HISTONE DEACETYLASE INHIBITION INDUCES ALTERED GENE EXPRESSION PROFILES IN PROSTATE CANCER

Introduction. Chromatin reorganisation associated withreversible epigenetic modifications is consideredresponsible for changes in gene expression observedduring prostate cancer progression. Histone acetylationis an important epigenetic modification promoting an‘open’ chromatin configuration and transcriptionalactivation. Trichostatin A (TSA), an inhibitor of HDACactivity, has been associated with a globalhyperacetylation that may lead to the disruption ofchromatin phenotype and the transcription of a selectionof genes that inhibit tumour growth. We have alreadyshown that at concentrations of >50ng/ml, TSA inducesapoptosis and G2M cell cycle arrest in these cell lines.However, even at low doses of TSA, which have noobvious impact on the cell cycle or apoptosis (sub-lethaldoses), subtle changes in chromatin phenotype areinduced. The aim of this study was to establish theunderlying alterations in gene expression in normalprostate cell line (PNT1A) and lymph node metastaticcell line (LNCaP) induced by Trichostatin A.Materials and Methods. Following treatment of PNT1Aand LNCaP cell lines with sublethal (12ng/ml) and lethal(100ng/ml) doses of TSA, total cellular RNA wasisolated from cell samples. cDNA was synthesised fromextracted RNA, and control and test samples weredifferentially labelled with fluorescent Cy3 and Cy5 dyesin situ for direct comparison. Dye swaps wereincorporated into the study design to ensure preciseinterpretation of expression profiles. Hybridisation ofappropriately labelled cDNA to MWG Human 30K arrayA containing 9,984 oligonucleotide probes, wasperformed under optimum binding conditions. Scanningand analysis of the microarray slide was completed usingGenePix and Acuity (Axon Instruments) software.Results. Gene expression was clearly disrupted in bothPNT1A and LNCaP in response to TSA inducedhyperacetylation. A significant trend for up-regulation ingene expression was identified in PNT1A following highconcentrations of TSA. This includes the increasedexpression of cell cycle genes such as a 2.27 foldincrease in cyclin g2, as well as a selection of genesrelated to the onset of apoptosis. This is likely to beassociated with increased apoptosis and cell cycle arrestseen at these concentrations. Interestingly, altered geneexpression was also evident in cells treated with sublethaldoses of TSA, where no cytotoxic effects of the treatmentare visible. Several functional gene groups were found tobe down-regulated across both cell lines in response toTSA, including a range of general transcription factors,along with genes involved in the MAP Kinase pathway.A consistent up-regulation can also be reported in both asubset of homeobox genes, and cytochrome c oxidasegenes. Also important is the observation that theKallikrein (prostate specific antigen) gene has beenfound to be down-regulated in both normal prostate andmetastatic cell lines in response to TSA treatment.Conclusions. Histone hyperacetylation and subsequentchromatin redistribution are associated with thedisruption of gene expression profiles in prostate cancer.Disruption in gene expression occurs in the absence ofcytotoxic effects which may be due to subtle chromatinphenotypic changes observed at sublethal doses of TSA,and may therefore be indicative of those genes directly orindirectly regulated by histone acetylation.