1. Targeting Eradication Of Leukemia Using Chimeric Oncolytic Adenovirus To Drive IL-24 Expression 2. A Study Of The Role Of Post-translational Modification In Ribonucleotide Reductase Proteins

Leukemia is the most commonly diagnosed malignant cancer. The thousands of patients die each year from leukemia cancer annually in China. Conventional tumor therapeutic approaches, including surgery and chemo radiation, have limited efficiency and are also harmfully to normal tissues and cells. Gene-therapy is a newly potential treatment of disease, which induces exogenous genes or other genetic materials into cells and thus correcting aberrant conducts of the host cells or tissues. And that a new strategy of targeting gene-virotherapy of cancer has been widely used.Virotherapy with oncolytic adenovirus is based on the use of tumor specific replicating adenovirus. The oncolytic adenovirus has a capacity of eliminating tumor cells, and producing new infectous viral particals. The new viruses can infect the turmor cells closed to them. So the whole tumor tissue collapses. At the same time, the oncolytic adenovirus could carry therapeutic genes and express them in the tumor cells. So, the aimed cancer cells will be treated by two efficient means.Studies have shown that constructed oncolytic adenovirus vector has a lower three ways:1, using tumor-specific or tissue-specific promoter to control the expression of genes necessary for viral replication; 2, the use of certain viral gene deletion, so that in normal cells copy necessary, but non-essential replication in tumor cells; 3, the transformation of the virus fibrin (fiber) to determine viral infection tropisms. Therefore, given the proliferation of the virus selectively target tumor cells.On this basis we constructed specifically targeting leukemia tumor cells oncolytic adenovirus AdCN205-11-gene. Selected on the basis of the molecular mechanism of action studies the therapeutic gene in leukemia cells.In this research, the chimeric oncolytic adenovirus Ad5/F11 vector, adenovirus E1A gene, lacking of 24 bp in the genome (8 amino acids), is controlled by the human telomerase reverse transcriptase (hTERT) promoter. CR2 region of adenoviral E1A protein can not attach to Rb protein without these 8 amino acids which are essential for the attachment. As a result, the chimeric of Ad5 skeleton and Adll fiber is safe, from Ad5, and targeted to the aimed cells, from Ad11.The anti-cancer gene, Mda-7/IL-24, a kind of secreted protein with general anti-tumor properties, may have potential "bystander effect", increase radiation lethality, induce immune-regulatory response, inhibit tumor angiogenesis and drive tumor cell to apoptosis.Studies have shown that AdCN205-11-IL-24 combines the advantages of organically-viral therapy and gene therapy, with tumor targeting, anti-tumor effect is remarkable characteristics such as leukemia cancer cure has brought new hope.Ribonucleotide reductase (RR) catalyzes the rate-limiting step in DNA precursor synthesis:the reduction of ribonucleotides to deoxyribonucleotides in all living cells. The crucial role of RR in DNA synthesis, cell proliferation, and malignant transformation makes it an important target for anticancer drugs. Studies on structure and function of RR will help clarify the mechanism of RR regulation and design novel RR inhibitors for cancer treatment. RR is divided into three classes based on different metal cofactors for catalytic activity. Class I RR is further divided into three subclasses (Ia, Ib, and Ic) according to their polypeptide sequence homology and overall allosteric regulation behavior. Human, mouse, and E.coli RRs belong to Class la subclasses. Most of the studies on structure and function of the class Ia RRs have been performed on E. Coli RR, which is a tetramer composed of two homodimeric subunits designated Rl (large subunit) and R2 (small subunit).In the recent years, the existence of two forms of RR in mammalian cells has been proved and major progress related with RR has been made by multi-disciplinary research.Post-translational modifications are crucial for regulating protein structure and function in different species. However, the post-translational modifications of RR are largely unknown. Here we first explored RR acetylation in the molecular mechanisms of hRR.Lysine (K) acetylation refers to the transfer of an acetyl moiety from acetyl-CoA to the e-amino group of a specific K residue,generating acetyl-lysine (AcK). This modification is also known as N-acetylation and is thus different from N-acetylation, which is typically co-translational and controls protein stability. N-acetylation is dynamic and reversible, with levels tightly governed by the opposing actions of K acetyltransferases (KATs) and deacetylases (KDACs), two families of enzymes that catalyze the fo rward and reverse reactions, respectively.The experment analysis using a pan-acetylation antibody showed that hRRM2 was acetylated. Immunoprecipitation (IP) experiments showed that CBP/P300 interact with hRRM2. The experiments result was indicating that hRRM2 acetylation was mediated by CBP.Mass spectrometry analysis recovered K23,K30,K46,K59,K61 of the hRRM2 subunit was the acetylation site. The hRRM2 were constructed in the mass spectrometer to five mutant lysine (K) are mutated to arginine (R) (K23R, K30R, K46R, K59R, K61R),and the detection of the expression of CBP to acetylation degree. The results indicated that the acetylation of hRRM2 significantly decreased when lysine (K23, K30, K46, K59, K61) were mutated to arginine (R) respectively. In addition, When we put all the detected mass acetylated lysine sites for the same mutation, and the detection of the expression of CBP and the degree of acetylation. Experiments show that, compared to wild type hRRM2, acetylation is not detected phenomenon. Altogether, our data indicated that the subunit of the hRRM2 (K23, K30, K46, K59, K61) were acetylated by CBP, which were probably related with RR’s function.Recent proteomic studies reveal that 5-10% of mammalian proteins undergo lysine acetylation, a post-translational modification that adds an acetyl group to the e-amino group of lysine residues.And our findings may be provide a new and valuble insight into the molecular mechanisms of in hRR, and help design novel hRR inhibitors as anticancer drugs.