Role for histone deacetylase 1 in human tumor cell proliferation

Abstract

Posttranslational modifications of core histones are central to the regulation of gene expression. Histone deacetylases (HDACs) repress transcription by deacetylating histones, and class I HDACs have a crucial role in mouse, Xenopus laevis, zebra fish, and Caenorhabditis elegans development. The role of individual class I HDACs in tumor cell proliferation was investigated using RNA interference-mediated protein knockdown. We show here that in the absence of HDAC1 cells can arrest either at the G(1) phase of the cell cycle or at the G(2)/M transition, resulting in the loss of mitotic cells, cell growth inhibition, and an increase in the percentage of apoptotic cells. On the contrary, HDAC2 knockdown showed no effect on cell proliferation unless we concurrently knocked down HDAC1. Using gene expression profiling analysis, we found that inactivation of HDAC1 affected the transcription of specific target genes involved in proliferation and apoptosis. Furthermore, HDAC2 downregulation did not cause significant changes compared to control cells, while inactivation of HDAC1, HDAC1 plus HDAC2, or HDAC3 resulted in more distinct clusters. Loss of these HDACs might impair cell cycle progression by affecting not only the transcription of specific target genes but also other biological processes. Our data support the idea that a drug targeting specific HDACs could be highly beneficial in the treatment of cancer.

FIG. 1.

siRNA-mediated knockdown of HDAC1, HDAC2, and HDAC3 in U2OS, MCF7, and MCF10A cells. The loss of both HDAC1 and HDAC2 produced a hyperacetylation of histones H3 and H4. (A to C) Relative (rel.) expression levels of the indicated mRNAs were determined by real-time PCR, as described in Materials and Methods. Averages of three independent experiments are shown, and standard deviations are indicated by the error bars. (D to F) Western blots of U2OS whole-cell extracts after transfection of siRNA HDAC1, HDAC2, HDAC3, and HDAC1 plus HDAC2. Cells were transfected and processed as described in Materials and Methods, and 30 μg was loaded per lane. As a control (CTRL), we used transfection of antiluciferase siRNA.

FIG. 2.

Absence of either HDAC1 or HDAC3 causes a defective proliferation. (A to C) Growth curves of U2OS, MCF7, and MCF10A transfected with HDAC siRNAs. The increase of absorbance at 595 nm is directly correlated with cell proliferation, as described in Materials and Methods. Each curve represents the average of three independent experiments, and the standard deviation is indicated by the error bars. (D to F) CFU assay of U2OS, MCF7, and MCF10A cells transfected with HDAC siRNAs. The ratio of the CFU number of each sample/CFU number of the control was calculated. Each column represents the average of three independent experiments, with the respective standard deviations. CTRL, control.

FIG. 3.

Absence of the mitotic marker H3-P in HDAC1-deficient cells. U2OS, MCF-7, and MCF10A cells were analyzed for the presence of H3-P. (Top) Distribution of the HDAC1 signal over the siRNA-control (CTRL)-transfected and siRNA-HDAC1-transfected populations. The dashed line represents the arbitrary threshold used to distinguish HDAC1-negative (gate I) from HDAC1-positive (gate II) cells. (Middle) Distribution of H3-P signals in gate I and gate II. The dashed line represents the arbitrary threshold used to distinguish between positive and negative H3-P cells. (Bottom) Scatter plot of HDAC1 signal versus H3-P signal. The percentages inside the plot indicate the percentages of double positive cells.

FIG. 4.

HDAC1-deficient cells arrest in G₁ or die during G₂/M transition. (A) Asynchronous (As) siRNA-control (CTRL)-transfected (black) and siRNA-HDAC1-transfected (gray) U2OS cells were blocked in G₁/S transition by use of thymidine and then released in fresh medium. (Top) DNA content was analyzed by FACS analysis of PI-stained cells at different time points after the release in fresh medium. (Bottom) Biparametric FACS analysis using anti-H3-P and PI staining was performed to calculate the percentages of cells in G₁, S, G₂, and M phases at each time point. (B) U2OS control- and HDAC1-interfered cells were blocked in G₁/S transition by use of thymidine and then released in medium containing nocodazole. (Top) DNA content was analyzed by FACS analysis of PI-stained cells at different time points. (Middle) Biparametric FACS analysis using anti-cyclin A (CYCA) and PI staining was performed to calculate the percentages of cells in G₁/S, G₂, and M phases at each time point. (Bottom) DNA content was analyzed by FACS analysis to evaluate the percentages of sub-G₁ cells. (C) U2OS control- and HDAC1-interfered cells were blocked in G₂/M transition by use of nocodazole and then released in fresh medium. (Top) DNA content was analyzed by FACS analysis on PI-stained cells at different time points. (Bottom) DNA content was analyzed by FACS analysis to evaluate the percentages of sub-G₁ cells. (D) (Top) Percentages of cells in the control- and HDAC1-interfered population able to undergo mitosis at least once in 30 h. (Bottom) Images of time-lapse experiments with U2OS siRNA-CTRL-transfected and siRNA-HDAC1-transfected cells. Control cells experience normal mitosis (white arrowheads), while HDAC1 knockdown cells cannot enter in mitosis and they die (yellow and black/white arrowheads).

FIG. 5.

HDAC1 knockdown cells undergo apoptosis. (A) Effect of siRNA HDAC1 on caspase-3 activation by FACS analysis. The bars represent the percentages of cleaved caspase-3-positive cells in control (CTRL) and siRNA-HDAC1-transfected U2OS cells after 96 h. The values correspond to the averages of three independent experiments, and the error bars indicate the associated standard deviations. (B) Western blots of U2OS whole-cell extracts after transfection of control or siRNA HDAC1. Cells were collected 120 h after transfection, and 50 μg of lysates was loaded per lane. As a control, we used an injection of antiluciferase siRNA. The anti-caspase-3 antibody used was able to recognize both full-length inactive and cleaved active forms.

FIG. 6.

Gene expression profiles. Supervised hierarchical clustering (according to Pearson's correlation) was performed using comparative expression values of the gene lists indicated above each dendrogram (for gene lists, see supplementary Table 1 posted at http://bio.ifom-ieo-campus.it/supplementary/MCB49407/). In all cases, the expression value for the control cells was used as the baseline (value of 1).

FIG. 7.

Gene expression profiles and quantitative real-time PCR. (A) Graphic representation of the expression patterns of genes functionally involved in apoptosis and proliferation regulated by ablation of HDAC1 or HDAC1 plus HDAC2 in U2OS cells. (B) Validation of microarray data by TaqMan quantitative real-time PCR. The error bars indicate the standard deviations from three independent experiments. CTRL, control.