Knockdown of DNMT3B results in resistance to low-dose 5-aza downstream of DNA damage and p53 protein induction.

<p><b>A</b>, DNMT3B knockdown in NT2/D1-R1 cells leads to resistance to low-dose 5-aza. Indicated doses of 5-aza were added fresh each day for 3 days to exponentially growing cultures of NT2/D1-R1 lentiviral control cells and NT2/D1-R1 cells stably expressing a lentiviral shDNMT3B construct. Viable cell growth and survival were measured. Data was normalized to no drug treatment. Error bars (within symbols) are standard deviation. * p = <0.05 compared to untreated controls. <b>B</b>, Low dose 5-aza induces DNA damage in NT2/D1-R1 cells independent of apoptosis. Cells were pretreated with vehicle or 20 µM Z-VAD-FMK (z-VAD) and treated with 100 nM 5-aza or 2 µM cisplatin for 1 day prior to Western analysis. Accumulation of pH2AX is indicative of double strand breaks. The arrow labeled cPARP indicates cleaved PARP that is indicative of apoptosis. Densitometry of pH2AX normalized to actin is shown. <b>C</b>, DNMT3B knockdown in NT2/D1-R1 cells results in resistant to low-dose 5-aza induced cell death and G2 cell cycle arrest. Cells were treated with 10 nM 5-aza for 3 days and then assayed for cell cycle analysis. Substantial cells in subG1 are indicative of cell death. <b>D</b>, Knockdown of DNMT3B in NT2/D1-R1 cells inhibits low-dose 5-aza mediated cell death but not p53 protein induction or induction of DNA damage. NT2/D1-R1 cells with no lentivirus and cells treated with sh-control lentvirus and sh-DNMT3B lentivirus were treated with 10 nM 5-aza for 3 days, 1 µM cisplatin for 12 hours or the combination. Western analysis was performed for p53, PARP and pH2AX.