent and -independent pathways and the impaired fidelity might contribute to the development of genetic instability found in HPV-associated cancer.

We investigated the phenotypic and molecular alterations in normal human oral keratinocytes (NHOK) during in vitro replication in two different culture conditions. The cells were cultured either in chemically defined Keratinocyte Growth Medium (KGM) without feeder layers or in serum-containing flavin-adenine dinucleotide (FAD) medium with feeder layers. Primary NHOK underwent 22 +/- 3 population doublings (PDs) in KGM and 42 +/- 4 PDs in FAD medium, reflecting 52% increase in replication capacity with feeder layers. In both culture conditions, exponentially replicating NHOK demonstrated telomerase activity and expression of human telomerase reverse transcriptase (hTERT) gene. Telomerase activity and hTERT expression were rapidly diminished in senescing NHOK, which exhibited small decrease of telomere length for the remaining limited cellular replications until the complete arrest of cell division. However, telomere length in senescent NHOK was 6.7 +/- 0.5 kilobase pairs (kbps), significantly longer than that (5.12 kbps) of senescent human fibroblasts. The onset of senescence was accompanied with marked induction of p16(INK4A), and this occurred in both culture systems using either KGM or FAD medium. These results indicate that replicative senescence of NHOK is associated with loss of telomerase activity followed by limited telomere shortening.

This study describes the first example of spontaneously immortalized, telomerase-negative hamster cells which replicated in the absence of telomere shortening. Primary hamster buccal pouch epithelial (HBPE) cells were subcultured for approximately 10 population doublings (PDs) before they ceased to divide. Among the non-replicating HBPE cells, a colony of proliferating cells had appeared. These cells named HBPE-SI (spontaneously immortalized) replicated with a mean doubling time of 27 h for 120 PDs in culture and are still replicating. Surprisingly, telomerase activity and ham TERT (hamster telomerase gene) expression were not detectable in HBPE-SI, although an exogenous hamTERT promoter showed promoter activity in these cells. HBPE-SI cells replicated in the apparent absence of apparent telomere shortening during their entire in vitro culture, indicating telomerase-independent mode of telomere maintenance. Analysis of the hamTERT promoter in HBPE-SI cells revealed hypermethylation. Exposure of the cells to 5-aza-2'-deoxycytidine (5-aza CdR) restored expression of endogenous hamTERT mRNA. These results indicated that HBPE-SI cells maintained their telomere length in the absence of telomerase activity, and that hamTERT gene expression in these cells was silenced by hypermethylation of the promoter.

Hereditary non-polyposis colorectal cancer (HNPCC), an inherited cancer predisposition syndrome, has been associated with germline mutations in DNA mismatch repair (MMR) genes. However, because all mutation carriers of hMLH1/hMSH2 do not account for CRC susceptibility, modifying genes may play a role in the variation of disease expression. In this study, we determined the GSTM1 and GSTT1 genotypes in 104 family members representing 19 Korean HNPCCs carrying hMLH1/hMSH2 mutation, and investigated the influence of GSTM1 and GSTT1 geno-/phenotype status on both age at diagnosis of CRC and cancer occurrence. The overall frequency of the GSTM1 and GSTT1 geno-/phenotype in 55 non-carriers, compared with that in mutation carriers (n=49), was not significantly different, and no significant correlation was found between mean age at diagnosis and the allelomorphs encoding the GSTM1 or GSTT1 enzymes. However, a comparison of the allele frequencies of GSTM1 in affected (n=30) and unaffected (n=19) mutation carriers revealed a significant difference, as the null allele was more prevalent in individuals with cancer (p=0.03; odds ratio, 3.7; 95% confidence interval, 1.1-12.7). Our results suggest that the genotypes of GSTM1 are associated with cancer occurrence in Korean HNPCC family members carrying the hMLH1/hMSH2 mutation. However, a bias due to the small sample size of this study cannot be rule out. Although evidence that GST genotypes are associated with increased cancer risk has often been controversial, the genotyping of GSTM1 could have implications for genetic counseling and the management of MMR gene mutation carriers.

PTEN, a tumor suppressor gene, has been found to be inactivated by structural abnormalities or epigenetic changes in several types of human cancers. Recently, several studies have also suggested the possibility that the PTEN gene is a target of genomic instability in human cancers displaying microsatellite instability (MSI). To investigate the role of PTEN in human oral squamous cell carcinomas, we screened the entire coding region sequences and examined the expression of the PTEN gene in 81 oral cancers displaying microsatellite stability (MSS) and 5 oral cancers displaying MSI. Mutation of the PTEN gene was identified in one MSS cancer (1/81; 1.2%) and three MSI cancers (3/5; 60%). The MSS cancer harbored a missense mutation from Ala (GCA) to Val (GTA) at codon 137. Of the MSI cancers containing the PTEN mutation, case 36 had a missense mutation from Lys (AAA) to Glu (GAA) at codon 254, case 43 contained a frameshift mutation (one A deletion) in a 6 bp poly(A) tract affecting codon 265-267, and case 64 harbored two missense mutations from Val (GTG) to Ala (GCG) at codon 222, and from Gly (GGA) to Arg (AGA) at codon 230 indicating biallelic mutation of PTEN. Genomic deletion of exon 5, resulting in loss of PTEN mRNA, was observed in two MSS cancers. In spite of an intact PTEN gene, one MSS and one MSI cancer lacked PTEN mRNA. These findings suggest that the inactivation of PTEN by either mutation or loss of transcript plays a role in the pathogenesis of some oral cancers (8/86; 9.3%). Furthermore, inactivation of PTEN was far more frequent in MSI oral cancers (4/5; 80%) than in MSS oral cancers (4/81; 4.9%).

To determine the etiologic factors of human oral cancer, we examined the prevalence of microsatellite instability (MSI), the inactivation of mismatch repair (MMR) genes, p53 mutation, and human papillomavirus (HPV) infection (HPV-16, -18, and -33) in 86 Korean oral cancer specimens, including 76 squamous cell carcinomas and 10 salivary gland tumors. MSI was observed in 3 of the 76 squamous cell carcinomas (4%) and 2 of 10 salivary gland tumors (20%). As MSI is a hallmark of the inactivation of the MMR genes, the genetic status of hMSH2 and hMLH1, and hypermethylation of the hMLH1 promoter region were investigated in oral cancers displaying MSI. Inactivation of the hMLH1 gene by either mutation or hypermethylation was observed 4 of the 5 MSI oral cancers. Mutation of the p53 gene was found in 11 of 76 squamous cell carcinomas (14.5%) but not in the salivary gland tumors. PCR assay revealed the presence of HPV DNA in 11 of the 76 squamous cell carcinomas (14.5%) and 4 of the 10 salivary gland tumors (40%). Type 18 HPV DNA was predominant in 11 of the HPV-infected squamous cell carcinomas (72.7%) and 4 of the HPV-infected salivary gland tumors (50%). Two squamous cell carcinoma tissues were found both to be HPV-infected and to harbor the p53 mutation. Our results suggest: i) that MSI plays a role in the pathogenesis of Korean oral cancers, squamous cell carcinomas (4%) and salivary gland tumors (20%); ii) that genetic alteration or hypermethylation of the hMLH1 gene may be the principal inactivating mechanism in Korean oral cancer with MSI; and iii) that inactivation of the p53 gene by either mutation or HPV infection is frequent in Korean squamous cell carcinomas (26%) and salivary gland tumors (40%).

Human oral squamous cell carcinoma cell lines (KOSCC-11, -25A, -25B, -25C, -25D, -25E, -33A, and -33B) were established by explantation culture from these oral squamous cell carcinomas. The histopathology of the primary tumors, in vitro growth characteristics, epithelial origin, in vitro anchorage-independency, in vivo tumorigenicity, the frequency of human papillomavirus (HPV) infections, and the status of proto-oncogenes, tumor suppressor genes, DNA mismatch repair genes, and microsatellite instability were investigated in the cell lines. KOSCC-11 is a well-differentiated oral squamous cell carcinoma (OSCC) derived from mandibular gingiva. KOSCC-25A, -25B, -25C, -25D, and -25E cell lines were derived from the same OSCC. KOSCC-33A and -33B were established from the same tumor that originated from the maxillary sinus. All tumor lines studied grew as monolayers and showed: i) epithelial origin by the presence of desmosome and keratin; ii) in vitro anchorage-independent growth ability; and iii) tumorigenic potential in nude mice. The cancer cell lines did not contain HPV DNA and did not express viral genes. Northern blot analysis revealed: i) overexpression of EGFR in four cell lines, ii) overexpression of c-H-ras in four cell lines, iii) overexpression of c-myc in three cell lines, iv) decreased expression of TGF-alpha in seven cell lines, and v) decreased expression of c-jun in five cancer cell lines compared with normal human oral keratinocytes. In all KOSCC cell lines and their corresponding tumor tissues, mutations were identified in highly-conserved functional regions of the p53 gene. The KOSCC-11 cell line contained a frameshift mutation and the other cell lines harbored an identical p53 mutation at codon 175 from CGC (Arg) to CTC (Leu). In five cell lines, a significant reduction of p21WAF1/Cip1 protein was evident. Cancer cell lines expressed higher level of Rb protein than normal human oral keratinocytes. DCC, a tumor suppressor gene, was not detected in KOSCC-25C. The KOSCC-33A cell line displayed microsatellite instability and showed a loss of hMSH2 expression. These well-characterized human OSCC cell lines should serve as useful tools for understanding the biological characteristics of oral cancer.

The human DNA mismatch repair genes hMSH2 and hMLH1 are responsible for the development of hereditary nonpolyposis colorectal cancer (HNPCC). Although genetic alteration of the coding region of hMSH2 and hMLH1 has been well investigated in HNPCC patients, the regulatory regions of these genes have been poorly investigated, though recent studies have defined and characterized the core promoter regions of hMSH2 and hMLH1. Therefore, to investigate the presence of germ-line mutations, we screened the core promoter regions of hMSH2 and hMLH1 from 157 nonmalignant control individuals, 40 cases of HNPCC, 56 suspected HNPCC cases, and 45 sporadic early onset colorectal cancer patients. Three novel germ-line mutations of the hMSH2 promoter were identified in two suspected HNPCC cases and one sporadic early onset colorectal cancer patient but not in the 157 nonmalignant controls, namely, an A insertion at position -80, a G-to-A transition at position -190, and a G-to-C transversion at position -225. Tumors from patients containing the promoter mutations displayed microsatellite instability. The A insertion at -80 is within a sequence homologous to the consensus sequence for E1AF and very close to the major transcription start point. Luciferase assay demonstrated that the -80A insertion and the -190A allele decreased the transcriptional efficiency by 82 and 77%, respectively, and the -225C allele increased the transcriptional efficiency by 466%. The -80A insertion allele was detected only in affected members within the family and showed novel transcription factor binding ability. Furthermore, the loss of single nucleotide polymorphism allelic expression was identified in blood of the patient containing the -80A insertion. Our results indicate that mutations in the promoter region of hMSH2 have a limited role in development of suspected HNPCC and sporadic early onset colorectal cancer.

Cancer is one of the most prevalent age-associated diseases in elderly populations and is largely attributed to accumulation of genetic mutations secondary to the loss of tumor suppressive functions. p53 is a potent tumor suppressor that is implicated in organismal aging in vivo and replicative senescence in vitro; however, its transcriptional regulation during replicative senescence in cells remain unclear. The current study was undertaken to examine the epigenetic regulation of p53 transcription during replicative senescence in normal human keratinocytes. Normal human oral keratinocytes (NHOKs) undergoing senescence upon serial subcultures exhibited a decrease in p53 gene expression. Methylation status of two CpG islands in the p53 promoter was evaluated by treating with 5-aza-CdR, a demethylating agent, and by using bisulfite methylation sequencing analysis. Status of histone modification was evaluated using Chromatin Immunoprecipitation (ChIP) assay with anti-H3K27me3, a repressive mark, and anti-H3Ac, an active mark at the 5 different sites of the p53 promoter regions. In the presence of 5-aza-CdR, there was no increase in p53 expression in senescent NHOK at both mRNA and protein level. Bisulfite-sequencing analysis revealed no methylation changes at the CpG islands in the p53 promoters from young and senescent NHOK. However, during replicative senescence in NHOK, there were notable enrichment of anti-H3K27me3 occupancy and significant decrease in anti-H3Ac occupancy in the p53 promoter regions, indicating that chromatin at the p53 locus undergo compaction during replicative senescence. Our study demonstrates that the diminution of p53 during replicative senescence is epigenetically regulated in human keratinocytes. Citation Format: Reuben H. Kim, Mo K. Kang, Terresa Kim, Paul Yang, Christine Hong, Ki-Hyuk Shin, No-Hee Park. p53 gene expression is epigenetically regulated during replicative senescence in keratinocytes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2256. doi:10.1158/1538-7445.AM2014-2256

High-risk human papillomaviruses (e.g., HPV-16 and HPV-18) are closely associated with the development of human head and neck squamous cell carcinomas (HNSCC), including malignant lesions in the oral cavity. However, less is known about the possible role and the underlying mechanisms of HPV in enhancing cancer progression and promoting the virulence of cancer, including oral squamous cell carcinoma (OSCC). Cancer stem cells (CSCs) play crucial role in cancer progression, metastasis and recurrence, and are epigenetically regulated by microRNAs (miRNAs). Recent studies demonstrated that miRNA expression is affected by HPV status in HNSCC, suggesting a possible role of HPV in epigenetic regulation of CSCs. In this study, we investigated the role of high-risk HPV on malignant and CSC phenotypes as well as epigenetic regulation in OSCC. Incorporation of HPV-16 whole genome into HPV-negative OSCCs resulted in enhancement of malignant phenotypes (e.g., increased anchorage independent growth, proliferation in organotypic epithelial raft culture, and tumorigenicity in nude mice) and CSC phenotypes (e.g., increased CSC-related markers, self-renewal, and migration/invasion). High-throughput miRNA expression analysis revealed that miRNA-181 family members were significantly underexpressed in the HPV16-harboring OSCCs. HPV-16 downregulated the promoter activity of miR-181a. Furthermore, restoration of miR-181 in the presence of HPV-16 suppressed CSC-like phenotype. Taken together our data indicate that high-risk HPV enhances stemness phenotypes in OSCC by epigenetic regulation of miR-181 expression. Citation Format: Sung Hee Hee Lee, Nicole Rigas, Chang-Ryul Lee, Jiho Han, Reuben Kim, Mo Kang, No-Hee Park, Ki-Hyuk Shin. Human papillomavirus enhances oral cancer stem cell phenotype by regulating microRNA-181. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1948. doi:10.1158/1538-7445.AM2014-1948

Little is known about genetic alterations of patients who present multiple primary cancers. We hypothesized that microsatellite instability (MSI) is one of the underlying genetic factors in the development of double primary cancers in colorectal cancer patients. We examined for MSI in 41 colorectal cancer patients who presented with extra-colonic primary cancers consisted of 17 gastric and 24 non-gastric cancers. Coincident MSI+ in tumors of two organs were observed in 3 (17.7%) of 17 patients with colon and stomach cancers and 0 of 24 patients with colon and non-gastric cancers (P =.03). In 17 patients with colon and stomach cancers, 6 (31.6%) of 19 colon cancers and 3 (17.7%) of 17 gastric cancers exhibited MSI+. Among four patients with metachronous colon cancers who were identified within the 41 double primary cancer patients, two patients were associated with the MSI+ phenotype. In summary, the prevalent coincidence of MSI suggests that genetic defect of mismatch repair deficiency may be responsible for a small subset of double primary cancers of the colorectum and stomach.

“High risk” human papillomaviruses (HPV) are known to cause or are closely associated with the development of human cervical, anal and some types of head and neck cancers. Integration of HPV DNA into host genome and subsequent expression of viral oncogenes, such as E6 and E7, are critical initial events for the HPV-associated cancer development; however, HPV infection alone is not sufficient to convert normal cells to malignant phenotypes, suggesting that other additional genetic and/or epigenetic events in the HPV-infected cells are required. The aim of the current study is to identify molecular determinants that are functionally linked to the pathogenesis of the HPV-associated cancers. We used HPV-16 harboring immortalized but non-transformed oral epithelial cells (HOK-16B), infected with high-throughput Decode RNAi lentiviral screening pools containing about 70,000 shRNAmir constructs capable of knocking down the target genes individually (3-4 constructs per gene), and selected them in DMEM-based high calcium/serum media in which HOK-16B cells are not permissive to survive. From the surviving colonies, we have identified several putative tumor suppressors including A Disintegrin and etalloproteinase domain-containing protein 23 (ADAM23). The function of ADAM23 was further validated such that singly knocking down in HOK-16B cells (HOK16B/ADAM23i) acquired proliferative potential in DMEM-based medium. HOK16B/ADAM23i cells also gained resistance to ionizing radiation (IR), cisplatin treatment, and differentiation as demonstrated by colonogenic assay, MTT assay, and the 3-dimensional raft culture systems, respectively. More importantly, the expression is largely diminished in squamous cell carcinoma (SCC) cell lines but more so in HPV-harboring SCC. We further found that ADAM23 expression is high in normal human oral epithelial tissues but low or none in HPV-positive OSCC tissue specimens as determined by immunohistochemical staining. Our data suggest that ADAM23 may play a role in development of HPV-associated cancers. Citation Format: Paul Yang, Drake W. Williams, Terresa Kim, Ki-Hyuk Shin, Mo Kang, No-Hee Park, Reuben H. Kim. Tumor suppressor ADAM23 mediates the HPV-associated carcinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1987. doi:10.1158/1538-7445.AM2013-1987

Zoledronic acid (ZA), a third generation of bisphosphonates, is known to have both anti-osteolytic and anti-tumor activities. It exerts anti-osteolytic function by interfering with mevalonate pathway and preventing protein prenylation required for growth and survival in osteoclast cells. Also, similar function has been found to induce cancer apoptosis and inhibit cancer growth in bone-related cancer, but its effect on other cancer types, including head and neck squamous cell carcinoma (HNSCC) is still unclear. In this study, we first monitored the effect of ZA on cancer growth in HNSCC cell lines in vitro. ZA significantly suppressed cancer growth, shown by decreased anchorage independent growth in soft agar assay and diminished cell growth in organotypic raft culture. Because cancer stem cell (CSC; also called tumor-initiating cell) plays a significant role in cancer growth, we further investigated the effect of ZA on CSC in HNSCC. ZA-treated HNSCC cells significantly decreased CSC phenotypes such as sphere forming ability and migratory ability. In SCC4, a tongue squamous cell carcinoma cell line, side population and expression of pluripotency marker genes were significantly decreased by ZA. Also, ZA inhibited the CSC-related Sonic Hedgehog pathway in SCC4 as demonstrated by decreased mRNA expression of transcriptional factor, Gli-1. Taken together, our data suggest that ZA targets CSC and hence inhibits cancer growth in HNSCC. Citation Format: Sung Hee Lee, Reuben Kim, Mo Kang, No-Hee Park, Ki-Hyuk Shin. Zoledronic acid inhibits cancer growth and cancer stem cell phenotypes in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3715. doi:10.1158/1538-7445.AM2013-3715

Bisphosphonates (BPs) are potent anti-resorptive agents that are widely used to treat bone-associated diseases including metastatic bone cancer. Many reports have indicated that long-term use of BPs can develop BP-related osteonecrosis of the jaw (BRONJ) that occurs largely at the site of tooth extraction; however, the pathophysiology of BRONJ is still poorly understood. Clinically, BRONJ is defined as exposed necrotic bone with unhealed and open oral mucosa, implying that the lack of wound closure may be caused either directly by BPs or indirectly via biological signals (i.e., cytokines) from the BP-affected surrounding structures. We hypothesize that oral mucosal tissues lose their capacity to close the wound properly at the affected sites and that the genes involved in wound healing processes are differentially expressed between BRONJ lesions and the normal counterparts. To test our hypothesis, we utilized the established BRONJ mice model. Zometa was administered via tail vein 1 week prior to tooth extraction. After extracting the upper first molar, the extracted socket was allowed to heal, and the overlaying oral mucosal tissues were harvested after 2 weeks from control and BRONJ groups (n=3). The fresh tissue samples were subjected to RNA isolation, cDNA sysnthesis, and cDNA hybridization onto microarray chip (Affymetrix MG 430 2.0). The data analysis by functions using IPA software revealed that the genes involved in skeletal and muscular disorders, dermatological diseases, cellular movement, cellular assembly and organization, and inflammatory disease were significantly associated with BRONJ. In particular, we found that growth factors (i.e., Igf2, Fgf7, Pdf2bp2), collagens (i.e., Col1a1, Col1a2, Col2a1, Col2a2, Col5a1, Col5a2, Col11a1, and Col11a2), and non-collagenous proteins (i.e., DMP-1, ITBP, and SPP1) were significantly down-regulated in BRONJ groups. Taken together, our study suggests that the diminution of growth factors as well as collagenous and non-collagenous structural proteins may be associated with the development of BRONJ. This study was supported by NIDCR/NIH (R03DE021114 and K08DE017121). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1257. doi:1538-7445.AM2012-1257

p63 is a p53 family protein required for morphogenesis and postnatal regeneration of epithelial tissues. Here we demonstrate that ΔNp63α, a p63 isoform lacking the N-terminal transactivation domain, induces epithelial-mesenchymal transition (EMT) in primary human keratinocytes in a TGF-β-dependent manner. Rapidly proliferating normal human epidermal keratinocytes (NHEK) were infected with retroviral vector expressing ΔNp63α or empty vector and serially subcultured until replicative senescence. No phenotypic changes were observed until the culture reached senescence. Then the ΔNp63α-transduced cells underwent morphological changes resembling mesenchymal cells and acquired the EMT phenotype. Treatment with exogenous TGF-β accelerated EMT in presenescent ΔNp63α-transduced cells, whereas the inhibition of TGF-β signaling reversed the EMT phenotype. TGF-β treatment alone led to growth arrest in control NHEK with no evidence of EMT, indicating that ΔNp63α altered the cellular response to TGF-β treatment. ΔNp63α-transduced cells acquiring EMT gained the ability to be differentiated to osteo-/odontogenic and adipogenic pathways, resembling mesenchymal stem cells. Furthermore, these cells expressed enhanced levels of Nanog and Lin28, which are transcription factors associated with pluripotency. These data indicate that EMT required ΔNp63α transduction and intact TGF-β signaling in NHEK.

p63 is a p53 family protein required for morphogenesis and postnatal regeneration of epithelial tissues. Here we demonstrate that ΔNp63α, a p63 isoform lacking N-terminal transactivation domain, induces epithelial-mesenchymal transition (EMT) in primary human keratinocytes in TGF-β-dependent manner. Rapidly proliferating normal human epidermal keratinocytes (NHEK) were infected with retroviral vector expressing ΔNp63α, ΔNp63α, ΔNp63α, or empty vector, and serially subcultured until replicative senescence. No phenotypic changes were observed until the culture reached senescence. Then, cells transduced with ΔNp63α and ΔNp63α demonostrated morphological changes resembling mesenchymal cells, while ΔNp63α did not trigger such change in phenotype. Treatment with exogenous TGF-β accelerated EMT in pre-senescent ΔNp63α-transduced cells, and inhibition of TGF-β signaling reversed the EMT phenotype. Molecular markers of EMT, such as loss of E-Cadherin and cytokeratin 14 expression, were observed in cells expressing ΔNp63α but not those expressing ΔNp63α. TGF-β treatment alone led to growth arrest in control NHEK with no evidence of EMT, indicating that ΔNp63α altered the cellular response to TGF-β treatment. ΔNp63α-transduced cells acquiring EMT gained ability to be differentiated to osteo/odontogenic and adipogenic pathways, resembling mesenchymal stem cells (MSCs). Furthermore, these cells expressed enhanced levels of Nanog and Lin28, which are transcription factors associated with pluripotency. These data indicate that ΔNp63α triggered EMT in TGF-β-dependent manner, while ΔNp63α only caused morphologically changes resembling mesenchymal cells. The current study suggests that mesenchymal transition of normal skin keratinocytes might be a novel approach to generate induced MSCs (iMSCs), which may be useful for regenerative therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3326. doi:1538-7445.AM2012-3326

Although there is an increasing evidence of chronic inflammation-associated tumorigenesis, the molecular and cellular mechanisms linking chronic inflammation to tumorigenesis have not yet been fully understood. Recent studies have uncovered and validated the pathophysiological role of self-renewing cells, named cancer stem cells (CSC; also called tumor-initiating cells), in long-term sustenance of cancers. In this study, we investigated the effect of chronic inflammation on CSC phenotype of oral squamous cell carcinoma cells (OSCC). We treated OSCC cell lines with tumor necrosis factor alpha (TNFα), a major proinflammatory cytokine, for extended periods and examined the effect of chronic TNFα on CSC characteristics, i.e., undifferentiated tumor sphere forming ability, stem cell-associated genes expression and chemo-radioresistance. Chronic treatment of OSCC with TNFα enhanced CSC phenotype, which is shown by increased tumor sphere forming ability, greatly increased tumorigenicity and chemo-radioresistance, and increased expression of stemness-related genes. Moreover, activation of Notch1 signaling was detected in the TNFα treated cell, and suppression of Notch1 signaling inhibited CSC phenotype. Furthermore, we demonstrated that inhibition of Hes-1, the downstream target of Notch1, led to suppression of CSC phenotype. Taken together, the data suggest that proinflammatory cytokine exposure can generate or/and enrich OSCC CSC population, in part, by activation of the Notch1 signaling pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3341. doi:1538-7445.AM2012-3341

The state of p53 tumour suppressor and the frequency of high-risk human papillomavirus (HPV) infections were studied in nine human oral cancer cell lines. Three cancer cell lines (SCC-4, Tu-177 and FaDu) had similar amounts of p53 transcripts to normal cells, but contained significantly higher levels of p53 protein than the normal control cells. Sequencing highly conserved open reading frames of the p53 gene of these cancer cells showed point mutations in the SCC-4 and Tu-177 cell lines, a base transition from CCC to TCC occurred at codon 151; and in the line FaDu, a mutation of CGG to CTG occurred at codon 248. The HEp-2 and 1483 cancer lines contained significantly lower levels of p53 protein compared to the normal counterpart. Sequencing of p53 cDNA for HEp-2 and 1483 lines showed no mutations, but northern analysis revealed that these cell lines expressed HPV-18 E6/E7 messages. Four cell lines (SCC-9, SCC-15, SCC-25, and Tu-139) expressed negligible amounts of p53 transcripts compared to the normal counterpart and undetectable levels of p53 protein. These cell lines contained mutations in the highly conserved open reading frames of the p53 gene as follows: the SCC-9 had a deletion of 32 base pairs between codons 274 and 285; the line SCC-15 had an insertion of five base pairs between codons 224 and 225; the line SCC-25 had a deletion of two base pairs in codon 209; and the Tu-139 line had a deletion of 46 base pairs between codons 171 and 186.(ABSTRACT TRUNCATED AT 250 WORDS)

While acute inflammation is a part of the defense response, there have been emerging lines of evidence that suggest a strong link between chronic inflammation and cancer. Human papillomaviruses (HPV) infection is one of key factors in cervical cancer development, and possible link between HPV and oral carcinogenesis have been proposed. However, mechanisms by which HPV and chronic inflammation contribute to oral carcinogenesis have not been fully understood. The objective of this study was to determine the effect of chronic inflammation on malignant progression of HPV-associated oral carcinogenesis. We treated HPV-immortalized oral keratinocytes (HOK-16B) with a major pro-inflammatory cytokine, TNF-α for 4 months and examined the effect of chronic TNF-α treatment on malignant behavior of HOK-16B. Chronic treatment of TNF-α resulted in reduction in differentiation potential, and induction in proliferation potential and in migration ability of the HPV-immortalized oral keratinocytes. cDNA microarray analysis revealed that a subset of genes was significantly downregulated in long-term TNF-α treated cells, and the genes that were involved in differentiation, tumor suppression, and cell-cell junction functions supported the three increased malignant phenotypes, respectively. Our results indicate that chronic inflammation promotes malignant behavior of HPV-immortalized oral keratinocytes, suggesting that chronic inflammation is an important co-carcinogenic factor for HPV-associated oral carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5542. doi:10.1158/1538-7445.AM2011-5542

Bisphosphonates (BPs) are potent anti-resorptive agents that are widely used to treat metastatic bone diseases in cancer patients. BPs target the mevalonate pathway and inhibit cholesterol synthesis and prenylation (e.g., geranylgernaylation and farnesylation) of small GTPases that are important for cell proliferation and survival. Long-term users of BPs are at the higher risk of developing osteonecrosis of the jaw (ONJ), which commonly occurs at the site of previous tooth extraction or other surgical interventions. Although bisphosphonate-related osteonecrosis of the jaw (BRONJ) is clinically defined as exposed necrotic bone with unhealed and open oral mucosa, the exact role of oral mucosa in the pathophysiology of BRONJ is not fully understood. The current study was undertaken to examine direct effects of BPs on the reepithelialization of oral mucosal cells. When normal human oral keratinocytes (NHOK) were treated with pamidronate (PAM), proliferation and migration were inhibited in dose-dependent manner as determined by MTT assay, proliferation assay, Western blotting against PCNA, and the scratch wound healing assay. Cell cycle analysis revealed increased S-phase content in PAM-treated NHOK, and this was associated with decreased mRNA and protein expressions of Cyclin A2 but not Cyclin D. PAM also induced premature senescence in NHOK as determined by increased β-galactosidase positivity, increased p16 expression, and phosphorylated p-38 MAP kinase pathway. PAM-induced premature senescence and proliferation inhibition were partially reversed by geranylgerniol (GGOH), but not farnesol (FOH), which reconstitute geranylgeranylation and farnesylation of the mevalonate pathway, respectively. Finally, using 3-dimensional (3D) raft culture system, we found that PAM-treated oral mucosal tissue constructs showed diminished immunohistochemical staining of PCNA and loss of reepithelialization in the wounded area, indicating that BPs impair proliferation and migration of oral epithelium constructs ex vivo. Our study demonstrates that BPs induce premature senescence and directly impair proliferation and migration of oral mucosal cells, which may explain the pathophysiology of BRONJ in cancer patients undergoing BP-treatment. This study was supported by NIDCR/NIH (DE017121). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3218.

MicroRNAs (miRNAs) are novel epigenetic regulators of eukaryotic gene expression and play key roles in many cellular processes. However, the contribution of miRNAs to replicative senescence remains largely unexplored. To understand the role of miRNA in replicative senescence of normal human epithelial cells, we examined the expression profiles of 847 miRNAs in non-senescent (young) and senescent (old) normal human oral keratinocytes (NHOKs). We identified 126 senescence-associated miRNAs (SA-miRs) whose expression was altered greater than 2-fold with senescence. Among SA-miRs, 117 miRNAs (93%) were up-regulated and 9 miRNAs (7%) were down-regulated in senescent NHOKs compared to non-senescent NHOKs. miR-137 and miR-668 were consistently up-regulated with senescence of three independent NHOK cultures and with ionizing irradiation, a premature senescence inducer of NHOKs. Overexpression of miR-137 and miR-668 significantly increased senescence-associated β-galactosidase (SA β-gal) activity and senescence markers, p16INK4A and p53 in non-senescing NHOKs, indicating that they are senescence-inducing miRNAs. Furthermore, we found that these senescence-inducing miRNAs were frequently downregulated in human head and neck squamous cell carcinoma cell lines. Since senescence is considered as a tumor suppressor mechanism, the newly identified senescence-inducing miRNAs need to be considered for therapeutic application for cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3205.

We recently identified Grainyhead-like 2 (GRHL2), a mammalian homologue of Grainyhead in Drosophila, to be a novel transcription factor that binds to and regulates the activity of the human telomerase reverse transcriptase (hTERT) gene promoter. In the current study, we investigated the biological function of GRHL2 in normal human keratinocytes (NHK) and elucidated the molecular mechanism underlying GRHL2 regulation of hTERT gene expression. Primary NHK were infected with retroviral vectors expressing GRHL2 or the empty vector. GRHL2 transduction led to significant extension of the replicative lifespan of NHK by evading the senescence block. However, GRHL2 failed to cause immortalization of NHK, alone or in combination with Bmi-1, a polycomb group protein that bestows extended replication capacity to NHK. Enhanced proliferation of cells transduced with GRHL2 was also observed in three dimensional organotypic raft culture. Telomerase activity was detected in replicating NHK while it was rapidly lost during senescence. GRHL2 transduction increased the expression of hTERT and maintained detectable telomerase activity for extended time period, suggesting that GRHL2 positively influences the hTERT expression and telomerase activity in NHK. Upon knockdown of endogenous GRHL2, hTERT mRNA expression and telomerase activity were notably reduced. We previously showed that hTERT expression in NHK is regulated in part by promoter methylation at the 5’ CpG island. Thus, we determined the effects of GRHL2 on the methylation status of hTERT promoter by methylation-specific PCR (MSP). Loss of hTERT expression in the control NHK occurred with hTERT promoter hypermethylation. On the contrary, GRHL2 transduction notably reduced the extent of promoter methylation in NHK. This effect was not linked with enhanced cell proliferation because Bmi-1 transduction in NHK, which allowed prolonged cell proliferation, showed no effect on the hTERT promoter methylation. These data indicate that GRHL2 inhibits the promoter DNA methylation, thereby delaying hTERT gene inactivation and senescence of NHK. This study was supported by the grants R01DE18295, K02DE18959 from NIDCR/NIH. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3213.

Bmi-1 is a polycomb group protein necessary for cellular proliferation and maintenance of stem cell phenotype. The current study was undertaken to determine the radioprotective effects of Bmi-1 in human epithelial cells. Primary normal human keratinocytes (NHK) from oral mucosa or epidermis were infected with retroviral vectors expressing Bmi-1. NHK infected with empty virus (NHK/B0) rapidly underwent premature senescence upon exposure to 5 or 10 Gy of ionizing radiation (IR). However, the cells transduced with Bmi-1 (NHK/Bmi-1) maintained cell proliferation and clonogenicity after IR. To determine the mechanism underlying the radioresistance conferred by Bmi-1, we compared the DNA damage response (DDR), production of reactive oxygen species (ROS), and DNA repair activities in both cell types. NHK/B0 and NHK/Bmi-1 cells demonstrated rapid induction of DDR after 5 Gy irradiation. ROS production was increased by irradiation, but Bmi-1 transduction suppressed the ROS production. Irradiation strongly induced the genes encoding the enzymes involved in ROS production, mainly of NADPH oxidase subunits, but Bmi-1 completely abolished the expression of these genes. Chromatin immunoprecipitation (ChIP) revealed binding of Bmi-1 to the promoter regions of these genes in vivo, suggesting transcriptional repression by Bmi-1. In vitro DNA end joining and in vivo double strand break (DSB) repair activities were enhanced in NHK/Bmi-1 cells compared with NHK/B0. These data indicate that Bmi-1 enhances the radioresistance of NHK in part by mitigating the genotoxicity of radiation. This study was supported in part by the grants, UCLA/CBPR (U19) from NIAID and R01DE18295/K02DE18959 from NIDCR/NIH. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1410.

Introducing highly active antiretroviral therapy (HAART) has significantly decreased the morbidity and mortality in human immunodeficiency virus-positive (HIV+) individuals by decreasing the viral loads and increasing the CD4+ T-cell counts. Subsequently, the occurrence of many HIV-associated diseases has been dramatically declined except human papillomavirus (HPV)-associated lesions. Such notion suggests that immune response is not a major determinant, and that the direct interaction between HIV and HPV may be involved in the HPV-associated pathogenesis. In the current study, we investigated whether HIV plays a direct role in HPV-associated oral carcinogenesis by using HIV-1 transactivator protein (Tat), which is known to have oncogenic properties. We found that HIV-1 Tat not only increased the expression of HPV-16 E6 and E7 oncogenes in human oral keratinocytes harboring the HPV type-16 genome (HOK-16B), but also notably enhanced the proliferative capacity of the cells in vitro. Moreover, HOK-16B cells expressing HIV-1 Tat was capable of inducing cystic nodules in nude mice, while the control HOK-16B cells failed to produce nodules in the mice. Our results indicate that HIV could play a role in the HPV-associated pathogenesis by exerting oncogenic stimulus via Tat protein.