Down-regulation of p53R2 enhances the sensitivity to DNA damaging agents in prostate cancer cells Free

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Introduction: p53R2 is a p53-inducible ribonucleotide reductase that contributes to DNA repair by supplying dNTP pools in response to DNA damage. The purpose of this study was to determine whether down-regulation of p53R2 sensitizes prostate cancer (CaP) cells to ionizing radiation (IR) or to doxorubicin (Dox). Methods: LNCaP, its sublines expressing dominant negative p53 mutant alleles (gain-of-function alleles: G245S, R248W and R273H; loss-of-function allele P151S), other prostate cancer cell lines (CWR22rv1, DU145 and PC3), and normal (PrSC) and immortalized (PZ-HPV-1 and RWPE-1) prostate epithelial cell lines were studied. Cells were treated with 2 or 10Gy IR, or Dox at 0.5ug/ml. Gene silencing of p53R2 was achieved using siRNA. Biological effects were investigated by Western blot, flow cytometry and clonogenic assays. Results: p53R2 was over-expressed in all the tumor cell lines compared to the benign cells. In LNCaP, upon 10Gy IR or Dox treatment, p53R2 protein was induced at 12hr and remained elevated up to 72hr. Anti-p53R2 siRNA down-regulated p53R2 to its basal level after 10Gy or Dox treatment. Silencing p53R2 combined with 10Gy IR or Dox enhanced sub-G1 content 4-fold (25%, 72hr) post-IR and 2-fold (54%, 48hr) post-Dox. Clonogenic assays showed a 7-fold reduction in colony formation following combined 2Gy IR and p53R2 silencing. LNCaP mutant p53 sublines expressed high p53R2 basal levels similar to the parental cell line LNCaP and also showed induction of p53R2 upon IR or Dox treatment with the exception of the R248W subline. Silencing of p53R2 combined with 10Gy IR enhanced radiation-induced apoptosis 2.3-fold (9% sub-G1) in R273H at 72hr post-IR, but had no effect in G245S, P151S and R248W cells. Silencing of p53R2 combined with Dox resulted in 1.7-fold (11%) and 1.6-fold (69%) increase of sub-G1 in R273H and P151S, respectively, but had no effect on G245S and R248W cells. Conclusions: p53R2 is over-expressed in CaP cells. Upon DNA damage, p53R2 is induced even in the presence of dominant-negative p53 mutants. Silencing of p53R2 sensitizes LNCaP to DNA damaging treatment, but is more selective in its killing in the mutant sublines, possibly related to basal p53R2 expression level and its induction status. This study demonstrates that p53R2 is involved in DNA repair in CaP and is a potential therapeutic target that could enhance the effectiveness of IR or chemotherapy in CaP.