Prostate derived factor (PDF) is a divergent member of the transforming growth factor-β (TGF-β) superfamily with limited expression in normal tissues, with the exception of placenta and prostate. Conversely, PDF overexpression has been observed in several cancers including colon and prostate carcinomas. The biological role of PDF is not yet fully understood, although a number of diverse functions have been identified, including involvement in foetal survival and bone formation. PDF expression has shown to be regulated by p53 and has been implicated in promoting both apoptosis and cell survival in a number of different biological systems. Using DNA microarray analysis and real-time RT-PCR we have demonstrated a time-dependent increase in PDF mRNA levels following treatment with IC50(72h) doses of both 5-fluorouracil (5-FU) and oxaliplatin in p53 wild-type HCT116 colorectal cancer (CRC) cells (∼3-fold @ 24h). In contrast, we saw no significant modulation of PDF in isogenic 5-FU- and oxaliplatin-resistant cell lines following exposure to drug. Of note, basal PDF mRNA levels were elevated in both 5-FU- and oxaliplatin-resistant cells relative to parental cells (∼3-fold). Using Western blot analysis we have also demonstrated inducible expression of PDF protein in HCT116 p53 wild-type cells following treatment with 5-FU, oxaliplatin and the topoisomerase I inhibitor SN38, which was attenuated in cells rendered resistant to each drug. In contrast, cytotoxic drug treatment in p53 null HCT116 cells saw no modulation of PDF expression. A marked decrease in basal PDF levels was also observed in p53 null cells compared to their p53 wild-type counterparts. To investigate the biological significance of PDF, siRNA was used to knock-down PDF protein expression in p53 wild-type HCT116 cells. PDF siRNA treatment alone had no affect on cell viability, however, the combination of siRNA and IC50(72h) doses of 5-FU, oxaliplatin and SN-38 resulted in a potentiation of cell death compared to chemotherapy alone, as demonstrated by loss of full-length PARP. In addition, flow cytometric analysis showed a marked increase in the sub-G0/G1 fraction of cells following co-treatment with PDF siRNA and chemotherapy. This potentiation of apoptosis was abrogated in p53 null HCT116 cells. Together these data suggest an anti-apoptotic role for PDF that is p53-dependent, and that acute induction of PDF following treatment with cytotoxic chemotherapy may result in upregulation of cell survival pathways that inhibit drug response.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]