The Action Mechanism of Omomyc Figure 9. Effect of Omomyc ectopic expression on proliferation and death of Rat1 and SH-SY5Y cells. A) Omomyc ability to hamper growth and induce apoptosis of Rat1 fibroblasts is Myc dependent. Growth curves (top panels) and percentage of death cells (bottom panels) of wild type (Rat1, left panels) and Myc (Rat1 myc 2/2; right panels) fibroblasts over expressing – or not (Ctr) – Omomer or c-Myc and Omomer (Myc+Omomer) [12]. B) Omomyc is able to reduce growth and promote apoptosis of the human neuroblastoma cell line SH- SY5Y. Growth curve (left) and percentage of cell death (right) of SH-SY5Y human neuroblastoma infected with control or Omomer (FLAG-Omomer) expressing lentiviruses. Cells (1.56105) were plated in multi-well plates in presence and absence of 4-OHT. Proliferation and death were assayed daily by cell count and vital staining with trypan blue. Data represent three independent biological repeats. doi:10.1371/journal.pone.0022284.g009 multiple interactions of Myc with epigenetic modulatory proteins remains to be determined. Omomyc strongly affected proliferation and apoptosis in two cell lines – immortalized Rat fibroblasts and a human neuroblastoma – in a way that was correlated to its expression level. Clearly, Omomyc itself is not a therapeutic agent, but serves as a tool that models the efficacy of future strategies that interfere with Myc function in tumorigenesis [60]. Our study has implications for the design of inhibitors that would target Myc for cancer therapy. The development of small-molecule inhibitors of protein-protein interactions is challenging, but significant progress is being made and bHLH-ZIP proteins are considered promising targets [13]. Most attempts to target Myc for cancer therapy focused on inhibiting the Myc/Max association and preventing Myc binding to E-boxes [12,13,61,62]. While this is clearly a crucial issue, our data indicate that this is likely not to be enough and that the repressing arm of Myc should be taken into account. It may be equally important to safeguard at least some aspects of the Myc transrepressive arm while disabling the activating one. This conclusion is in agreement with evidence indicating that transrepression has an important role in apoptosis, senescence and tumorigenesis. The particular type of perturbation introduced by Omomyc in the Myc interactome may turn the transrepressive arm in a powerful tumor suppressor, promoting cancer cell death. Materials and Methods Cell culture Rat1 fibroblasts expressing Omomer and c-Myc + Omomer, and c-Myc Rat1 (myc 2/2) fibroblasts expressing Omomer were described previously [10]. Rat1 (myc 2/2) fibroblasts were obtained by J. Sedivy [63]. Rat1 and HEK 293T cells [64] were cultured in complete DMEM medium and SH-SY5Y neuroblas- toma cells [65] in DMEM F12, supplemented with 10% foetal calf serum (EuroClone) and 1% penicillin/streptomycin at 37uC in 5% CO2. For Omomer induction, cells were treated with 4-OHT (Sigma), added to the culture medium at a final concentration of 561027 M. For serum induction, cells were grown for 48 h in 0.1% serum (serum-starvation) and switched to media with 10% serum in the absence or presence of 4-OHT (added 4 h before switching to 10% serum). Transfections were performed using Lipofectamine 2000 (Invitrogen). Plasmids Omomyc and Omomer expressing plasmids (pCS-Omomyc and pBP-Omomer) were described previously [9,10]. c-Myc, FLAG-c-Myc and FLAG-N-Myc expressing vectors (cbS-c-Myc, cbSFLAG-c-Myc and cbSFLAG-N-Myc) were from M. Cole; the Miz-1 expressing vector (pCMV-Miz-1) was from M. Eilers and the Hif-1a vector (pCMV- Hif-1a) was obtainded from A. Levi. GST-HEB and GST-ID1 expressing plasmids were previously described [65]; the GST-MAD plasmid was from R. Eisenman and the His-Max expressing plasmid was from L. Lania. Luciferase reporters of the nucleolin and p21 promoters (pNucL14 and p21Cip1-Luc) were from B. Amati. The cbSFLAG-Omomyc expression plasmid and the Tween lentiviral vector [66] expressing FLAG-Omomer were assembled according to standard proce- dures by means of the following oligonucleotide primers: cbSFLAG-Omomyc: 59-GGCCCCCGGGACCGAGGAGAA- TGTCAAGAGG-39 (forward) and 59-GGCCAAGCTTTTACG- CACAAGAGTTCCGTAG-39 (reverse). Tween-FLAG-Omomer 59-GGCCGTCGACATGGACTAC- AAGGACGATGAT-39 (forward) and 59-GGCCGATATCAC- TAGTAGGAGCTCTCAGAT-39 (reverse). Pull-down assay Pull-down assays with GST-linked proteins (Heb, Id1, Max, MAD) were performed as previously described [65]. Coimmunoprecipitation pBP-Omomer, pCMV-Miz-1, cbSFLAG-c-Myc, cbSFLAG-N- Myc, pJ4V-Max, pCMV- Hif-1a and cbSFLAG-Omomyc plasmids were transfected into 293T cells. After 2 days, cells were collected and processed as previously described [65]. Immunoblotting Western blot analysis was performed as described previously [65], using the following antibodies and reagents: ER (MC20, Santa Cruz Biotechnology; 1:1000 dilution), Miz-1 (H190, Santa Cruz Biotechnology; 1:1000), Hif-1a (clone-54, BD Transduction Laboratories; 1:1000), Max (c-124, Santa Cruz Biotechnology; 1:1000 dilution), and FLAG (M2, Sigma; 1:2500) antibodies; peroxidase conjugated rabbit anti-mouse IgG (Chemicon; 1:10000), peroxidase conjugated protein A (Sigma; 1:10000). Densitometric analysis of immunoblots was done by the ImageJ program. Immunofluorescence Cells grown on glass chamber slides were washed in PBS, fixed for 10 min in 4% paraformaldehyde, treated for 5 min with 0.2% Triton X-100 and processed for immunofluorescence as previously described [65]. The following antibodies were used: H3acK9 (04- 1003, Millipore; 1:200 dilution), H3diMeK9 (07-212, Millipore; 1:200), ER (MC20, Santa Cruz; 1:200), c-Myc (N-262, Santa Cruz; 1:200), Miz-1 (H190, Santa Cruz; 1:200), FLAG (M2, Sigma Aldrich; 1:1000), FITC conjugated rabbit anti-mouse IgG (Chemicon; 1:1000) and Rhodamine conjugated goat anti-rabbit IgG (Chemicon; 1:1000). DAPI (200 ng/ml) was used for staining of nuclei. Images were acquired by a Nikon fluorescence light microscope, through NIS-Elements 3.1 software. Fluorescence intensity was determined using ImageJ software. mRNA expression profiling and data analysis Total RNA was extracted with TRIzol reagent (GIBCO-BRL) according to the manufacturer’s protocol. RNA was checked for quantity, purity, and integrity by gel electrophoresis and UV spectrophotometric measurements: 10 mg of total RNA were used as starting material for preparing cDNA. Preparation of biotinylated target RNA – synthesis and cleanup of double- stranded cDNA followed by synthesis, cleanup and fragmentation PLoS ONE | www.plosone.org 13 July 2011 | Volume 6 | Issue 7 | e22284