Abstract
Purpose :
Previous data from our lab revealed histone deacetylase 1 and 2 (HDAC1/2) expression was significantly reduced in human retinal pigment epithelium (RPE) with advanced dry AMD. Cellular response to HDAC inhibition is complex and the pharmacological HDAC1/2 inhibitor, romidepsin, induces widespread cellular changes including cell death, cell cycle arrest, autophagy, and altered protein acetylation. In this study, we have evaluated the effects of HDAC1/2 inhibition on cell viability and histone H3 and H4 acetylation (H3ac/H4ac) in RPE cell lines as compared to numerous other cell types.
Methods :
ARPE-19, primary human RPE (hRPE), HMVEC (endothelial cells), HMC-3 (microglial cells), and THP-1 (monocyte) cell cultures (Lonza, ATCC) were treated with 0.5, 1.0, and 5.0 ng/ml romidepsin (Selleckchem, S3020, 24/48/72h). Cell viability was measured (MTS, Promega, G3580) at 0, 24, 48, and 72h, and statistical analyses was performed (Kruskal-Wallis). Histone acetylation (H3Ac/H4Ac) after romidepsin treatment was evaluated by Western blot.
Results :
Romidepsin treatment significantly reduced RPE cell (ARPE19/hRPE) viability, and increased H3/H4 hyperacetylation. At 72h, romidepsin treated ARPE-19 cells (1.0 ng/ml) exhibited significantly decreased cell viability (69.04±1.97%). Similarly decreased cell viability was observed in hRPE (35.7±8.9%), HMVEC (46.6±1.09%) and HMC3(59.2±4.49%) cells. THP-1, a monocytic cell line, showed the least effect with romidepsin with enhanced cell viability at 24h post-treatment. THP-1 cells were resistant to romidepsin treatment and at 72h the loss in cell viability was only 6.56±2.22%. Densitometry analyses revealed romidepsin (5ng/mL) induced the highest levels of H3ac (22-fold) and H4ac (4-fold) in ARPE19, followed by hRPE (H3ac,14-fold; H4ac, 13.3-fold). In contrast, romidepsin treatment of THP-1 cells induced significantly less H3ac (8-fold) and H4ac (2.8-fold).
Conclusions :
The cellular changes associated with HDAC1/2 inhibition are cell-type dependent. We observed variable loss of cell viability and global acetylation in numerous different cell types. The RPE is exquisitely sensitive to HDAC1/2 inhibition with robust loss of cell viability and significantly increased H3ac/H4a. Thus, loss of HDAC1/2 function could play an important role in the RPE degeneration in patient with atrophic dry AMD.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.