• Biomedical Science Letters
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• v.11 no.4
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• pp.421-427
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• 2005

To evaluate the diagnostic significance of p16 overexpression in human hepatocellular carcinoma (HCC), we analyzed p16 status and growth inhibitory patterns by p16 overexpression in HCC cell lines having different pRE status. SKHep1 and SNU449 cells show homozygous deletion of p16. The p16 gene in SNU398 cell is inactivated at posttranscription level. Adenovira1-p16 (Ad-p16) infection inhibits the cell growth in Hep3B, SNU398, and SNU449. Failure of growth inhibition in SKHepl results from the low transduction efficiency of adenovirus. The p16-mediated growth inhibition shows G 1 phase arrest in pRE-positive SNU449 but not in pRE-negative Hep3B. These results suggest that therapeutic efficacy of p16 gene might be considered on the transduction efficiency and the toxicity of adenoviral vector. Beside, growth inhibitory effect of p16 could be exerted through either pRE-dependent or -independent pathway.

• Microbiology and Biotechnology Letters
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• v.32 no.1
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• pp.72-77
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• 2004

Previously, we synthesized a novel Cyclin-dependent kinase inhibitor, MCS-5A. Also, we investigated the involvement of cell cycle regulatory events during MCS-5A-mediated apoptosis in HL-60(+p16/-p53) cells with up-regulation of p16 protein expression. In contrast, apoptosis was not observed in A549(-p16/+p53) cells. Therefore we propose that $p16^{INK4A}$ is a key enzyme for inducing apoptosis. In the present studies, we have explored the mechanism of $p16^{INK4A}$ -mediated cytotoxicity and the role of p16.sup INK4A/ overexpression in the induction of apoptosis in human tumor cells. The tumor suppressor gene $p16^{INK4A}$ is known as a cyclin-dependent kinase inhibitor (CKI) and cell cycle regulator. We expressed wild type $p16^{INK4A}$ in pcDNA3.1 vector and then transfected into non-small cell lung cancer (NSCLC) cell expressing different statue of p16$^{INK4A}$, p53 gene〔A549(-p16/+p53), H1299(-p16/-p53) and HeLa(+pl6/+p53) cell line〕. TUNEL assay (including propidium iodide staining following transfection of these cell line with pcDNA3.1-pl6) indicate that p16$^{INK4A}$-mediated cytotoxicity was associated with apoptosis. This is supported by studies demonstrating an induction of caspase 3 cleavage due to the transfection of A549, H1299 and HeLa cells with pcDNA3.1-pl6. These results suggest that p16$^{INK4A}$ has a new function of inducing apoptosis which is not related with the function of tumor suppressor gene p53.

• Tuberculosis and Respiratory Diseases
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• v.44 no.4
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• pp.796-805
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• 1997

Background : It is clear that deregulation of cell cycle progression is a hallmark of neoplastic transformation and genes involved in the $G_1$/S transition of the cell cycle are especially frequent targets for mutations in human cancers, including lung cancer. p16 gene product, one of the G1 cell-cycle related proteins, that is recently identified plays an important role in the negative regulation of the the kinase activity of the cyclin dependent kinase (cdk) enzymes. Therefore p16 gene is known to be an important tumor suppressor gene and is also called MTS1 (multiple tumor suppressor 1). No more oncogenes have been reported to be frequently related to multiple different malignancies than the alterations of p16 gene. Especially when it comes to non-small cell lung cancer, there was no expression of p16 in more than 70% of cell lines examined. And also it is speculated that p16 gene could exert a key role in the development of non-small cell lung cancer. This study was designed to evaluate whether p16 gene could be used as a candidate for gene therapy of non-small cell lung cancer. Methods : After the extraction of total RNA from normal fibroblast cell line and subsequent reverse transcriptase reaction and polymerase chain reaction, the amplified p16 cDNA was subcloned into eukaryotic expression plasmid vector, pRC-CMV. The constructed pRC-CMV-p16 was transfected into the NCI-H441 NSCLC cell line using lipofectin. The changes of G1 cell-cycle related proteins were investigated with Western blot analysis and immunoprecipitation after extraction of proteins from cell lysates and tumor suppressive effect was observed by clonogenic assay. Results : (1) p16(-) NCI-H441 cell line transfected with pRC-CMV-p16 showed the formation of p16 : cdk 4 complex and decreased phosphorylated Rb protein, while control cell line did not. (2) Clonogenic assay demonstrated that the number of colony formation was markedly decreased in p16(-) NCI-H441 cell line transfected with pRC-CMV-p16 than the control cell line. Conclusion : It is confirmed that the expression of p16 protein in p16 absent NSCLC cell line with the gene transfection leads to p16 : cdk4 complex formation, subsequent decrease of phosphorylated pRb protein and ultimately tumor suppressive effects. And also it provides the foundation for the application of p16 gene as a important candidate for the gene therapy of NSCLC.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.19
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• pp.8351-8359
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• 2014

Background: Recent epidemiological data have implicated human papilloma virus (HPV) infection in the pathogenesis of head and neck cancers, especially oropharyngeal cancers. Although, HPV has been detected in varied amounts in persons with oral dysplasia, leukoplakias and malignancies, its involvement in oral tongue carcinogenesis remains ambiguous. Materials and Methods: HPV DNA prevalence was assessed by PCR with formalin fixed paraffin embedded sections (n=167) of oral tongue squamous cell carcinoma patients and the physical status of the HPV16 DNA was assessed by qPCR. Immunohistochemistry was conducted for p16 evaluation. Results: We found the HPV prevalence in tongue cancers to be 51.2%, HPV 16 being present in 85.2% of the positive cases. A notable finding was a very poor concordance between HPV 16 DNA and p16 IHC findings (kappa<0.2). Further molecular classification of patients based on HPV16 DNA prevalence and p16 overexpression showed that patients with tumours showing p16 overexpression had increased hazard of death (HR=2.395; p=0.005) and disease recurrence (HR=2.581; p=0.002) irrespective of their HPV 16 DNA status. Conclusions: Our study has brought out several key facets which can potentially redefine our understanding of tongue cancer tumorigenesis. It has emphatically shown p16 overexpression to be a single important prognostic variable in defining a high risk group and depicting a poorer prognosis, thus highlighting the need for its routine assessment in tongue cancers. Another significant finding was a very poor concordance between p16 expression and HPV infection suggesting that p16 expression should possibly not be used as a surrogate marker for HPV infection in tongue cancers. Interestingly, the prognostic significance of p16 overexpression is different from that reported in oropharyngeal cancers. The mechanism of HPV independent p16 over expression in oral tongue cancers is possibly a distinct entity and needs to be further studied.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.38 no.2
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• pp.101-109
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• 2012

Objectives: The inactivation of the tumor suppressor gene $p16^{INK4a}$ plays an important role in the development of malignant tumors, including oral squamous cell carcinoma. The p16 gene is involved in the p16/cyclin-dependent kinase/retinoblastoma (Rb) gene pathway of cell cycle control. The p16 protein is considered a negative regulator of this pathway. The p16 gene encodes an inhibitor of cyclin-dependent kinases 4 and 6 which regulate the phosphorylation of the retinoblastoma gene and G1 to S phase transition in the cell cycle. However, the p16 gene can lose its functionality through point mutations, loss of heterozygosity or methylation of its promoter region. Materials and Methods: In this study, the authors analyzed the correlation between various clinicopathological findings- patient age, gender and smoking, disease recurrence, tumor size, stage, and differentiation- and p16 protein expression or p16 promoter hypermethylation in 59 cases of head and neck squamous cell carcinoma. Results: The results revealed p16 protein expression and p16 promoter hypermethylation in 28 cases (47.5%) and 21 cases (35.6%), respectively, of head and neck squamous cell carcinoma. However, neither p16 protein expression nor p16 promoter hypermethylation had any statistical influence on clinicopathological findings or survival rate. Conclusion: This data, and a review of the literature, suggest that p16 promoter hypermethylation cannot yet be used as an independent prognostic factor influencing carcinogenesis, but must be considered as an important factor along with other genetic alterations affecting the pRb pathway.

• Clinical and Experimental Pediatrics
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• v.53 no.7
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• pp.753-758
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• 2010

Purpose: It has been suggested that p16 has a role in glucocorticoid (GC)-related apoptosis in leukemic cells, but the exact mechanisms have yet to be clarified. We evaluated the relationship between the GC response and p16 expression in a lymphoma cell line. Methods: We used p16 siRNA transfection to construct p16-inactivated cells by using the B-cell lymphoblast cell line NC-37. We compared glucocorticoid receptor (GR) expression, apoptosis, and cell viability between control (p16+NC-37) and p16 siRNA-transfected (p16-NC-37) cells after a single dose of dexamethasone (DX). Results: In both groups, there was a significant increase in cytoplasmic GR expression, which tended to be higher for p16+NC-37 cells than for p16- NC37 cells at all times, and the difference at 18 h was significant (P<0.05). Similar patterns of early apoptosis were observed in both groups, and late apoptosis occurred at higher levels at 18 h when the GR had already been downregulated ($P$<0.05). Cell viability decreased in both groups but the degree of reduction was more severe in p16+NC-37 cells after 18 h ($P$<0.05). Conclusion: These results suggest a relationship between GR expression and cell cycle inhibition, in which the absence of p16 leads to reduced cell sensitivity to DX.

• Toxicological Research
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• v.20 no.2
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• pp.109-116
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• 2004

For the safety evaluation of adenovirus-mediated gene transfer, we investigated differential gene expressions after transfecting adenoviral vector containing p16 tumor suppressor gene (Ad5CMV-p16) into human non-small cell lung cancer cells. In the previous study, we showed adenovirus-mediated $p16^{INK4a}$ gene transfer resulted in significant inhibition of cancer cell growth. We investigated gene expression changes after transfecting Ad5CMV-p16, Ad5CMV (null type, a mock vector) into A549 cells by using cDNA chip and oligonucleotide microarray chip (1200 genes) which carries genes related with signal transduction pathways, cell cycle regulations, oncogenes and tumor suppressor genes. We found that $p16^{INK4a}$ gene transfer down regulated 5 genes (cdc2, cyclin D3, cyclin B, cyclin E, cdk2) among 26 genes involved in cell cycle regulations. Compared with serum-free medium treated cells, Ad5CMV-p16 changed 27 gene expressions, two fold or more on oligonucleotide chip. In addition, Ad5CMV-p16 did not seem to increase the tumorigenicity-related gene expression in A549 cells. Further studies will be needed to investigate the effect of Ad5CMV-p16 on normal human cells and tissues for safety evaluation.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.3
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• pp.1165-1168
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• 2015

Objective: To investigate the incidence of malignant transformation and P53 and P16 expression in teratomatous skin of ovarian mature cystic teratoma. Materials and Methods: Data on ovarian teratoma specimens in nearly 10 years were reviewed. P53 and P16 expression were detected by immunohistochemistry in 25 cases of teratomatous skin of ovarian mature cystic teratoma, 20 cases of squamous cell carcinoma and 2 cases of squamous cell carcinoma originated from teratomatous skin. Results: Of 1913 cases of ovarian mature cystic teratoma in nearly 10 years, only two cases of squamous cell carcinoma were found in teratomatous skin, with malignant transformation rate of 0.1045%. P53 expression was detected in 2 cases squamous cell carcinoma originated from teratomatous skin and P16 overexpression in one. There were no expressions of P53 and P16 in 25 cases of teratomatous skin of ovarian mature cystic teratoma. Of 20 cases of squamous cell carcinoma P53 overexpression (positive rate of 55%) was detected in 11 cases, P16 overexpression (positive rate of 35%) in 7 cases. The positive rates of P53 and P16 expression in squamous cell carcinomas were significantly higher than that in the teratomatous skins (p< 0.001, p= 0.002). Conclusions: There was low risk of malignant transformation in teratomatous skin of ovarian mature cystic teratoma which can be explained by lower P53 and P16 expressionin teratomas than that in squamous cell carcinoma.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.8
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• pp.4049-4057
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• 2016

Background: High-risk human papillomaviruses (HR-HPV), particularly types 16 and 18, have been found to play an important role in head and neck cancer, including oropharyngeal squamous cell carcinoma (OPSCC) and oral squamous cell carcinoma (OSCC). p16, a cell cycle inhibitor, has been postulated as a surrogate marker for HR-HPV, since p16 is aberrantly overexpressed in such lesions, especially in HR-HPV-positive OPSCC. However, p16 as a surrogate marker for HR-HPV infection in cancers of the oral cavity remains controversial. Objective: The objectives of the study were to investigate the expression of p16 and the presence of HR-HPV in OSCC and oral verrucous carcinoma (VC) and to determine if p16 could be used as a surrogate marker for HR-HPV. Materials and Methods: Forty one formalin-fixed, paraffin-embedded tissues of OSCC (n=37) or VC (n=4) with clinical and histopathologic data of each case were collected. Expression of p16 was determined by immunohistochemistry, focusing on both staining intensity and numbers of positive cells. The presence of HPV types 16 and 18 was detected by polymerase chain reaction (PCR). Descriptive statistics were employed to describe the demographic, clinical, and histopathologic parameters. Associations between p16 overexpression, HR-HPV and all variables were determined by Fisher's exact test, odds ratios (ORs) and corresponding 95% confidence intervals (CIs). In addition, the use of p16 as a surrogate marker for HR-HPV was analyzed by sensitivity and specificity tests. Results: p16 was overexpressed in 8/37 cases (21.6%) of OSCC and 2/4 cases (50%) of VC. HPV-16 was detected in 4/34 OSCC cases (11.8%) and HPV-18 was detected in 1/34 OSCC cases (2.9%). Co-infection of HPV-16/18 was detected in 1/4 VC cases (25%). Both p16 overexpression and HR-HPV were significantly associated with young patients with both OSCC and VC (p<0.05, OR 20, 95% CI 1.9-211.8; p<0.05, OR 23.3, 95% CI 2.4-229.7, respectively). p16 was able to predict the presence of HPV-16/18 in OSCC with 40% sensitivity and 79.3% specificity and in VC with 100% sensitivity and 66.7% specificity, respectively. Conclusions: p16 overexpression was found in 24.4% of both OSCC and VC. HR-HPV, regardless of type, was detected in 15.8% in cases of OSCC and VC combined. The results of sensitivity and specificity tests suggest that p16 can be used as a surrogate marker for HR-HPV in OSCC and VC.

• IMMUNE NETWORK
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• v.4 no.4
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• pp.237-243
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• 2004

Background: The normal functions of the cell cycle inhibitor p16INK4a are frequently inactivated in many human cancers. Over 80% of hepatocellular carcinoma (HCC) cases lack a functional p16/Rb pathway. p16/Rb pathway, as well as p53 pathway, is considered as one of key components of tumor suppression. Methods: To study the roles of p16INK4a in HCC, a stable cell line expressing exogenous p16 was generated from SNU-449 hepatocellular carcinoma cells lacking endogenous p16, and suppression subtractive hybridization (SSH) was performed in parallel with the control cells. Results: 1) SSH identifies fibronectin (FN1), crystallin ${\alpha}B$ (CRYAB), Rac1, WASP, RhoGEF, and CCT3 as differentially-expressed genes. 2) Among the selected genes, the up-regulation of FN1 and CRYAB was confirmed by Northern blot, RT-PCR and by proteomic methods. Conclusion: These genes are likely to be associated with the induction of stress fiber and stabilization of cytoskeleton. Further studies are required to clarify the possible role of p16 in the signal transduction pathway.