p53-Mediated Apoptosis in Multiple Myeloma Cells: Evidence for a Transcription-Independent Pathway
MN Saha, A Asuma-Mukai, H Chang. University Health Network, University of Toronto, Toronto, Canada
Background: The p53 protein plays a key role in the apoptotic response of various hematological malignancies. Although p53-mediated apoptosis is conventionally followed by transcriptional regulation of p53-target genes, non-transcriptional mitochondrial p53 mediated apoptotic pathway has been identified in cancer cells. As the functional consequence p53 signaling in multiple myeloma (MM) is not clearly understood, here we explore the molecular mechanisms of apoptosis in myeloma cells mediated through activation of the p53 pathway by an MDM2 antagonist, nutlin.
Design: Human MM cell lines (MM1.S and H929 cell lines harboring wild type p53; and LP1 and U266 cell lines harboring mutant p53) and primary MM samples were exposed to nutlin, and assessed for cell viability, cell cycle analysis and apoptosis.
Results: Treatment of MM cells with nutlin resulted in a reduction in cell viability, an increase in the apoptotic fraction, and cell cycle arrest in MM1.S and H929 as well as 3 of the 5 primary MM samples tested. These effects were accompanied by up-regulation of p53 and induction of p53-dependent proteins p21, MDM2, puma, bax and Bak; and down-regulation of anti-apoptotic proteins, Bcl2 and survivin. By contrast, no effects on cell cycle arrest or apoptosis were found in MM cell lines harboring mutant p53. Aside from nuclear stabilization, nutlin caused cytoplasmic p53 accumulation and translocation to mitochondria, associated with cytochrome C release that precedes the induction of p53 target genes. Importantly, although pifithrin-α (PFT-α), an inhibitor of p53-mediated transcription, inhibited the nutlin-induced up-regulation of p53-transcriptional targets, surprisingly augmented apoptosis by ∼ 2 fold. Furthermore, immunoprecipitation with an anti-Bcl2 antibody showed co-precipitation of Bcl2 and p53 from lysates (mitochondrial fractions) of MM1.S cells treated with nutlin.
Conclusions: Our data suggest that a transcription-independent pathway involving direct binding of p53 to mitochondrial anti-apoptotic proteins plays a major role in p53-mediated apoptosis in MM cells; the transcriptional targets of p53 may include proteins that inhibit non-transcriptional pathway. Thus, therapeutic strategies aimed at inhibiting up-regulation of p53-mediated transcription together with administrating non-genotoxic drugs such as nutlin may hold promise for the treatment of MM patients who are relatively drug resistant.
Wednesday, March 24, 2010 9:30 AM
Poster Session V # 220, Wednesday Morning