Enhancement of DNA transfection by NP, a highly basic and reversibly phosphorylated peptide derived from the N-terminal region of sea urchin sperm histone variant SpH1, was investigated in HEK293 cell cultures. NP and its corresponding C-terminal peptide CP were prepared by digestion of purified SpH1 with Staphylococcus aureus V8 protease followed by separation of the resulting N-terminal and C-terminal peptides using hydroxylapatite chromatography. Transfection vectors containing NP or CP, NP or CP mixed with polyethylenimine (NP-PEI and CP-PEI, respectively) and NP or CP crosslinked to PEI (NPxPEI and CPxPEI, respectively) were generated and mixed with a plasmid bearing a FLAG-tagged beta-2-adrenergic-receptor gene (FLAG-β_2AR) to create the corresponding transfection complexes. Free peptides (NP and CP) didn’t enhance transfection, rather they suppress transfection compared to PEI alone. Transfection efficiency of chemically crosslinked NPxPEI-DNA enhances transfection rate up to 1.4 fold increase compared to PEI-DNA. The data shows that the NPxPEI vehicle had an improved condensing capability than that of PEI alone at same mass ratio. Our results demonstrate that NP is a potential transfection vehicle when crosslinked with PEI.
Cancer is the second leading cause of death in the United States and accounts for 25% of deaths, which are roughly 1,600 deaths per day, and almost 587,000 deaths per year. Cells become cancerous due either to changes to their DNA or epigenetic alterations that cause misregulation of histone modifications. The acetylation alterations of H2A, H2B, H3, and H4 histone were also screened by two-dimensional gel electrophoresis. The retinoblastoma binding protein 2 (RBP2), a histone demethylase belongs to the JARID1 protein family and is known to demethylate the H3K4 methyl groups. First, Wbras and H2009 cells lines will be screened for expression of RBP2 by western blot analysis. The histone deacetylation drugs, Vorinostat, MS-275 and 4-Phenyl-3-Butenoic Acid (PBA) were used at varying concentrations ranging, to test its effect on the expression of RBP2 and H3K4 methylation marks in H2009 and WBras1 cells. Results indicate that PBA showed the ability to increase covalent histone modification of H3, H4 and H2B in WBras1 cells while only modifying H3 and H4 in H2009 cells, and very similar migration patterns can be seen with it structurally similar compound Vorinostat (SAHA). RBP2 expression was decreased when treated with MS-275, SAHA and PBA, which lead to an increase in H3K4me2 and H3K4me3 expression.||