Mutations in the p53 gene are the most common genetic abnormality in human cancers. P53 is a tumor suppressor gene, which regulates a wide variety of target genes controlling cell cycle and apoptosis. Arsenic Trioxide (ATO) is an effective chemotherapeutic agent for the treatment of acute promyelocytic leukemia and is being tested in phase II studies in various types of hematological malignancies and solid tumors. We have previously shown that ATO is a potent inducer of apoptosis in multiple myeloma cells, engaging the intrinsic apoptotic pathway in cells expressing w.t. p53. In contrast, in cells expressing mutant p53, both the intrinsic and extrinsic apoptotic pathways are engaged. Our finding in this respect were further supported by our recent study using a p53 temperature sensitive (p53Ts) mutant cell line, BRK, expressing w.t. p53 at 32°C and mutant p53 phenotype at 37°C (
Akay et al.,AACR; Abstract #5344, 2005
). We hypothesized that knocking out of p53 or p21 mRNA will result in modifying ATO-induced apoptotic pathways as observed using the p53-Ts conditional mutant of BRK cells. Thus, IM9 cells (expressing w.t.p53) were transfected [AMAXA Nucleoporator, ~60% transfection by green fluorescence protein (GFP) tag] with a GFP-SiRNA expressing plasmid co-expressing anti-sense RNA sequences for p53 (GFP/Sip53) and p21 (GFP/Sip21). As a control, GFP expressing plasmid not containing SiRNA (GFP/cont) was used. We observed in GFP/cont-transfected cells a G1 arrest through activation of p21 and moderate apoptosis, primarily through activation of procaspase 9, with no activation of BID and procaspase 8. In cells transfected with GFP/Sip53 or GFP/Sip21 plasmids we observed rapid mitochondrial membrane depolarization and apoptosis primarily through activation of the extrinsic apoptotic pathway with downregulation of FLIP, activation of procaspase 8 and BID, enhanced release of AIF from mitochondria and rapid apoptosis. Depletion of glutathione was observed with or without silencing of p21 or p53. Furthermore, silencing of p53 or p21 resulted with a decrease in ATO-induced activation of c-jun and Erk and in a decrease in the activation of Akt. These results further substantiate the model we put forward for the role of p53 in ATO-induced apoptosis in myeloma and other cancer cell lines.
