Inhibition of histone deacetylase in cancer cells slows down replication forks, activates dormant origins, and induces DNA damage

Abstract

Protein acetylation is a reversible process regulated by histone deacetylases (HDAC) that is often altered in human cancers. Suberoylanilide hydroxamic acid (SAHA) is the first HDAC inhibitor to be approved for clinical use as an anticancer agent. Given that histone acetylation is a key determinant of chromatin structure, we investigated how SAHA may affect DNA replication and integrity to gain deeper insights into the basis for its anticancer activity. Nuclear replication factories were visualized with confocal immunofluorescence microscopy and single-replicon analyses were conducted by genome-wide molecular combing after pulse labeling with two thymidine analogues. We found that pharmacologic concentrations of SAHA induce replication-mediated DNA damage with activation of histone gammaH2AX. Single DNA molecule analyses indicated slowdown in replication speed along with activation of dormant replication origins in response to SAHA. Similar results were obtained using siRNA-mediated depletion of HDAC3 expression, implicating this HDAC member as a likely target in the SAHA response. Activation of dormant origins was confirmed by molecular analyses of the beta-globin locus control region. Our findings demonstrate that SAHA produces profound alterations in DNA replication that cause DNA damage, establishing a critical link between robust chromatin acetylation and DNA replication in human cancer cells.

Figure 1

Time- and concentration-dependent induction of γH2AX in response to SAHA. A, Breast carcinoma MCF-7 cells were exposed to 1.25 μM SAHA for 1 to 24 hours. On the left, representative Western blots showing acetylation of histone H3 on lysine 9 (H3K9ac) and γH2AX induction. Total H2AX was examined in parallel. Tubulin was used as a loading control. On the right, representative images of γH2AX observed by immunofluorescence microscopy in interphase nuclei. Nuclear outlines are shown. B, MCF-7 cells were treated for 4 hours with the indicated SAHA concentrations. On the left, concentration-dependent induction of H3K9ac and γH2AX. Actin was used as a loading control. On the right, representative images of γH2AX foci observed by immunofluorescence microscopy. Nuclear outlines are shown. C, Quantitation of γH2AX induction by SAHA in MCF-7 cells. On the left, quantification of γH2AX foci induced by increasing concentrations of SAHA for 4 hours. In the middle and right, quantification of γH2AX foci induced by 1.5 μM SAHA for the indicated times. Error bars represent the SD of three independent experiments. D, Induction of γH2AX by SAHA in breast carcinoma MDA-231 cells (1.25 μM for 4 hours; left) and in colon carcinoma HCT116 cells treated with 1.25 μM SAHA for the indicated times (right). Experiments have been repeated at least 3 times with consistent results.

Figure 2

Induction of DNA double-strand breaks (DSB) by SAHA. A, Colocalization of γH2AX foci (middle) with phospho-53BP1 (left) foci in MCF-7 cells treated with SAHA (10 μM) or camptothecin (CPT; 1 μM) for 4 hours. Nuclear outlines are shown with dotted lines. B, Representative images of COMET tails obtained in an untreated sample and in MCF-7 cells treated with 10 μM SAHA for 16 hours. C, Quantification of the COMET tail length in cells treated for 4 or 16 hours with 10 μM SAHA (a.u. = arbitrary unit; see Material and Methods). The assay was repeated three times. The graph shows the result of a representative experiment. The statistical significance was calculated with the Mann-Whitney test. D, Quantification of the COMET tail moment for samples including at least 35 cells each. In the Box Plot the gray area boxes represent the interval where the middle 50% of the data lies and the vertical line in the grey box represents the median. The horizontal bars extending from the boxes encompass the data set within a 95% confidence interval. The dots are outliers.

Figure 3

Single cell analyses of DNA replication-associated γH2AX induction by SAHA. A, MCF-7 cells were treated with 1.25 μM SAHA for 4 hours and pulse-labeled with CldU during the last hour of treatment to label replication factories (33) (schematized at top). CldU and γH2AX were detected by immunofluorescence in red and green, respectively. Representative cells treated with SAHA show the colocalization of γH2AX with DNA replication factories (CldU). B, MCF-7 cells were pre-treated with 1μM aphidicolin (APH) followed by 4 hours co-exposure with APH and SAHA (scheme at the top). Bottom, quantification of the γH2AX response to 1.25 or 10μM SAHA in the absence or presence of APH. Bars represent SD for at least 3 independent determinations. C, FACS profile of cells treated for 4 hours with APH or SAHA at the indicated concentrations and pulse-labeled with IdU for the last 5 min of the treatment. The horizontal dotted lines show the decreased incorporation of IdU in APH- and SAHA-treated cells. D, Immunofluorescence analyses of DNA replication dynamics in individual cells treated with SAHA. MCF-7 cells were pulse-labeled for 45 min with IdU, treated for 4 hours or 45 min with SAHA and pulse-labeled with CldU during the last 45 min. IdU and CldU were then detected in green and red, respectively. Representative nuclei from untreated (control) and SAHA-treated cells are shown. Bottom left, Quantification of the CldU/IdU intensity ratio. The control data have been normalized to 1 and the error bar in the SAHA-treated sample represents the SD of three independent experiments.

Figure 4

Reduction of replication fork velocity by SAHA. A, Cell treatment protocol: MCF-7 cells were treated with 1.5 or 10 μM SAHA for 4 hours; the IdU pulse was performed in the last 20 minutes of treatment; after wash cells have been pulsed with further 20 minutes with CldU. B, Representative image and fork velocity analysis on individual combed molecules from untreated cells (top) and cells treated with 1.5 μM (middle) or 10 μM SAHA (bottom). Fork velocity was measured during the IdU (green) and CldU (red) pulses separately. n: number of signals scored; S.D.: standard deviation. For each treatment, at least three independent combing experiments have been performed, showing consistent results. C, Western blotting analyses demonstrate no detectable effect of SAHA on ribonucleotide reductase large and small subunits (RR1 and RR2, respectively) and thymidylate synthase (TS) after exposure to 10 μM SAHA for the indicated times. Histone hyperacetylation (H3K9ac) and γH2AX induced by SAHA are also shown at the same time points.

Figure 5

Reduction of replication fork velocity by HDAC3 downregulation. A, Western blotting showing the down-regulation of HDAC3 by siRNA in MDA-MB-231 cells. B, FACS analysis of cells transfected for three days with a control or HDAC3 siRNA. C, Fork velocity measured in cells treated with a control siRNA (top) and the HDAC3 siRNA (bottom). The median values of measured fork speeds are indicated for the two samples. Similar results were obtained in two independent transfection experiments. Representative image are shown at right.

Figure 6

Dormant origins of replication are activated and average inter-origin distance is reduced after SAHA treatment. A, MCF-7 cells were treated with 1.5 or 10 μM SAHA for 4 hours and replication forks were analyzed using a dual pulse with IdU and CldU. B, Inter-origin distance measured on individual molecules from untreated cells (No SAHA), and cells treated with 1.5 μM and 10 μM SAHA. C, Schematic representation of the human β-globin region (LCR: Locus Control Region) and position of the primers used for the RT-PCR. D, Origin activity analyzed in the LCR and human β-globin replicator (hBG) by RT-PCR on nascent strand DNA. The bars represent standard deviation.