Nancy C Joyce1, Deshea L Harris, Cheng C Zhu. 1. Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts 02114, USA. nancy.joyce@schepens.harvard.edu
Abstract
PURPOSE: Nuclear oxidative DNA damage increases with age in human corneal endothelial cells (HCECs) and contributes to their decreased proliferative capacity. These studies investigated whether HCECs respond to this damage by upregulating their expression of oxidative stress and DNA damage-signaling genes in an age-dependent manner. METHODS: HCECs were dissected from the corneas of young (30 years and younger) and older (50 years and older) donors. Total RNA was isolated and reverse-transcribed. Oxidative stress and DNA damage-signaling gene expression were analyzed using commercial PCR-based microarrays. Western blot analyses were conducted on selected proteins to verify the microarray results. Nuclear DNA damage foci were detected in the endothelium of ex vivo corneas by immunostaining for H2AX-Ser139. RESULTS: Four of 84 genes showed a statistically significant age-related difference in the expression of oxidative stress-related genes; however, Western blot analysis demonstrated an age-related increase in only 2 (cytoglobin and GPX-1) of 11 proteins tested. No age-related differences were detected in the expression of DNA damage-signaling genes. Western blot analysis of seven DNA damage-related proteins verified this finding. Intense nuclear staining of DNA damage foci was observed in nuclei within the central endothelium of older donors. Central endothelium from young donors consistently showed a low level of positive staining. CONCLUSIONS: HCECs respond to age-related increases in oxidative nuclear DNA damage by forming DNA damage repair foci; however, they do not vigorously defend against or repair this damage by upregulating the expression of multiple oxidative stress or DNA damage-signaling genes.
PURPOSE: Nuclear oxidative DNA damage increases with age in human corneal endothelial cells (HCECs) and contributes to their decreased proliferative capacity. These studies investigated whether HCECs respond to this damage by upregulating their expression of oxidative stress and DNA damage-signaling genes in an age-dependent manner. METHODS: HCECs were dissected from the corneas of young (30 years and younger) and older (50 years and older) donors. Total RNA was isolated and reverse-transcribed. Oxidative stress and DNA damage-signaling gene expression were analyzed using commercial PCR-based microarrays. Western blot analyses were conducted on selected proteins to verify the microarray results. Nuclear DNA damage foci were detected in the endothelium of ex vivo corneas by immunostaining for H2AX-Ser139. RESULTS: Four of 84 genes showed a statistically significant age-related difference in the expression of oxidative stress-related genes; however, Western blot analysis demonstrated an age-related increase in only 2 (cytoglobin and GPX-1) of 11 proteins tested. No age-related differences were detected in the expression of DNA damage-signaling genes. Western blot analysis of seven DNA damage-related proteins verified this finding. Intense nuclear staining of DNA damage foci was observed in nuclei within the central endothelium of older donors. Central endothelium from young donors consistently showed a low level of positive staining. CONCLUSIONS: HCECs respond to age-related increases in oxidative nuclear DNA damage by forming DNA damage repair foci; however, they do not vigorously defend against or repair this damage by upregulating the expression of multiple oxidative stress or DNA damage-signaling genes.
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