Increased Levels of the Heat Shock Protein (hsp) 70 Contribute as a Pro-Survival and Pro-Growth Mechanism in the Transformation of Bone Marrow Progenitor Cells.

Hsp70 is an ATP-dependent molecular chaperone that assists in the folding of native proteins into active conformation and prevents aggregation of misfolded and mutated abnormal proteins. In normal non-transformed cells, the expression of hsp70 is low and largely stress-inducible due to misfolded and denatured proteins. Recent studies in our laboratory have demonstrated that human acute leukemia cells abundantly express hsp70, which exerts strong antiapoptotic effects upstream and downstream of the mitochondria. Additionally, as compared to the controls, the mouse myeloid 32D or BaF3 cells transformed by Bcr-Abl or FLT-3 also show increased expression of hsp70. To further elucidate the pro-survival and pro-growth effects of hsp70 and its role in the leukemia transformation, we created stable hsp70 transfectants of the IL-3-dependent 32D and BaF3 (normally maintained in culture in IL-3 containing 10% WEHI medium), i.e., 32D/hsp70 and BaF3/hsp70 cells. These cells displayed 3 to 5 fold higher levels of hsp70, as compared to the control 32D or BaF3 cells. Both 32D/hsp70 and BaF3/hsp70 cells showed significantly improved growth and survival supported by 10% WEHI medium. Following culture in 0%, and less so in 1%, WEHI medium for 24 hours, 32D and BaF3 cells undergo cell cycle G1 phase accumulation, with corresponding decline in the % of cells in the S phase. Following this exposure, they also show markedly increased apoptosis and loss of clonogenic survival, as determined by the colony growth assays in methylcellulose. In contrast, under similar conditions of exposure to reduced % of WEHI conditioned medium, 32D/hsp70 and BaF3/hsp70 cells displayed significantly less accumulation in G1 phase, as well as reduced loss of clonogenic survival and apoptosis (p<0.05). This was also associated with reduced loss of mitochondrial membrane potential and increased accumulation of reactive oxygen species. Notably, following IL-3 withdrawal, exposure to 10 ng/ml of G-CSF for 72 hours induced significantly less differentiation of 32D/hsp70 versus 32D cells, as determined by increase in the % of cells expressing CD11b and GR1 (determined by specific antibody staining and flow cytometry) or by evaluation of the morphologic features of differentiation (p<0.05). Western blot analyses demonstrated that both 32D/hsp70 and BaF3/hsp70 cells, compared to their controls, possessed significantly higher expression of IL-3β receptor (R) and pSTAT5. Importantly, the supernatants of hsp70 overexpressing cells, compared to their controls, also showed higher levels of IL-3, as detected by an ELISA. In addition, BaF3/hsp70, versus the control cells, showed increased DNA binding activity and transactivation by the AP1 transcription factor, utilizing a protein/DNA binding array assay (Panomics, Redwood City, CA) and AP-1-luciferase cis-reporting analysis (Stratagene, La Jolla, CA), respectively. These findings strongly suggest that increased hsp70 levels confer a growth and survival advantage through an IL3- IL-3βR-STAT5-dependent mechanism in the marrow progenitor cells, which may contribute to the transformation induced by leukemia associated oncoproteins.