Inhibition of human bladder cancer growth by a suicide gene delivered by JC polyomavirus virus-like particles in a mouse model.

PURPOSE: Bladder cancer is one of the most common cancers of the urinary tract. The poor 5-year survival rate of invasive bladder cancer represents a challenge for bladder cancer treatment. Previous studies demonstrated that human urothelial carcinoma is susceptible to infection by JC polyomavirus. We used JC polyomavirus virus-like particles to deliver genes into human urothelial carcinoma cells for possible therapeutic investigation. MATERIALS AND METHODS: Reporter plasmids (pEGFP-N3) for expressing green fluorescent protein, LacZ expression plasmids bearing cytomegalovirus or Muc1 promoter and a functional plasmid (pUMVC1-tk) for expressing thymidine kinase were packaged into JC polyomavirus virus-like particles. Plasmid DNAs were transduced via the JC polyomavirus virus-like particles into human urothelial carcinoma cells in vitro and into xenografted human bladder tumor nodules in vivo. RESULTS: pEGFP-N3 DNA was delivered and green fluorescent protein was expressed in human urothelial carcinoma cells in vitro and in the tumor nodules of mice in vivo. The thymidine kinase transgene also functioned in vitro and in vivo after JC polyomavirus virus-like particle transduction. The thymidine kinase gene transduced urothelial carcinoma nodules were drastically reduced in the presence of acyclovir. In addition, we noted selective Muc1-LacZ expression in human urothelial carcinoma cells transduced by JC polyomavirus virus-like particles. CONCLUSIONS: These findings provide a possible future approach to human urothelial carcinoma gene therapy using JC polyomavirus virus-like particles.

Inhibition of human diffuse large B-cell lymphoma growth by JC polyomavirus-like particles delivering a suicide gene.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is one of the most common types of aggressive B-cell non-Hodgkin lymphoma. About one-third of patients are either refractory to the treatment or experience relapse afterwards, pointing to the necessity of developing other effective therapies for DLBCL. Human B-lymphocytes are susceptible to JC polyomavirus (JCPyV) infection, and JCPyV virus-like particles (VLPs) can effectively deliver exogenous genes to susceptible cells for expression, suggesting the feasibility of using JCPyV VLPs as gene therapy vectors for DLBCL. METHODS: The JCPyV VLPs packaged with a GFP reporter gene were used to infect human DLBCL cells for gene delivery assay. Furthermore, we packaged JCPyV VLPs with a suicide gene encoding thymidine kinase (TK) to inhibit the growth of DLBCL in vitro and in vivo. RESULTS: Here, we show that JCPyV VLPs effectively entered human germinal center B-cell-like (GCB-like) DLBCL and activated B-cell-like (ABC-like) DLBCL and expressed the packaged reporter gene in vitro. As measured by the MTT assay, treatment with tk-VLPs in combination with gancyclovir (GCV) reduced the viability of DLBCL cells by 60%. In the xenograft mouse model, injection of tk-VLPs through the tail vein in combination with GCV administration resulted in a potent 80% inhibition of DLBCL tumor nodule growth. CONCLUSIONS: Our results demonstrate the effectiveness of JCPyV VLPs as gene therapy vectors for human DLBCL and provide a potential new strategy for the treatment of DLBCL.

H3K9 histone methyltransferase, KMT1E/SETDB1, cooperates with the SMAD2/3 pathway to suppress lung cancer metastasis.

Aberrant histone methylation is a frequent event during tumor development and progression. KMT1E (also known as SETDB1) is a histone H3K9 methyltransferase that contributes to epigenetic silencing of both oncogenes and tumor suppressor genes in cancer cells. In this report, we demonstrate that KMT1E acts as a metastasis suppressor that is strongly downregulated in highly metastatic lung cancer cells. Restoring KMT1E expression in this setting suppressed filopodia formation, migration, and invasive behavior. Conversely, loss of KMT1E in lung cancer cells with limited metastatic potential promoted migration in vitro and restored metastatic prowess in vivo. Mechanistic investigations indicated that KMT1E cooperates with the TGFß-regulated complex SMAD2/3 to repress metastasis through ANXA2. Together, our findings defined an essential role for the KMT1E/SMAD2/3 repressor complex in TGFß-mediated lung cancer metastasis.

Rab5 and class III phosphoinositide 3-kinase Vps34 are involved in hepatitis C virus NS4B-induced autophagy.

Autophagy has been shown to facilitate replication or production of hepatitis C virus (HCV); nevertheless, how HCV induces autophagy remains unclear. Here, we demonstrate that HCV nonstructural protein 4B (NS4B) alone can induce autophagy signaling; amino acid residues 1 to 190 of NS4B are sufficient for this induction. Further studies showed that the phosphorylation levels of S6K and 4E-BP1 were not altered, suggesting that the mTOR/S6 kinase pathway and mTOR/4E-BP1 pathway did not contribute to NS4B- or HCV-induced autophagy. Inhibition of Rab5 function by silencing Rab5 or overexpressing dominant-negative Rab5 mutant (S34N) resulted in significant reduction of NS4B- or HCV-induced autophagic vesicle formation. Moreover, the autophagy induction was impaired by inhibition of class III phosphoinositide 3-kinase (PI 3-kinase) Vps34 function. Finally, the coimmunoprecipitation assay indicated that NS4B formed a complex with Rab5 and Vps34, supporting the notion that Rab5 and Vps34 are involved in NS4B-induced autophagy. Taken together, these results not only reveal a novel role of NS4B in autophagy but also offer a clue to the mechanism of HCV-induced autophagy.

mRNA guanylation catalyzed by the S-adenosylmethionine-dependent guanylyltransferase of bamboo mosaic virus.

The S-adenosylmethionine-dependent guanylyltransferase of bamboo mosaic virus belongs to a novel class of mRNA capping enzymes distantly conserved in Alphavirus-like superfamily. The reaction sequence of the viral enzyme has been proposed comprising steps of 1) binding of GTP and S-adenosylmethionine, 2) formation of m7GTP and S-adenosylhomocysteine, 3) formation of the covalent (Enzyme-m7GMP) intermediate, and 4) transfer of m7GMP from the intermediate to the RNA acceptor. In this study the acceptor specificity of the viral enzyme was characterized. The results show that adenylate or guanylate with 5'-diphosphate group is an essential feature for acceptors, which can be RNA or mononucleotide, to receive m7GMP. The transfer rate of m7GMP to guanylate is greater than to adenylate by a factor of approximately 3, and the K(m) value for mononucleotide acceptor is approximately 10(3)-fold higher than that for RNA. The capping efficiency of the viral genomic RNA transcript depends on the length of the transcript and the formation of a putative stem-loop structure, suggesting that mRNA capping process may participate in regulating the viral gene expression.

Critical residues for GTP methylation and formation of the covalent m7GMP-enzyme intermediate in the capping enzyme domain of bamboo mosaic virus.

Open reading frame 1 of Bamboo mosaic virus (BaMV), a Potexvirus in the alphavirus-like superfamily, encodes a 155-kDa replicase responsible for the formation of the 5' cap structure and replication of the viral RNA genome. The N-terminal domain of the viral replicase functions as an mRNA capping enzyme, which exhibits both GTP methyltransferase and S-adenosylmethionine (AdoMet)-dependent guanylyltransferase activities. We mutated each of the four conserved amino acids among the capping enzymes of members within alphavirus-like superfamily and a dozen of other residues to gain insight into the structure-function relationship of the viral enzyme. The mutant enzymes were purified and subsequently characterized. H68A, the mutant enzyme bearing a substitution at the conserved histidine residue, has an approximately 10-fold increase in GTP methyltransferase activity but completely loses the ability to form the covalent m(7)GMP-enzyme intermediate. High-pressure liquid chromatography analysis confirmed the production of m(7)GTP by the GTP methyltransferase activity of H68A. Furthermore, the produced m(7)GTP sustained the formation of the m(7)GMP-enzyme intermediate for the wild-type enzyme in the presence of S-adenosylhomocysteine (AdoHcy), suggesting that the previously observed AdoMet-dependent guanylation of the enzyme using GTP results from reactions of GTP methylation and subsequently guanylation of the enzyme using m(7)GTP. Mutations occurred at the other three conserved residues (D122, R125, and Y213), and H66 resulted in abolition of activities for both GTP methylation and formation of the covalent m(7)GMP-enzyme intermediate. Mutations of amino acids such as K121, C234, D310, W312, R316, K344, W406, and K409 decreased both activities by various degrees, and the extents of mutational effects follow similar trends. The affinity to AdoMet of the various BaMV capping enzymes, except H68A, was found in good correlations with not only the magnitude of GTP methyltransferase activity but also the capability of forming the m(7)GMP-enzyme intermediate. Taken together with the AdoHcy dependence of guanylation of the enzyme using m(7)GTP, a basic working mechanism, with the contents of critical roles played by the binding of AdoMet/AdoHcy, of the BaMV capping enzyme is proposed and discussed.

Analysis of DNA-binding activity of the JC virus minor capsid protein VP2.

To investigate the DNA binding activity of the JC virus minor capsid protein, VP2, both wild-type and mutant VP2 were cloned and expressed in Escherichia coli. Southwestern blotting was employed for the DNA-binding assay. The results showed that VP2 was able to bind to DNA, except when either the last 13 or the last 29 amino acids were truncated. The results indicate that the DNA-binding domain of VP2 is located within the last 13 amino acids. Furthermore, we also demonstrated that Lys(332) and Lys(336) within the DNA-binding domain are crucial for DNA binding. The findings may provide further information for understanding the mechanism of virion assembly of the JC virus.