Research On Histone H4 Fusion Protein Vector In Cancer Gene Therapy

Protein/peptide-mediated gene delivery has recently emerged as a powerful approach in non-viral gene transfer. In previous studies, several groups found that histones can efficiently mediate gene transfer (histonefection). Wheat histone H4 is a nucleosome core histone whose gene was cloned and structure determined by Tabata et al. It is relatively conservative in eukaryotic core histones. There are only two amino acid differences at sites 61 and 78 between human histone H4 and that of wheat, which allows for a good candidate for gene transfer. For these reasons, we have constructed recombinant wheat histone H4 as a new gene delivery carrier.Protein transduction domain TAT (PTD-tat) is one of the most well-studied and efficient cell-penetrating peptides. It contains an 11-amino acid peptide of the human immunodeficiency viruses type 1 (HIV-1) Tat Protein. The amino acids of PTD-tat are highly basic, YGRKKRRQRRR, which is crucial for penetrating the plasma membrane. Because of the potential of PTD-tat for mediating cellular uptake of small and large molecules, in this study we genetically incorporated PTD-tat into the C-terminus of wheat histone H4 to allow H4 to enhance penetration of the plasma membrane via the PTD-tat pathway. In addition, a targeted drug delivery system must achieve high cell permeability and retention by the specific cell population. Target cell specificity is achieved by attaching molecular targeting moieties to the drug delivery system that interact precisely with receptors of cancer cell surface. This ensures that adverse side effects on healthy tissues will be minimized. Receptors for the luteinizing hormone-releasing hormone (LHRH) are overexpressed in the plasma membrane of cancer cells, and expression is not detected in normal visceral organs. Therefore, LHRH can potentially be used as the targeting moiety to deliver anticancer drugs specifically to cancer cells and facilitate their cellular uptake.Here, we report the generation of plasmid constructs that overproduce H4-TAT-LHRH (H4TL) as heterologous, His-tagged fusion proteins. The wheat genome was extracted from wheat root tissue. The putative fusion H4TL gene containing the wheat histone H4 gene with PDT-tat (YGRKKRRQRRR) and LHRH (QHWSYGLRPG) gene sequences was cloned from the Triticum aestivum genome and amplified by overlap extention polymerase chain reaction (OE-PCR). Every PCR fragment was determined by cloning it into pMD18-T vector and sequenced. The sequence of modified wheat histone H4 gene was in alignment with the GenBank sequence. The last amplified sequence was digested using EcoR I-Not I, cloned into pET28a (++), transferred E.coli JM109 cells, and identified by PCR and restriction digest. Correct constructs were identified by sequencing. E.coli BL21(DE3) containing the pET28a(++)-H4TL recombinant plasmid were induced by isopropyl-β-D-thiogalactopyranoside (IPTG) .The expression positive cells were induced using shake-flask fermentation and collected by centrifugation (7000xg at 4 oC for 15 min), washed twice with PBS and lysed by sonication. The clear supernatant was collected by centrifugation (10000xg at 4oC for 15 min), then obtained by filtration through a 0.22μm Millipore membrane. The supernatant proteins were denaturated by 6 M guanidine hydrochloride and loaded onto a nickel column, (0.7x2.5 mm, GE Healthcare). Chromatography operation was performed on an AKTA^TM Explorer Fast Protein Liquid Chromatography system (GE Healthcare). Ni⁺ -metal affinity chromatography was used to purify His-tagged H4TL fusion proteins from supernatant proteins by guanidine hydrochloride denaturation.Purified proteins were desalted using a HiTrap desalt column (GE Healthcare) according to the manufacturer's instructions, concentrated using an Amicon Ultra-15 Centrifugal Filter Units (Milipore, UK), and buffer-exchanged with 5% dextrose in 20 mM Tris-HCl pH 7.4. The H4TL fraction was freeze-dried and stored at -70oC for further use.The modified H4TL gene encoded a recombinant protein of 141 amino acids with an approximate molecular weight of 15.5 kDa. Gel electrophoresis mobility shift assays demonstrated that the purified modified H4 protein had high affinity for DNA. Most significantly, complexing plasmid pEGFP/C1 with H4TL was transfected with high efficiency into MCF-7, HO8910, LNCap, A549 and HeLa cells in vitro. The transfection efficiency mediated by histones is dependent on the relative concentration (DNA: histone ratio), type of cells and the incubation time.The chicken anaemia virus-derived protein apoptin is a tumour-specific cell-killing agent. This 14 kDa protein selectively induces apoptosis in a wide variety of transformed cells but not in primary cells. The cancer-selective toxicity of apoptin correlates with its cellular localization. Apoptin enters the nuclei of cancerous cells, whereas in nontransformed cells it remains in the cytoplasm. The current hypothesis is that nuclear trafficking and tumor specific phosphorylation of apoptin at Thr-108 are essential for the induction of apoptosis. Recently, Rohn and colleagues reported that phosphorylation of apoptin is required for apoptosis, but that abolishing the phosphorylation site of apoptin does not significantly disrupt its nuclear input into tumor cells.In the present study, we attempted to use H4TL with naked plasmid DNA, which contains a apoptin tumor suppressor gene, to evaluate the transfection efficiency and antitumor activity in human ovarian carcinoma (HO8910) and breast cancer (MCF-7) cells in vitro. We observed that apoptin delivery resulted in a more differential growth inhibition pattern in cancer cells in vitro. In this study, the wild-type apoptin gene was transfected into cancer cells using the H4TL as a vector, and the expression level and the activity of apoptin gene were evaluated in vitro. Gene expression was determined by classic reverse transcriptase-PCR and western blotting analysis. The cellular growth inhibition of H4TL-mediated apoptin transfection were assessed by MTT ([3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrasolium bromide] benzene sulfonic acid hydrate) assay. The apoptosis induced by apoptin were measured by Annexin V–FITC (fluorescein isothiocyanate) /PI staining, DNA ladder and lost of mitochondrial membrane potential. The results showed that remarkable apoptotic characteristics such as nuclear shrinkage appeared in tumor cells, but few apoptotic characteristics were observed in control groups. The apoptosis present were higher than control, the cell cycle were exchanged also detected by FACS. It was suggested that the recombination protein H4TL could mediate apoptin's transfection and could promote them expression and induce apoptosis in different tested tumor cells effectively. Our data suggest that H4TL can mediate apoptin gene transfer in vitro with high efficiency and is a promising new strategy for apoptin gene therapy. These results demonstrate that the targeting of non-viral vectors to tumor-specific receptors provide a cheap, simple, highly efficient tool for gene delivery.