Sulforhodamine B Growth Inhibition Assays
Cells were seeded into 96-well plates at a density appropriate for exponential growth at the beginning of the assay. After overnight attachment, cells were treated with NU9056 over a range of concentrations for 3 days (equivalent to approximately 3 population doubling times). Cells were then fixed in 10% (w/v) TCA and stained with sulforhodamine B (SRB) as previouslycH2AX Immunofluorescence293T cells were seeded onto coverslips in 6 well plates and allowed to adhere for 24 hours. Figure 7. NU9056 inhibits Tip60 accumulation and ATM phosphorylation in response to ionising radiation. To demonstrate the inhibition of Tip60 activity by NU9056, levels of pATM, Tip60 and cH2AX were investigated in response to IR in the presence and absence of drug. LNCaP cells were treated with 24 mM NU9056 or vehicle control for 1 hour prior to 0.5 Gy IR. Protein lysates were collected at various time points post-IR and analysed by Western blotting for (A) pATM and (B) Tip60 levels. (C) 293T, (D) LNCaP and (E) LNCaP-CdxR cells were pretreated with NU9056 (24 mM) or vehicle control for 1 hour prior to 2 Gy IR. Cells were fixed and stained for cH2AX foci over time and foci determined by immunofluorescence for 293T cells and flow cytometry for LNCaP and LNCaP-CdxR. Experiments were repeated 3 times with representative images shown and quantified data shown as mean % cells stained for cH2AX 6 standard deviation. applied to the cells for 1 hour prior to exposure to 2 Gy IR. Cells were then fixed in 4% (v/v) paraformaldehyde and stained for cH2AX as previously described [39] using primary anti-phosphohistone H2AX (Ser139) antibody (clone JBW301, mouse monoclonal antibody; Upstate, Millipore Corp).
cH2AX FACS Analysis
LNCaP cells were seeded onto 6 well plates and allowed to attach overnight. The appropriate concentration of NU9056 was then applied for the indicated time. Medium containing any floating cells was removed and retained, and cells were trypsinised from the plates and added to the removed media. Cells were fixed using 50 ml cytofix/ cytoperm (BD) then incubated in primary anti-phospho-histone H2AX (Ser139) antibody for 2 hours. Cells were then washed twice in 1 X Permwash (BD) and incubated in secondary rabbit anti-mouse FITC conjugated antibody (DAKO) for 2 hours in the dark. Fluorescence was detected on the FL-1 channel of FACSCalibur (BD).
Figure S5 Caspase 9 cleavage in LNCaP cells. LNCaP cells were seeded onto 6 well plates for 24 hours, then NU9056 was applied for 1? days. All cells were collected and fixed with cytofix/cytoperm (BD) then caspase 9 assay kit (BD) was used to assess caspase cleavage activity by flow cytometry. Fluorescence was detected on the FL-1 channel of FACSCAN. Experiments were repeated 3 times. Data shown are dot plots of 10,000 events for 1 representative experiment. (TIF) Figure S6 Caspase 3 cleavage in LNCaP cells. LNCaP cells were seeded onto 6 well plates for 24 hours, then NU9056 was applied for 1? days. All cells were collected and fixed with cytofix/cytoperm (BD) then caspase 3 assay kit (BD) was used to assess caspase cleavage activity by flow cytometry. Fluorescence was detected on the FL-1 channel of FACSCAN. Experiments were repeated 3 times. Data shown are dot plots of 10,000 events for 1 representative experiment. (TIF) Figure S7 Caspase 3 cleavage in LNCaP cells in response to HAT inhibition. LNCaP cells were seeded out onto 6 well plates and incubated with GI50 concentrations of HAT inhibitors for 96 hours. Cells were then harvested and caspase 3 cleavage detected using an anti-cleaved caspase 3 FITC conjugated antibody and flow cytometry. (A) Mean fold change of 3 experiments 6 standard deviation is shown. (B) Dot plots of 10,000 events are shown for 1 representative experiment. Docetaxol was included as a positive control to induce apoptosis.
Abstract
The notion that epigenetic alterations in neoplasia are reversible has provided the rationale to identify epigenetic modifiers for their ability to induce or enhance tumor cell death. Histone deacetylase inhibitors (HDACi) represent one such class of anti-neoplastic agents. Despite great interest for clinical use, little is known regarding the molecular targets important for response to HDACi-based cancer therapy. We had previously shown that interferon regulatory factor (IRF)-8, originally discovered as a leukemia suppressor gene by regulating apoptosis, also regulates Fas-mediated killing in non-hematologic tumor models. Furthermore, we and others have shown that epigenetic mechanisms are involved in repression of IRF-8 in tumors. Therefore, in our preclinical tumor model, we tested the hypothesis that IRF-8 expression is important for response to HDACi-based antitumor activity. In the majority of experiments, we selected the pan-HDACi, Trichostatin A (TSA), because it was previously shown to restore Fas sensitivity to tumor cells. Overall, we found that: 1) TSA alone and more so in combination with IFN-c enhanced both IRF-8 expression and Fas-mediated death of tumor cells in vitro; 2) TSA treatment enhanced IRF-8 promoter activity via a STAT1-dependent pathway; and 3) IRF-8 was required for this death response, as tumor cells rendered IRF-8 incompetent were significantly less susceptible to Fas-mediated killing in vitro and to HDACimediated antitumor activity in vivo. Thus, IRF-8 status may underlie a novel molecular basis for response to HDACi-based antitumor treatment.
Citation: Banik D, Khan ANH, Walseng E, Segal BH, Abrams SI (2012) Interferon Regulatory Factor-8 Is Important for Histone Deacetylase Inhibitor-Mediated Antitumor Activity. PLoS ONE 7(9): e45422. doi:10.1371/journal.pone.0045422 Editor: Joao P.B. Viola, National Cancer Institute (INCA), Brazil Received April 24, 2012; Accepted August 22, 2012; Published September 19, 2012 Copyright: ?2012 Banik et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported, in part, by Grant Number R01CA140622 from the National Cancer Institute (to SIA), an Alliance Developmental Award from the Roswell Park Alliance Foundation (to SIA), and National Cancer Institute Cancer Center Support Grant to Roswell Park Cancer Institute (CA016056). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.
