Figure 5.

(A) Left: Indicated MEFs were transfected as described in Material and Methods. Cells were then stimulated for approximately 36 hours with 1nM DHT or ethanol control and relative luciferase activity determined. Normalized AR activity in the absence of ligand in the wild type MEFs was set to “1”. Data shown reflects the mean of at least nine independent biological replicates ±SE. Middle: PARP-1^−/− MEFs were transfected as above, with the addition of either wild type PARP-1 or a PARP-1 catalytic domain point mutant allele, then treated, processed, harvested, and analyzed as above. *=p<0.05, **=p<0.01, and ***=p<0.001 Right: Parp-1 ^−/− MEFs were transfected as before, except cells were harvested, lysed, and total protein was separated by SDS-PAGE, transferred to PVDF, and immunoblotted for indicated proteins. (B) Upper panel: Schematic: VCaP xenografts were established for 4 weeks prior to treatment, at which time the mice received a single dose of either Casodex or Olaparib (100mg/kg), tumors were harvested 4 hours later and qPCR analyses were performed for indicated target genes. Data reflect average and standard deviation of at least three independent xenograft tumors, each performed with technical triplicates. Results were normalized to β-actin and control is set to “1”. (lower panel). (C) Tumors were established as in B, except mice were treated with ABT888 (100mg/kg twice daily). 72h later, tumors were harvested and qPCR analyses were performed for indicated target genes. Data reflect average and standard deviation of at least three independent experiments, each performed with technical triplicates. Results were normalized to 18S and control is set to “1”. (D) Upper panel: VCaP xenograft tumors were established as in B and C. Treatment was initiated when tumors reached 150mm³, and consisted of: control, castration alone (Cx), ABT888 (100mg/kg twice daily)(PARPi), and castration + ABT888 (Cx+PARPi.) Tumor volumes were assessed three times each week. The cumulative incidence plot depicts the percent of tumors in each treatment group that have doubled in volume, as a function of time. Each treatment group is significantly different than the control group. The combined treatment group is significantly different than the individual treatment groups as determined by with log-rank (Mantel-Cox) analysis. Lower panel: Median time elapsed before tumor volume doubled for each treatment group. (E) Upper panel: C4-2 xenografts were established as in B. Treatment was initiated when tumors reached 150mm³, and consisted of: castration alone (Cx) as control, and castration + ABT888 (Cx+PARPi). Tumor volumes were assessed daily until animals were sacrificed. Lower panel: tumors from upper panel were excised, homogenized in Trizol, cDNA was generated, and qPCR for the indicated mRNA was performed. Data are presented as mean ± SD of at least three xenografts from each treatment group. Statistical significance was determined using Student’s t test. *=p<0.05, **=p<0.01, and ***=p<0.001.