Identification of the new molecular components of the DNA damage signaling

Identification of the new molecular components of the DNA damage signaling cascade opens novel avenues to enhance efficacy of chemotherapeutic drugs. interfering RNAs. Using the cell proliferation assay we found that knockdown of HMGB1-associated proteins resulted in 8-50-fold decreased chemosensitivity of A549 cells to cytarabine. Western blot analysis and immunofluorescent microscopy were used to evaluate genotoxic stress markers in knocked down cancer cells after 24-72 hr incubation with 1 μM cytarabine. Our results dissect the functions of HMGB1-associated proteins in DNA damage response: HMGB1 and HMGB2 facilitate p53 phosphorylation after exposure to genotoxic stress; and PDIA3 has been found essential for H2AX phosphorylation (no γ-H2AX was accumulated after 24-72 hr incubation with 1 μM cytarabine in PDIA3 knockdown cells). We conclude that phosphorylation of p53 and phosphorylation of H2AX occur in two distinct branches of the DNA damage response. These findings identify new molecular components of the DNA damage signaling cascade and provide novel promising targets for chemotherapeutic intervention. drug concentration data decided in triplicate from three impartial experiments. Cell Growth and Viability Cell viability and cell count were determined by flow cytometry using ViaCount reagent with Guava Personal Cell Analyzer (Guava Technologies CA). Clonogenic assay was performed after cell treatment for 3 days cell growth for 10 days and staining with methylene blue(17). For the MTT (3-(4 5 5 assay (CellTiter 96 cell proliferation kit Promega WI) A549 and UO31 cells (250 cells per well) were plated into 96-well plates and cultured for 3-5 days in varying concentrations of the following drugs: 0-100 μM MP 0 μM araC and 0-100 Itgb5 μM FU. RNA interference with short duplex RNA RNAi experiments were performed using the pre-designed Stealth RNA (Invitrogene Carlsbad CA) (HMGB1 – HSS142453 HSS142454 HSS142455; HMGB2 – HSS104854 HSS104855 HSS104856; GAPDH Validated Stealth RNAi DuoPak duplexes 1 and 2; PDIA3 – HSS142315 HSS142316 CEP-18770 HSS142317; and HSPA8 – HSS105082 HSS105083 HSS105084). Effective siRNA were selected using a lac-Z reporter system (BLOCK-iT RNAi Target Screening System Invitrogen). Scrambled Unfavorable Stealth RNAi control (Invitrogen) was used as unfavorable control in all siRNA experiments. Analysis of mRNA expression by Real-Time CEP-18770 PCR Total cellular RNA was extracted with TriReagent (GIBCO BRL/Invitrogen Carlsbad CA) from A549 and UO31 cells (about 5×106 cells per experiment 3 replicates) reverse transcribed using the TaqMan Reverse Transcription kit (Applied Biosystems CA) according to manufacturer’s instructions. The level of mRNA was evaluated using Relative Quantification protocol with human β-actin as a normalization standard on ABI 7300 Real Time PCR instrument (Applied Biosystems CA) according to manufacturer’s instructions. Data were collected from 3 impartial experiments for CEP-18770 each sample. Western analysis was performed as described earlier (Krynetski et al. 2003 The subcellular fractionation into cytosolic and nuclear fractions was performed using NE-PER Extraction reagent (Pierce Biotechnology Rockford IL) according to manufacturer’s instructions. The protein concentration was decided in cellular extracts using PlusOne2D Quant kit (Amersham Biosciences NJ). Electrophoretic separation was performed using 16% PAGE gels for analysis of γ-H2AX HMGB1 and HMGB2; 12% PAGE gels for analysis of phosphorylated p53; and gradient 4-12% PAGE gels for analysis of PARP PDIA3 and HSPA8 (PageGel San Diego CA). Membranes were developed with rabbit polyclonal antibodies specific to HMGB1 at 1:1000 dilution and HMGB2 at 1:500 dilution (Abcam Cambridge MA); rat anti-HSPA8 monoclonal Ab at 1:5000 dilution (Stressgen Ann Arbor MI); rabbit anti-GAPDH polyclonal Ab at 1:10000 dilution (Santa Cruz Santa Cruz CA); rabbit anti-PDIA3 polyclonal Ab (Rockland Gilbertsville PA) were generated as described CEP-18770 earlier and used at 1:5000 dilution(4); rabbit anti-Ser15-phosphorylated p53 polyclonal Ab at 1:1000 dilution and rabbit anti-γ-H2AX (H2AX phosphorylated at Ser139) polyclonal Ab at 1:500 dilution (Calbiochem La Jolla CA); rabbit anti-PARP polyclonal Ab at 1:1000 dilution (Cell Signaling Technology Danvers MA); rabbit anti-85 kDa PARP fragment polyclonal Ab at 1:500 dilution (Abcam Cambridge MA) and mouse anti-β-Actin monoclonal Ab (loading control) at dilution 1:10000 (Sigma St. Louis MO). Bands were visualized with secondary antibody – IRDye680 donkey anti-mouse antibody and IRDye680 goat anti-rabbit.