The long-term goal of our research is to understand the molecular mechanism of senescence and immortalization in human oral epithelial cells. Cellular senescence is an irreversible arrest of cell divisions that results in normal somatic cells after completing a finite number of cell divisions. Several theories of senescence have been proposed, including DNA damage theory, mitochondrial theory, and telomere hypothesis. These theories of aging focus either on (1) extrinsic environmental factors or (2) intrinsic genetic programming as the cause of cellular senescence. Among the extrinsic factors, oxidative stress imposes the most detrimental effect on cells and subcellular macromolecues due to generation of reactive oxygen species (ROS). In contrast, the telomere hypothesis maintains the important role of telomere shortening in senescence of cells. Both theories of senescence mechanism have been experimentally validated to some extent, although exceptions do exist in some cell systems. To determine the mode of senescence in normal human oral keratinocytes (NHOK), we established an in vitro model of senescence, in which the cells undergo 22+/- 3 cell divisions before senescence. Unlike fibroblasts, replicating NHOK demonstrated telomerase activity and maintained constant length of telomeres, suggesting telomere length-independent mode of senescence in these cells. In our recent study, we also found that senescence of NHOK enhanced expression of mitochondrial proteins and increased mitochondrial mass, which reflect oxidative stress response in cells. Simutaneously, the expression levels of DNA repair genes were down-regulated in senescing NHOK. Therefore, our data point to the linkage between oxidative stress, DNA damage, and senescence in NHOK. The central hypothesis of the current proposal is: Senescence of NHOK is triggered by accumulation of DNA damage that results from accumulation of oxidative stress and impaired DNA repair capacity of cells. To test this hypothesis, we propose to: (1) determine the phenotypic and molecular alterations of NHOK in response to oxidative stress, (2) investigate the changes in the DNA repair capacities of NHOK during in vitro life span with or without oxidative stress, and (3) determine the effect of disrupting the DNA repair pathways on phenotypic and molecular alterations of NHOK in response to oxidative stress. With these Aims, we will address the questions: What is the effect of oxidative stress on replication and senescence of NHOK? Is senescence associated with reduction in DNA repair activities and accumulation of DNA damage in NHOK? What is the role of mitochondria in senescence of these cells? Can we disrupt the DNA repair capacities in NHOK and trigger premature aging in cells? Can the cells with impaired DNA repair activities be rescued from premature senescence by antioxidants?

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Career Transition Award (K22)
Project #
5K22DE015316-03
Application #
6909101
Study Section
Special Emphasis Panel (ZDE1-LK (48))
Program Officer
Hardwick, Kevin S
Project Start
2003-08-04
Project End
2007-05-31
Budget Start
2005-06-01
Budget End
2006-05-31
Support Year
3
Fiscal Year
2005
Total Cost
$135,000
Indirect Cost
Name
University of California Los Angeles
Department
Dentistry
Type
Schools of Dentistry
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Kang, X; Chen, W; Kim, R H et al. (2009) Regulation of the hTERT promoter activity by MSH2, the hnRNPs K and D, and GRHL2 in human oral squamous cell carcinoma cells. Oncogene 28:565-74
Coppe, Jean-Philippe; Boysen, Megan; Sun, Chung Ho et al. (2008) A role for fibroblasts in mediating the effects of tobacco-induced epithelial cell growth and invasion. Mol Cancer Res 6:1085-98
Shin, Ki-Hyuk; Kang, Mo K; Park, No-Hee (2008) Heterogeneous nuclear ribonucleoprotein G, nitric oxide, and oral carcinogenesis. Nitric Oxide 19:125-32
Yip, Felix K; Kang, Mo K; Park, No-Hee (2007) Microarray analysis of Bmi-1 downstream genes in normal human oral keratinocytes. J Calif Dent Assoc 35:858-64
Kang, Mo K; Park, No-Hee (2007) Extension of cell life span using exogenous telomerase. Methods Mol Biol 371:151-65
Kim, Reuben H; Kang, Mo K; Shin, Ki-Hyuk et al. (2007) Bmi-1 cooperates with human papillomavirus type 16 E6 to immortalize normal human oral keratinocytes. Exp Cell Res 313:462-72
Kang, M K; Kim, R H; Kim, S J et al. (2007) Elevated Bmi-1 expression is associated with dysplastic cell transformation during oral carcinogenesis and is required for cancer cell replication and survival. Br J Cancer 96:126-33
Shin, Ki-Hyuk; Kang, Mo K; Kim, Reuben H et al. (2006) Heterogeneous nuclear ribonucleoprotein G shows tumor suppressive effect against oral squamous cell carcinoma cells. Clin Cancer Res 12:3222-8
Kang, Mo K; Shin, Ki-Hyuk; Yip, Felix K et al. (2005) Normal human oral keratinocytes demonstrate abnormal DNA end joining activity during replicative senescence. Mech Ageing Dev 126:475-9
Kang, Mo K; Kim, Reuben H; Shin, Ki-Hyuk et al. (2005) Senescence-associated decline in the intranuclear accumulation of hOGG1-alpha and impaired 8-oxo-dG repair activity in senescing normal human oral keratinocytes in vivo. Exp Cell Res 310:186-95