title: p53-Associated Parkin-like cytoplasmic protein (Parc) short-interfering RNA (siRNA) alters p53 location and biology of Peyronie's disease fibroblasts.
abstract: OBJECTIVE • 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 • Plaque tissue was excised from men with stable PD undergoing penile reconstructive surgery and used to produce cultured PD plaque-derived fibroblasts. • 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. • Nuclear fractionation studies were used to assess the chronology of translocation of p53 from cytoplasm to nucleus on Parc knockdown. • The terminal transferase dUTP Nick end labelling (TUNEL) assay was used to assess the apoptotic indices of the PD fibroblasts after Parc knockdown. RESULTS • 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. • 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. • 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. • On TUNEL assay, apoptotic indices increased dramatically after Parc siRNA. CONCLUSIONS • 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. • These findings support Parc as a novel target for PD pharmacotherapy, perhaps using human siRNA technologies once commercially available.
authors.affiliation: Department of Urology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.
authors.forename: John P
authors.initials: JP
authors.lastname: Mulhall
authors.forename: Jennifer
authors.initials: J
authors.lastname: Barnas
authors.forename: Keith
authors.initials: K
authors.lastname: Kobylarz
authors.forename: Alexander
authors.initials: A
authors.lastname: Mueller
mesh_terms: D002352:Carrier Proteins / Q000378:metabolism*; D003593:Cytoplasm; D015180:Electrophoresis, Gel, Two-Dimensional; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D020287:In Situ Nick-End Labeling; D008297:Male; D010411:Penile Induration / Q000209:etiology / Q000378:metabolism* / Q000473:pathology; D034741:RNA, Small Interfering / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014166:Transferases
chemical_list: D002352:Carrier Proteins; D034741:RNA, Small Interfering; C400192:Cul9 protein, human; D014166:Transferases