p53-Associated Parkin-like cytoplasmic protein (Parc) short-interfering RNA (siRNA) alters p53 location and biology of Peyronie's disease fibroblasts

OBJECTIVE center dot To evaluate the impact of p53-associated Parkin-like cytoplasmic protein (Parc) short-interfering RNA (siRNA) on the location of p53 as well as the biology of Peyronie's disease (PD) plaque-derived fibroblasts after Parc knockdown. PATIENTS AND METHODS center dot Plaque tissue was excised from men with stable PD undergoing penile reconstructive surgery and used to produce cultured PD plaque-derived fibroblasts. center dot Immunofluorescence (IF) and reverse transcription-polymerase chain reaction (RT-PCR) were then used to define the location of p53 and Parc before and after siRNA. center dot Nuclear fractionation studies were used to assess the chronology of translocation of p53 from cytoplasm to nucleus on Parc knockdown. center dot The terminal transferase dUTP Nick end labelling (TUNEL) assay was used to assess the apoptotic indices of the PD fibroblasts after Parc knockdown. RESULTS center dot IF and PCR showed high cytoplasmic levels of p53 and Parc before siRNA. On IF, there was little or no p53 present within the nucleus before Parc knockdown. center dot After Parc siRNA, IF showed translocation of p53 to the fibroblast nucleus, while Parc levels dropped significantly, but what Parc remained was confined to the cytoplasm with none present in the nucleus. center dot Nuclear fractionation studies using RT-PCR confirmed this translocation phenomenon and showed the chronology of the event. All p53 had moved from the cytoplasm to the nucleus within 16 h of Parc siRNA. center dot On TUNEL assay, apoptotic indices increased dramatically after Parc siRNA. CONCLUSIONS center dot These data prove that Parc is a cytoplasmic anchor for p53 in PD plaque-derived fibroblasts and may be the primary cause of the stabilization and defunctionalization of p53 in these cells. center dot These findings support Parc as a novel target for PD pharmacotherapy, perhaps using human siRNA technologies once commercially available.