Rad51 overexpression contributes to chemoresistance in human soft tissue sarcoma cells: a role for p53/activator protein 2 transcriptional regulation

Abstract

We investigated whether Rad51 overexpression plays a role in soft tissue sarcoma (STS) chemoresistance as well as the regulatory mechanisms underlying its expression. The studies reported here show that Rad51 protein is overexpressed in a large panel of human STS specimens. Human STS cell lines showed increased Rad51 protein expression, as was also observed in nude rat STS xenografts. STS cells treated with doxorubicin exhibited up-regulation of Rad51 protein while arrested in the S-G 2 phase of the cell cycle. Treatment with anti-Rad51 small interfering RNA decreased Rad51 protein expression and increased chemosensitivity to doxorubicin. Because we previously showed that reintroduction of wild-type p53 (wtp53) into STS cells harboring a p53 mutation led to increased doxorubicin chemosensitivity, we hypothesized that p53 participates in regulating Rad51 expression in STS. Reintroduction of wtp53 into STS cell lines resulted in decreased Rad51 protein and mRNA expression. Using luciferase reporter assays, we showed that reconstitution of wtp53 function decreased Rad51 promoter activity. Deletion constructs identified a specific Rad51 promoter region containing a p53-responsive element but no p53 consensus binding site. Electrophoretic mobility shift assays verified activator protein 2 (AP2) binding to this region and increased AP2 binding to the promoter in the presence of wtp53. Mutating this AP2 binding site eliminated the wtp53 repressive effect. Furthermore, AP2 knockdown resulted in increased Rad51 expression. In light of the importance of Rad51 in modulating STS chemoresistance, these findings point to a potential novel strategy for molecular-based treatments that may be of relevance to patients burdened by STS. [Mol Cancer Ther 2007;6(5):1650 -60]

Combining PCI-24781, a Novel Histone Deacetylase Inhibitor, with Chemotherapy for the Treatment of Soft Tissue Sarcoma

“…Rad51 promoter sequence (from −403 to +63 bp of the transcriptions start site -numbered according to Entrez GeneID 5888, gi:19924132) cloned into pGL3 as well as deletion mutants were cotransfected into SKLMS1, RD, and HT1080 cells using Fugene 6 transfection reagent (Roche Diagnostics GmbH) per the manufacturer's instructions, as described (18). The Rad51 reporter construct was further mutated at the E2F proximal binding site using Stratagene Quick Change mutagenesis kit (Stratagene) per the manufacturer's instruction and used for transfection as above.…”

Structure-Function Relationship of DNA Repair Proteins: Lessons from BRCA1 and RAD51 Studies

“…Moreover, in cases of either TP53 deletion or some TP53 point mutations Rad51 expression up-regulation is detected. The Transcriptional activator protein 2 (AP2), in combination with p53 suppresses rad51 transcription (Hannay et al, 2007). Abl kinase phosphorylates Rad51 in Tyr315 and in cases of the presence of the oncogenic constituvely active BCR/Abl fusion tyrosine kinase (i.e.…”

Novel Therapeutic Targets in Soft Tissue Sarcomas

“…In the presence of wild type p53 through activator protein 2, RAD51 expression was downregulated through decrease in promotor activity. 198 …”