Here we show that the adenovirus early region 4 (E4) open reading frame 4 (ORF4) protein autoregulates its own transcription by inhibiting adenovirus E1A-induced activation of E4 transcription both in transient transfection experiments and during lytic virus growth. The inhibitory activity of E4-ORF4 was selective for E1A-CR3-dependent transactivation and had no effect on CR1 transactivation. The inhibitory activity of E4-ORF4 was relieved by okadaic acid treatment, which inhibits the cellular protein phosphatase 2A (PP2A), suggesting that E4-ORF4 controls the phosphorylated status of transcription factors important for E4 promoter activity. This conclusion agrees with previous demonstrations that E4-ORF4 associates with PP2A and causes a partial dephosphorylation of certain transcription factors, including E1A (U. Müller, T. Kleinberger, and T.

Previous studies have shown that the cell cycle-regulated E2F transcription factor is subjected to both positive and negative control by phosphorylation. Here we show that in transient transfection experiments, adenovirus E1A activation of the viral E2 promoter is abrogated by coexpression of the viral E4 open reading frame 4 (E4-ORF4) protein. This effect does not to require the retinoblastoma protein that previously has been shown to regulate E2F activity. The inhibitory activity of E4-ORF4 appears to be specific because E4-ORF4 had little effect on, for example, E4-ORF6/7 transactivation of the E2 promoter. We further show that the repressive effect of E4-ORF4 on E2 transcription works mainly through the E2F DNA-binding sites in the E2 promoter. In agreement with this, we find that E4-ORF4 inhibits E2F-1/DP-1-mediated transactivation. We also show that E4-ORF4 inhibits E2 mRNA expression during virus growth. E4-ORF4 has previously been shown to bind to and activate the cellular protein phosphatase 2A. The inhibitory effect of E4-ORF4 was relieved by okadaic acid, which inhibits protein phosphatase 2A activity, suggesting that E4-ORF4 represses E2 transcription by inducing transcription factor dephosphorylation. Interestingly, E4-ORF4 did not inhibit the transactivation capacity of a Gal4-E2F hybrid protein. Instead, E4-ORF4 expression appears to result in reduced stability of E2F/DNA complexes.

AIM:To evaluate the ability of anti-ricin A-chain antibodies, delivered intracellularly, to protect against ricininduced cytotoxicity in RAW264.7 cells. METHODS:Anti-deglycosylated ricin A-chain antibody and RAC18 anti-ricin A-chain monoclonal antibody were delivered intracellularly by encapsulating in liposomes or via conjugation with the cell-penetrating MTS-transport peptide. RAW264.7 cells were incubated with these antibodies either before or after ricin exposure. The changes in cytotoxicity were estimated by MTT assay. Co-localization of internalized antibody and ricin was evaluated by fluorescence microscopy. RESULTS:Internalized antibodies significantly increased cell viability either before or after ricin exposure compared to the unconjugated antibodies. Fluorescence microscopy confirmed the co-localization of internalized antibodies and ricin inside the cells.

We have shown that anti-RTA RNA aptamer can protect against ricin ribotoxicity in cell-based luciferase and cell cytotoxicity assays. Hence, RNA aptamer that inhibits RTA enzymatic activity represents a novel class of nucleic acid inhibitor that has the potential to be developed as a therapeutic agent for the treatment of ricin intoxication.

Hodgkin/Reed Sternberg (H/RS) cells are the neoplastic cells in classical Hodgkin lymphoma (HL). They are thought to resemble post-germinal center (GC) B cells with expression of markers associated with late stage of B-cell differentiation, for example, interferon regulatory factor -4/multiple myeloma-1 (IRF4/MUM1) and syndecan 1 (CD138). The PR (PRDI-BF1-RIZ) domain zinc finger protein 1 (PRDM1), a transcription repressor with a master regulatory role in plasma cell differentiation, is normally co-expressed with IRF-4/MUM-1 in plasma cells and in a subset of activated GC cells committed to plasma cell fate. We studied expression of PRDM1α, the functional isoform of PRDM1, in 14 classical HL cases [including 3 positive for Epstein-Barr-virus (EBV)] and 4 HL cell lines by immunohistochemistry and Western blotting, respectively. H/RS cells in primary HL cases are negative for PRDM1α, implying a desynchrony in expression between IRF-4/MUM1 and PRDM1. While the myeloma cell line U266 expresses relatively abundant PRDM1α, it was undetectable by Western Blotting in all HL cell lines tested, except for the EBV-positive HL cell line L591 which, unlike in vivo H/RS cells, has a Type III EBV latency pattern. PRDM1α expression in L591 but not in vivo H/RS cells suggests that PRDM1 expression may be modulated by latency type-specific EBV-encoded gene products or the B-cell phenotype exhibited by the cell line. The lack of PRDM1α protein in H/RS cells is not due to impaired gene transcription, since real-time quantitative PCR revealed similarly abundant PRDM1α transcripts in the HL cell lines as U266. In the absence of mutation in the PRDM1 coding region, these results suggest that failure to accumulate PRDM1α protein in H/RS cells is likely due to abnormal translation repression or protein turnover. Loss of functional PRDM1 as a result of translational or post-translational deregulation may represent a novel molecular lesion in HL.