leupeptins and Adenoviridae-Infections

The adenovirus vector-based cancer gene therapy is controversial. Low transduction efficacy is believed to be one of the main barriers for the decreased expression of coxsackie and adenovirus receptor (CAR) on tumor cells. However, the expression of CAR on primary tumor tissue and tumor tissue survived from treatment has still been not extensively studied. The present study analyzed the adenovirus infection rates and CAR expression in human lung adenocarcinoma cell line A549 and its cisplatin-resistant subline A549/DDP. The results showed that although the CAR expression in A549 and A549/DDP was not different, compared with the A549, A549/DDP appeared obviously to reject adenovirus infection. Moreover, we modified CAR expression in the two cell lines with proteasome inhibitor MG-132 and histone deacetylase inhibitor trichostatin A (TSA), and analyzed the adenovirus infection rates after modifying agent treatments. Both TSA and MG-132 pretreatments could increase the CAR expression in the two cell lines, but the drug pretreatments could only make A549 cells more susceptible to adenovirus infectivity.

Topics: A549 Cells; Adenocarcinoma; Adenocarcinoma of Lung; Adenoviridae; Adenoviridae Infections; Cell Line, Tumor; Cisplatin; Coxsackie and Adenovirus Receptor-Like Membrane Protein; Drug Resistance, Neoplasm; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Leupeptins; Lung Neoplasms; Proteasome Inhibitors

Successful viral replication entails elimination or bypass of host antiviral mechanisms. Here, we show that shRNA-mediated knockdown of murine double minute (Mdm2) and its paralog Mdm4 enhanced the expression of early and late viral gene products during adenovirus (HAdV) infection. Remarkably, whereas the expression of HAdV genes was low in p53-deficient mouse embryonic fibroblasts (p53KO MEFs), the HAdV early gene products were efficiently expressed in Mdm2/p53 double-knockout (DKO) and Mdm4/p53 DKO MEFs, and viral capsid proteins were produced in Mdm2/p53 DKO MEFs. Thus, Mdm2 and Mdm4 seem to have potent antiviral property. In cells infected with wt HAdV or a mutant virus lacking the E1B-55K gene (dl 1520), both Mdm2 and Mdm4 were rapidly depleted, whereas replication-deficient mutant viruses (Ad-GFP) or ΔpTP with deletions within the coding sequence of preterminal binding protein failed to induce their downregulation. Reduced expression of Mdm2 and Mdm4 was not due to general shutoff of host protein synthesis. Additionally, expression of a dominant-negative mutant of Cul5 did not affect Mdm2/Mdm4 downregulation. Thus, viral replication but not the presence of E1B-55K is required for Mdm2/Mdm4 degradation. Surprisingly, treatment of HAdV-infected cells with proteasome inhibitor MG132 only partially restored the protein levels of Mdm2 and Mdm4, suggesting that they may also be downregulated through an additional mechanism independent of proteasome. Interestingly, cyclin D1 and p21 appear to be downregulated similarly during HAdV infection. Collectively, our work provides the first biochemical evidence for antiviral function of Mdm2 and Mdm4 and that viruses employ efficient countermeasure to ensure viral replication.

Topics: Adenoviridae; Adenoviridae Infections; Adenovirus E1B Proteins; Animals; Cell Line; Cullin Proteins; Cyclin D; Cyclin-Dependent Kinase Inhibitor p21; Cysteine Proteinase Inhibitors; Down-Regulation; Fibroblasts; Gene Expression Regulation, Viral; HCT116 Cells; Humans; Leupeptins; Mice; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; RNA Interference; RNA, Small Interfering; Tumor Suppressor Protein p53; Ubiquitin-Protein Ligases; Virus Replication