December 2002
Volume 43, Issue 13
ARVO Annual Meeting Abstract  |   December 2002
Measurement of p16 in Canine Lens Epithelial Cells in Response to Oxidative Stress
Author Affiliations & Notes
  • C Colitz
    Ohio State University Columbus OH
  • A Whittington
    Louisiana State University Baton Rouge LA
  • J Warren
    Louisiana State University Baton Rouge LA
  • Footnotes
    Commercial Relationships   C. Colitz, None; A. Whittington, None; J. Warren, None. Grant Identification: NIH Grant K8EY00414A
Investigative Ophthalmology & Visual Science December 2002, Vol.43, 2356. doi:
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      C Colitz, A Whittington, J Warren; Measurement of p16 in Canine Lens Epithelial Cells in Response to Oxidative Stress . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2356.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract: : Purpose:We previously demonstrated that lens epithelial cells (LEC) have telomerase activity. Neoplastic cells can have a reciprocal relationship between telomerase activity and levels of p16. Telomere length, development of senescence and p16 expression are influenced by oxidative stress. Since oxidative stress is implicated in cataractogenesis, the purpose of these experiments was to determine how exposure of LEC to tertiary butyl-hydroperoxide (TBHP) induced oxidative stress affects telomerase activity and p16 expression. Methods:Fresh canine lenses were incubated in keratinocyte growth medium and M199 with serum. In the first experiment, lenses were exposed to media with or without 0.5mM TBHP for varying time periods (8 lenses per group; 15, 25, 40, 60, 150 minute and overnight) In the second experiment, lenses were exposed to media containing 0.5 mM TBHP for one hour and then recovered in media without TBHP for varying time periods (8 lenses per group; 5, 60, 180 minute and overnight recoveries, no recovery, control group without treatment). Following these treatments, the anterior lens capsules with adhered LEC were collected. P16 levels in LEC were measured by ELISA using a dig- labeled antibody to p16. Telomerase activity was measured by TRAP-ELISA. Results:There was no change in p16 levels following direct exposure to TBHP. Telomerase activity was significantly up-regulated at the 150 minute point (p<0.01). The second group had significantly lower p16 levels (0.179-0.280) than the control group (0.441, p<0.05) at 15, 25, 40, 60, 180 minute and overnight recovery. Telomerase activity was significantly elevated at the 15, 60 and 180 minute and overnight recovery times (2.173-2.268) vs control (1.547, p<0.02). Conclusion:p16 is a cell cycle inhibitor that is down-regulated when telomerase activity is up-regulated in many tumors contributing to unregulated growth. Acute exposures with TBHP did not alter p16 or telomerase activity (except at 150 minutes) which may indicate that either the length of exposure may too short to cause changes in the proteins or the proteins' half-lives may be too long for changes to be seen in this experiment. In the recovery experiment, telomerase activity was up-regulated and p16 was down-regulated, similar to tumor cells. The recovery process following oxidative stress in LEC may involve proliferation of cells to replace damaged or dead cells. Future experiments will further examine the proliferative capacity of LEC under similar conditions and the response of p16 when LEC are chronically exposed to oxidative stress.

Keywords: 504 oxidation/oxidative or free radical damage • 338 cataract 

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