Abstract
We have recently found that the trimeric transcription factor NF-Y is a direct stimulator of hematopoietic stem cell (HSC) renewal, activating the transcription of several genes involved in HSC self-renewal and differentiation including the Hox4 paralogs HoxB4, HoxC4, HoxD4 as well as LEF1, Notch1, p27 and telomerase (Zhu J. et al. PNAS, 102:11728–11733; 2005). HIV-TAT peptide transduction offers a distinct therapeutic advantage over viral vectors due to the temporary and regulatable nature of the induction. We have previously modeled the delivery of NF-YA protein fused with the protein transduction domain of the HIV-1 TAT protein to K562 cells, demonstrating upregulated HOXB4 to levels similar to that achieved by retroviral overexpression (Domashenko A. et al. Blood, 104:964a; 2004). We have now asked whether TAT-NF-YA protein induction can be modified for direct application to normal human CD34+ cells. Recombinant protein was designed by fusion of the human NF-YA sequence with the TAT protein transduction domain and a GST-tag at the N-terminus, and purified under native conditions. Binding activity of fusion protein to HOXB4 promoter probe was confirmed by electrophoretic mobility shift assay (EMSA). Human peripheral blood CD34+ cells were cultured with GST-TAT-NF-YA for 1.5h and displayed bright fluorescence in the cytoplasm and nucleus after staining with anti-GST-Alexa Fluor 488 Ab. To evaluate the effect of TAT-NF-YA fusion protein on HOXB4 promoter activity in primary hematopoietic cells, the level of HOXB4 mRNA was measured by real-time PCR with normalization to GAPDH. It was found that delivered NF-YA was able to increase endogenous HOXB4 message up to 3.5-fold in 2h after incubation with the protein. To minimize the recombinant protein toxicity and maximize the efficiency of delivery we investigated the possibility of having the same functional effect at low protein concentration using lysosomotropic agents (chloroquine and sucrose), to facilitate fusion protein nuclear uptake. The effect of varying concentrations of each agent on protein delivery and cell survival was analyzed by determination of endogenous HOXB4 message using quantitative PCR and trypan blue staining. Both chloroquine and sucrose increased the effect of upregulation of the HOXB4 message by GST-TAT-NF-YA 1.5 – 2.5 fold. Chloroquine exhibited substantial toxicity to the cells whereas sucrose was improving cell survival; therefore we have used sucrose in subsequent experiments. A sustained several fold activation of HOXB4 expression in CD34+ cells exposed to GST-TAT-NF-YA (40 nM) and sucrose (250 mM) has been observed for at least 24 h following exposure to the protein. In conclusion, our results showed the ability to transduce functionally active NF-YA protein into human primary hematopoietic cells. These findings support the further employment of protein transduction techniques for development of new therapeutic strategies.
PP-070 Generation of protective CTLs against the influenza A virus using single matrix epitope fused to protein transduction domain of Tat of HIV