Effect Of Tat-CT3-cMyc Poly-peptide On Human Promyeloid Leukemia HL-60 Cells

Leukemia is a type of cancer of the blood or bone marrow characterized by an abnormal increase of white blood cells. Leukemia is a broad term covering a spectrum of diseases. In turn, it is part of the even broader group of diseases called hematological neoplasms. Despite that visual masses might not be observedthroughout the whole course of leukemia; this disease has plenty shared characteristics with other cancer, such as uncontrolled proliferation, differential disorder, hindered apoptosis, etc. Additionally, as leukemia is a systematic disease, the specific conceptions like metastasis or spreading that may be appropriate for solid cancers were not involved in leukemia. As for the therapy, the hematopoietic stem cell transplant is the only way that could totally cure the disease,despite the difficulties brought by strict HLA matching or the costly therapy terms, all of which makes it almost impossible for every patient with leukemia. The current research on leukemia is focusing on the systematical review on all diseases of human body, trying to figure out the origin and development of leukemia andexploring the possible target-specific molecular medication for the disease.Leukemia inhibitory factor (LIF), as a member of the IL-6 family of cytokines, produces biological effects via binding to its receptor, which consists of a low-affinity LIF receptorα-chain (referred to as LIFRα) and a high-affinity subunit (referred to as gp130), both of which are shared with other IL-6 family cytokines. It has been reported that the amino-acid 136-145 region of the distal C-terminal in cytoplasm, which contains 5-tyrosine residues (Y5) and several YXXQ motifs, is genetically conserved among gp130, LIFRα, and G-CSFR in numerous known cell lines. These motifs could, thus, initiate intracellular signaling by triggering LIFRα-gp130 heterodimerization via Janus kinase-signal transducer and activator of transcription (Jak/STAT) and subsequently influence the growth or differentiation of leukemia cells. Intriguingly, because previous findings have confirmed that the enrichment of triple YXXQ motifs of the LIFRαcytoplasmic domain (LIFRα-CT3) via liposome transfection can inhibit proliferation and induce differentiation in human promyeloid leukemia HL-60 cells, we hypothesize that the LIFRα-CT3 polypeptide could also intracellularly achieve a therapeutic effect, thus elucidating a potential future peptide-targeting leukemia therapy.Advances in protein delivery and the identification of several protein transduction domains have facilitated the delivery of proteins/peptides to cells or organs. The HIV-trans-activating transduction domain (TAT-PTD) has 11 amino acids (aa; TAT-PTD49-57:YGRKKRRQRRR) and is capable of the intracellular delivery of proteins across the plasma membrane. Although the mechanism of TAT-facilitated cellular uptake remains controversial and uncertain, TAT has been widely applied as a tool for protein transduction in vivo and in vitro after fusion with various full-length or truncated peptides.The technology of generating TAT fusion proteins requires the synthesis of a fusion protein in which TAT is linked to the molecule of interest via the use of a bacterial expression vector. In general, the TAT fusion protein is also linked to some sort of tag so as to facilitate its subsequent purification. The purified recombinant fusion protein could be directly added to mammalian cells in culture or injected in vivo into an animal.The technique above is generally highly applicable but laborious; in addition, a protein that is derived from a prokaryotic expression system is potentially more limited by its lack of splicing and the associated post-transcription processing systems or post-translation modifying systems in comparison to eukaryotic expression systems.In this manuscript, we have developed an alternative technology that offers advantages in terms of the application of TAT-mediated transduction techniques. We fused TAT-PTD49-57 with LIFRα-CT3 in recombinant plasmid pcDNA3.0-ss-TAT-CT3-cMyc with an inserted a signal peptide (ss) on the N-terminal. Next, the ss-TAT-CT3-cMyc fusion protein was expressed in Chinese Hamster Ovary (CHO) cells before cell lysates were purified via an anti-cMyc agarose affinity column. By comparing the ss-TAT-CT3-cMyc fusion protein to its ss-CT3-cMyc counterpart, the ss-TAT-CT3-cMyc fusion protein was capable of being secreted from CHO cells and subsequently demonstrated a distinct capacity to be delivered into human myeloid leukemia HL-60 cells. Furthermore, our in vitro experiments suggested that targeting the TAT-CT3-cMyc and related pathways may be a promising novel approach in the future treatment of leukemia. We also demonstrate that intracellular enrichment of LIFRα-CT3 polypeptide in human promyeloid HL-60 cells enhances STAT3 phosphorylation in a Jak2-independent manner, which confirmed the bioactivity of phosphorylated STAT3 (pSTAT3) in the induction of HL-60 myeloid differentiation.