Acute promyelocytic leukemia (APL) is a particular subset of acute myeloid leukemia (AML) and is genetically characterized by the reciprocal translocation between chromosomes 15 and 17, which fuses the promyelocytic leukemia (PML) gene and the retinoic acid receptor alpha (RAR-\#945;) gene. This fusion protein inhibits differentiation of myeloid cells. APL patients respond to all-trans retinoic acid (ATRA) through complete remissions associated with the degradation of the PML-RAR\#945; that accelerate the leukemic cells differentiation and induce apoptosis of the differentiated granulocyte. ATRA-induced remissions are transient and relapsed APL is often ATRA-resistant. The mechanisms of ATRA resistance are largely unknown. In this study, we used stable isotope coding quantitative proteomics approach to determine differences in the protein expression levels in ATRA-sensitive NB4 APL cells versus NB4-R1 (a de novo ATRA-resistance derivative of NB4 cell line). While NB4-R1 cells were grown in normal RPMI medium, NB4 cells were grown in RPMI medium in which normal leucin was replaced with deuterium labeled leucin. Protein extracts were mixed in a 1:1 ratio and then separated by 1-dimentional SDS-PAGE. Selected bands were excised and trypsin digested and later separated and identified using a high performance quadrupole time-of-flight (QqTOF) mass spectrometer with a mass range of m/z 5-40,000. Proteomics profile revealed that several autophagy-related proteins, including, LAMP-2A, HSP-90, HSP-75, HSP-70, Bcl-2, and 14-3-4 zeta were differentially regulated in NB4 vs. NB4-R1 cells. Western blot experiments showed that HSP-90 is regulated in a time- and dose-dependent manner after treatment of NB4 cells with pharmacological doses of ATRA. We are currently investigating whether suppression of these proteins (based in the proteomic data) attenuate the resistance of NB4-R1 cells to the ATRA treatment. Overall our results indicate that autophagy-related proteins could be involved in development of ATRA-resistance, and in the regulation of apoptotic/autophagy signaling in APL.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 4490.

100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO

American Association for Cancer Research
2009