September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Paxillin regulates UM cell survival by PKC-delta signaling
Author Affiliations & Notes
  • Vanessa Marie Morales
    Ophthalmology, University of Tennessee Health Science Center, Memphis, Tennessee, United States
    Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, Tennessee, United States
  • Sumana R Chintalapudi
    Ophthalmology, University of Tennessee Health Science Center, Memphis, Tennessee, United States
  • Zachary Goldsmith
    Ophthalmology, University of Tennessee Health Science Center, Memphis, Tennessee, United States
  • Bradley Gao
    Ophthalmology, University of Tennessee Health Science Center, Memphis, Tennessee, United States
  • Pia Mendoza
    Ophthalmology, Emory University, Atlanta, Georgia, United States
  • Hans E Grossniklaus
    Ophthalmology, Emory University, Atlanta, Georgia, United States
  • Matthew W Wilson
    Ophthalmology, University of Tennessee Health Science Center, Memphis, Tennessee, United States
    Surgery, St. Jude Children's Research Hospital, Memphis, Tennessee, United States
  • Footnotes
    Commercial Relationships   Vanessa Morales, None; Sumana Chintalapudi, None; Zachary Goldsmith, None; Bradley Gao, None; Pia Mendoza, None; Hans Grossniklaus, None; Matthew Wilson, None
  • Footnotes
    Support  Research to Prevent Blindness, SJCRH Endowment, West Cancer Center
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 4120. doi:
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    • Get Citation

      Vanessa Marie Morales, Sumana R Chintalapudi, Zachary Goldsmith, Bradley Gao, Pia Mendoza, Hans E Grossniklaus, Matthew W Wilson; Paxillin regulates UM cell survival by PKC-delta signaling. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4120.

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

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Abstract

Purpose : Protein Kinase C (PKC) members play a role in cell proliferation and apoptosis in mammalian cells. One of its isoforms, PKC-delta, is considered to be involved in anti- and pro-apoptotic pathways, being potential molecular target to study in tumor cells. In this study we investigate the expression of PKC-delta in healthy and uveal melanoma (UM) eyes and how manipulation of the cytoskeleton in UM cell lines regulates PKC-delta and ultimately, UM cell survival.

Methods : PKC-delta expression was analyzed from primary (Mel270, 92.1) and metastatic (OMM2.5) UM cell lines by quantitative PCR (qPCR) analysis. Protein expression of phosphorylated and total PKC-delta was evaluated by Western blot and flow cytometry analyses (phospho-PKC delta: Ser643, both antibodies from Cell Signaling Technology). We culture UM cell lines with compound 6-B345TTQ at 10uM for the downregulation of the cytoskeletal adaptor paxillin. Apoptosis was measured by Annexin V/PI labeling in UM cell lines and BAX expression of ocular tissue by immunohistochemistry (D2E11, Cell Signaling Technology).

Results : We found higher expression of PKC-delta protein in UM eyes compared to healthy eyes (n=5 patients). UM cell lines show high percentage of phopho-PKC delta. The phosphorylation status of all tested UM cell lines was reduced when the paxillin inhibitor was used (untreated versus treated: 90±1 versus 12±8, p=2.9 x 10-5). The use of the paxillin inhibitor caused a decrease in cell viability by augmentation of apoptosis. Apoptosis measurement by Bax labeling increased in the choroid area of the UM patient and in some areas of the sclera and the retina.

Conclusions : In this study, phosphorylation of PKC-delta was found higher in eyes with UM than in their healthy counterparts. Inhibition of the cytoskeletal adaptor paxillin reduced phosphorylation in PKC-delta, accompanied by an increase in cell death. These results illustrate a potential therapy for UM and a novel target fir next-generation drug development. Paxillin regulates UM cell survival by PKC-delta signaling

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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