Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2005 Mar 8;102(10):3697-702.
doi: 10.1073/pnas.0500369102. Epub 2005 Feb 28.

Identification and Functional Significance of Genes Regulated by Structurally Different Histone Deacetylase Inhibitors

Affiliations
Free PMC article

Identification and Functional Significance of Genes Regulated by Structurally Different Histone Deacetylase Inhibitors

Melissa J Peart et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Histone deacetylase inhibitors (HDACis) inhibit tumor cell growth and survival, possibly through their ability to regulate the expression of specific proliferative and/or apoptotic genes. However, the HDACi-regulated genes necessary and/or sufficient for their biological effects remain undefined. We demonstrate that the HDACis suberoylanilide hydroxamic acid (SAHA) and depsipeptide regulate a highly overlapping gene set with at least 22% of genes showing altered expression over a 16-h culture period. SAHA and depsipeptide coordinately regulated the expression of several genes within distinct apoptosis and cell cycle pathways. Multiple genes within the Myc, type beta TGF, cyclin/cyclin-dependent kinase, TNF, Bcl-2, and caspase pathways were regulated in a manner that favored induction of apoptosis and decreased cellular proliferation. APAF-1, a gene central to the intrinsic apoptotic pathway, was induced by SAHA and depsipeptide and shown to be important, but not essential, for HDACi-induced cell death. Overexpression of p16(INK4A) and arrest of cells in G(1) can suppress HDACi-mediated apoptosis. Although p16(INK4A) did not affect the genome-wide transcription changes mediated by SAHA, a small number of apoptotic genes, including BCLXL and B-MYB, were differentially regulated in a manner consistent with attenuated HDACi-mediated apoptosis in arrested cells. We demonstrate that different HDACi alter transcription of a large and common set of genes that control diverse molecular pathways important for cell survival and proliferation. The ability of HDACi to target multiple apoptotic and cell proliferation pathways may provide a competitive advantage over other chemotherapeutic agents because suppression/loss of a single pathway may not confer resistance to these agents.

Figures

Fig. 1.
Fig. 1.
Numbers of genes up- and down-regulated at each time point after treatment with SAHA or depsipeptide. Numbers of genes that were differentially expressed by at least 1.5-fold from time 0 h to at least one later time point in response to SAHA or depsipeptide are shown.
Fig. 2.
Fig. 2.
Hierarchical cluster analysis of gene expression profiles induced by SAHA and depsipeptide in CEM cells. Genes selected as differentially expressed by either SAHA or depsipeptide based on a 1.5-fold up- or down-regulation from time 0 h to at least one later time point were clustered based on standard correlation coefficients by using SAHA expression ratios (Left), and this gene order was retained for the depsipeptide cluster (Right). Each row represents a separate element on the microarray, and each column represents time in culture with SAHA or depsipeptide. The scale in this and other figures shows the level of expression, where red indicates increased gene expression, green indicates decreased expression, and the intensity of color correlated to the magnitude change. Black indicates no change.
Fig. 3.
Fig. 3.
Gene expression profiles of SAHA- and depsipeptide-selective genes. Genes identified as selectively regulated by SAHA (n = 150) (A) or depsipeptide (n = 364) (D) are shown, with their corresponding expression in response to the other HDACis shown in C and B, respectively. Up-regulated genes are depicted in red, down-regulated genes are depicted in green, and each line corresponds to a single gene. Genes were considered to be selectively regulated by SAHA if they (i), were significant for SAHA; (ii), were not significant for depsipeptide; and (iii), showed a significant difference in response to SAHA relative to depsipeptide. The third condition was also tested by using an F statistic and was used to eliminate genes that responded similarly to the two compounds but narrowly failed to reach significance for one. The reverse criterion was used to select genes specifically regulated by depsipeptide.
Fig. 4.
Fig. 4.
Functional clustering of SAHA- and depsipeptide-regulated cell proliferation genes. (A) Genes that encode early regulated antiproliferative proteins. (B) Genes that encode cell cycle-related proteins. For this and all similar data, genes were hierarchically clustered based on standard correlation coefficients.
Fig. 5.
Fig. 5.
Functional clustering of SAHA- and depsipeptide-regulated apoptosis genes. (A) Genes that encode members of the intrinsic death pathway. (B) Genes that encode apoptotic proteins mentioned in the text. (C) Genes that encode members of the death receptor pathway.
Fig. 6.
Fig. 6.
Validation of microarray data by SYBR Green quantitative real-time PCR. (A) SAHA-mediated gene expression changes obtained from microarray assays. (B) The same RNA as used for SAHA microarray experiments was subjected to quantitative real-time PCR. Values for each gene were normalized to expression levels of L32, and fold induction at each time point was calculated relative to time 0 h. Data are representative of at least three independent experiments.
Fig. 7.
Fig. 7.
Knockdown of APAF1 expression attenuates SAHA-induced apoptosis. (A) CEM and Jurkat cells transfected with no siRNA (lanes 1 and 4), 1.25 μM control (GFP) (lanes 2 and 5), or APAF1 siRNA (lanes 3 and 6) were analyzed 48 h after transfection for APAF1 expression by Western blot. The blot was reprobed for α-tubulin as a loading control. (B) At 48 h after electroporation, CEM and Jurkat cells were treated with SAHA (2.5 μM) for 18 h, then collected and scored for apoptosis by propidium iodide staining. Data are expressed as the mean ± SD of three separate experiments. Statistical differences between samples (P < 0.05) were determined by using the Mann-Whitney U test and are denoted by an asterisk.
Fig. 8.
Fig. 8.
Functional clustering of specific SAHA-regulated apoptosis genes in cycling and arrested cells. Expression profiles of apoptosis-related genes identified as specifically regulated by SAHA in cycling (A) or arrested cells (B).

Similar articles

See all similar articles

Cited by 182 articles

See all "Cited by" articles

Publication types

LinkOut - more resources

Feedback