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Y Guo, SL Bernstein; HSC70 Down-Regulation in a Cell Culture Model of Age-Related Retinal Gene Expression . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1448.
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Purpose:Age-related gene expression changes have been documented in many systems, but their functional effects are largely unknown. We recently reported that the mRNA for the constitutively-expressed form of heat shock-70 (HSC70) decreases during aging in human and rhesus retina. HSC70 plays an important role in the cellular stress response, and regulation of normal levels of active and denatured proteins. Prior attempts to knock out HSC70 gene function have been unsuccessful. We wished to evaluate the functional effects of age-related HSC70 down-regulation during aging. Methods:We used the spontaneously derived human retinal pigment epithelial cell (D407) line, and the rat pheochromocytoma (PC-12) cell lines. Cells were incubated with 30, 50 and 60 uM morpholino antisense or control oligonucleotides (Genetools, OR), generated from sequences derived from Genbank human HSC70 (Y00371) and rat HSC70 (Y00054). The protein levels of HSC70, HSP70 and ubiquitin were analyzed by Western analysis. Cell messenger RNA level was analyzed by using semi-quantitative RT-PCR. The apoptotic response to HSC70 down-regulation was monitored using TUNEL assay. Results:Antisense to HSC70 specifically down regulates HSC70 protein levels. At highest levels, HSC70 protein showed a 30% down regulation in D407 cells, and 40-70% down regulation in PC12 cells. Interestingly, there was an increase in PC-12 cell apoptosis at the greatest levels of HSC70 down regulation, compared to control cells. There was a reactive increase in HSP70 protein. Conclusion:HSC70 protein can be reduced in vitro to levels similar to those seen in elderly primate retina. The induction of apoptosis at reduced HSC70 levels suggests that HSP70 species cannot substitute for all HSC70 functions. The role of age-related reduction of HSC70 in maintaining normal levels of ubiquitinated proteins and lipofuscin will be discussed.
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