A truncated isoform of the PP2A B56g regulatory subunit reduces irradiation-induced Mdm2 phosphorylation and could contribute to metastatic melanoma cell radioresistance

Abstract

F10, a subline of the B16 mouse melanoma cell line, is itself the parent of the more metastatic BL6 line. BL6 cells differ from F10 cells by an alteration of the gene encoding the B56g regulatory subunit of protein phosphatase 2A (PP2A), which results in mRNA encoding a truncated variant of the subunit (Dg1). Expression of Dg1 protein is detectable only when BL6 cells are transplanted into mice and then g-irradiated. Recently, B56g subunit-containing PP2A holoenzymes have shown to dephosphorylate Mdm2, a negative regulator of p53. Thus, we assessed whether the expression of Dg1 affects irradiation-induced phosphorylation of Mdm2 and radioresistance of melanoma cells by perturbing the regulation of p53. Western blot analyses revealed that irradiated COS-7 and NIH3T3 cells stably expressing Dg1 showed significantly less irradiation-induced Mdm2 phosphorylation. Mdm2 phosphorylation reduces the ability of Mdm2 to target p53 for degradation, which probably explained why p53 protein levels in NIH3T3 cells expressing Dg1 were not significantly elevated by irradiation, unlike in wild-type cells. This was also true for F10 cells transfected with Dg1 (F10Dg1) when the cells expressed Dg1 after being irradiated in vivo. p53 mRNA levels in irradiated wild-type and Dg1-expressing cells were both only slightly elevated, suggesting that Mdm2 regulates p53 levels by a post-transcriptional mechanism. p53-mediated induction of the pro-apoptotic gene encoding Bax was also significantly lower in F10Dg1 cells irradiated in vivo. Moreover, F10Dg1 and BL6 cells were less apoptotic than F10 cells when the cells were irradiated in vivo. The p53 in F10 cells appears to be as functional as that in NIH3T3 cells because irradiation-induced expression of p53-target genes was comparable in both cells. Collectively, Dg1 appears to reduce irradiation-induced Mdm2 phosphorylation, which then blocks irradiation-stimulated p53 accumulation. Defects, such as Dg1, in PP2A may thus contribute to melanoma cell radioresistance.