September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Analysis of ultraviolet-B irradiation induced changes in the hTCEpi corneal epithelial cell line including effect of recovery time on mRNA and protein expression
Author Affiliations & Notes
  • Kristin Olsson
    Integrative Physiology and Pathobiology, Tufts University, Boston, Massachusetts, United States
  • Christopher Talbot
    Integrative Physiology and Pathobiology, Tufts University, Boston, Massachusetts, United States
  • James Kenneth Kubilus
    Integrative Physiology and Pathobiology, Tufts University, Boston, Massachusetts, United States
  • Thomas Linsenmayer
    Integrative Physiology and Pathobiology, Tufts University, Boston, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Kristin Olsson, None; Christopher Talbot, None; James Kubilus, None; Thomas Linsenmayer, None
  • Footnotes
    Support  NIH Grant R01 EY 013127
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 4374. doi:
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    • Get Citation

      Kristin Olsson, Christopher Talbot, James Kenneth Kubilus, Thomas Linsenmayer; Analysis of ultraviolet-B irradiation induced changes in the hTCEpi corneal epithelial cell line including effect of recovery time on mRNA and protein expression. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4374.

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

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Abstract

Purpose : Corneal epithelial cells are constantly exposed to UV irradiation. Previous studies focused on chicken corneal epithelial cells and the molecule, ferritin, and its chaperone molecule, ferritoid, which lend protection upon UV-B exposure. Current studies transitioned to human telomerase-immortalized corneal epithelial (hTCEpi) cells and focused on determining the normal apoptotic response of these cells to UV-B irradiation. Future studies will seek to elucidate protective capabilities of ferritin and ferritoid in hTCEpi cells transfected with the molecules and exposed to UV-B.

Methods : hTCEpi cells were exposed to 12.5mJ/cm2 of UV-B radiation based on previous studies. mRNA and protein isolated from cells exposed to 12.5mJ/cm2 UV-B and from unexposed cells were used in PCR arrays targeting human apoptotic markers and in western blot analysis. Follow-up studies repeated these studies, but with various recovery times (3, 7, 10, and 24 hours), to better determine potential translational regulation.

Results : Initial PCR arrays showed changes in several apoptotic markers, such as CD27, TNFRSF21, and NFkB1. Follow-up, targeted qPCR analysis demonstrated results that mirrored initial PCR array results with elevations in CD27 expression and decreases in TNFRSF21 and NFkB1 levels. However, western blot against these targets showed the opposite effect of UV-B irradiation for CD27 protein levels and reduced amounts of change in TNFRSF21 and NFkB1 protein levels. Additional studies using different recovery times after UV-B radiation exposure allowed for better determination of the translational regulation that affects these pathways.

Conclusions : Based on previous studies and those described here, we have shown that a dose of UV-B irradiation of 12.5mJ/cm2 causes cell damage and alters mRNA levels of factors involved in apoptosis. However, the protein levels of these genes do not always directly correlate with the mRNA levels. To better determine the effect of translational regulation of these cells following UV-B exposure, various recovery times post-exposure were employed. Future studies will determine if UV-B exposed hTCEpi cells transfected with ferritin and ferritoid receive protection from these molecules or if the apoptotic response demonstrated here in non-transfected cells persists.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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