VP22 fusion protein-based dominant negative mutant can inhibit hepatitis B virus replication

AIM： To investigate the inhibitory effect of VP22 fusion protein-based dominant negative （DN） mutant on Hepatitis Bvrus （HBV） replication. METHODS： Full-length or truncated fragment of VP22 was fused to C terminal of HBV core protein （HBc）, and subcloned into pcDNA3.1 （-） vector, yielding eukaryotic expression plasmids of DN mutant. After transfection into HepG2.2.15 cells, the expression of DN mutant was identified by immunofluorescence staining. The inhibitory effect of DN mutant on HBV replication was indexed as the supernatant HBsAg concentration determined by RIA and HBV-DNA content by fluorescent quantification-PCR （FQ-PCR）. Meanwhile, metabolism of HepG2.2.25 cells was evaluated by MTI- colorimetry. RESULTS： VP22-based DN mutants and its truncated fragment were expressed in HepG2.2.15 cells, and had no toxic effect on host cells. DN mutants could inhibit HBV replication and the transduction ability of mutantbearing protein had a stronger inhibitory effect on HBV replication. DN mutants with full length of VP22 had the strongest inhibitory effect on HBV replication, reducing the HBsAg concentration by 81.94%, and the HBV-DNA content by 72.30%. MTT assay suggested that there were no significant differences in cell metabolic activity between the groups. CONCLUSION： VP22-based DN mutant can inhibit HBV replication effectively.

Aberrant expression and function of TCF4 in the proliferation of hepatocellular carcinoma cell line BEL-7402

Wnt signaling pathway is essential for development and tumorigenesis,however,this signaling pathway in the progress of hepatocellular carcinoma (HCC) remains unclear. In this paper,we studied the function of human T-cell transcription factor-4 (TCF4),a key factor of Wnt signaling pathway,on the proliferation of HCC cell line. We showed that the expression of TCF4 mRNA in HCC cell line BEL-7402 was higher than that in immortalized normal liver cell line L02. Blockage of Wnt pathway by △NTCF4,a dominant negative TCF4,could suppress BEL-7402 cells growth and decrease the expression of cyclin D1 and c-myc,two of target genes of Wnt pathway. On the other hand,stimulating Wnt pathway by introducing a degradation-resistant β-catenin S37A could increase BEL-7402 cells proliferation. But all the treatments had no effect on L02 cells. Our data indicated that TCF4 might be another key factor in Wnt pathway involved in HCC cells proliferation and TCF4 could be an effective therapeutic target for suppressing the growth of hepatocellular cancers.

Gene therapeutic approaches to inhibit hepatitis B virus replication

Acute and chronic hepatitis B virus(HBV) infections remain to present a major global health problem. The infection can be associated with acute symptomatic or asymptomatic hepatitis which can cause chronic inflammation of the liver and over years this can lead to cirrhosis and the development of hepatocellularcarcinomas. Currently available therapeutics for chronically infected individuals aim at reducing viral replication and to slow down or stop the progression of the disease. Therefore, novel treatment options are needed to efficiently combat and eradicate this disease. Here we provide a state of the art overview of gene therapeutic approaches to inhibit HBV replication. We discuss non-viral and viral approaches which were explored to deliver therapeutic nucleic acids aiming at reducing HBV replication. Types of delivered therapeutic nucleic acids which were studied since many years include antisense oligodeoxynucleotides and antisense RNA, ribozymes and DNAzymes, RNA interference, and external guide sequences. More recently designer nucleases gained increased attention and were exploited to destroy the HBV genome. In addition we mention other strategies to reduce HBV replication based on delivery of DNA encoding dominant negative mutants and DNA vaccination. In combination with available cell culture and animal models for HBV infection, in vitro and in vivo studies can be performed to test efficacy of gene therapeutic approaches. Recent progress but also challenges will be specified and future perspectives will be discussed. This is an exciting time to explore such approaches because recent successes of gene therapeutic strategies in the clinic to treat genetic diseases raise hope to find alternative treatment options for patients chronically infected with HBV.

Proline-rich tyrosine kinase 2 and its inhibitor PRNK

Proline-rich tyrosine kinase 2 (Pyk2) is a nonreceptor protein tyrosine kinase,which is also known as Ca2 +-dependent tyrosine kinase or related adhesion focal tyrosine kinase.Pyk2 activation exerts a critical regulatory mechanism for various physiological processes including cytoskeleton function,regulation of cell growth and death,modulation of ion channels and multiple signaling events.However,mechanisms underlying the functional diversity of Pyk2 are not clear.A Pyk2 isoform that encodes only part of the C-terminal domain of Pyk2,named as PRNK (Pyk2-related non-kinase),acts as a dominant-negative inhibitor of Pyk2-dependent signaling by displacing Pyk2 from focal adhesions.Research on functional PRNK probably provides new potential inhibitory tool targeting Pyk2 and makes it possible to explore more of Pyk2 pathological mechanism.PRNK is a promising candidate targeting Pyk2 modulation.This review focuses on the functional investigation of Pyk2 and its structure and localization,including recent research with inhibitory strategies targeting Pyk2 by the method of PRNK.

Inhibition of Cell Growth and Telomerase Activity in Osteosarcoma Cells by DN-hTERT

In order to study the effects of dominant negative human telomerase reverse transcriptase （DN-hTERT） on cell growth and telomerase activity in osteosarcoma cell line MG63, MG63 cells were transfected with DN-hTERT-IRES2-EGFP9 （DN） or IRES2-EGF （I, blank vector） with lipofectamine 2000. The stably transfected cells were selected with G-418. Cell growth properties were examined under a fluorescence microscope. The hTERT mRNA expression was detected by reverse transcription-polymerase chain reaction （RT-PCR）. Telomerase activities were measured by TRAP-ELISE. The tumorigenicity was studied with tumor xenografts by subcutaneous injection of cancer cells into nude mice. The results showed that cell growth was suppressed in MG63 cells transfected with DN-hTERT. The hTERT mRNA was increased in N-hTERT transfected-MG63 cells （MG63/DN）. The telomerase activity was 2.45±0.11 in MG63/DN cells, while 3.40±0.12 in the cells transfected with blank vector （MG63/I）, （P〈0.05）; DN-hTERT-expressing clones did not form tumors in 2 weeks, but the ratio of tumorigenesis was 30 % in nude mice bearing MG63/I （P〈0.01）. It was concluded that DN-hTERT could specifically inhibit the cell growth and telomerase activity in MG63 cells.

Histone 3 lysine 36 to methionine mutations stably interact with and sequester SDG8 in Arabidopsis thaliana

Post-transcriptional modifications of histones play important roles in various biological processes. Here, we report that Arabidopsis plants overexpressing histone H3 lysine to methionine mutations at histone H3.1K36(H3.1K36M) and H3.3K36(H3.3K36M) have serious developmental defects with early-flowering and change in the modifications of endogenous histone H3, including acetylation at lysine 9(H3K9ac), trimethylation at lysine 27(H3K27me3), di-and tri-methylation at lysine 36(H3K36me2 and H3K36me3). In addition, H3K36M mutation alters its subcellular localization and interacts with H3K36 methyltransferase SDG8. Our results support a model in which H3K36M stably interacts with SDG8, and inhibits the activity of SDG8 by sequestering SDG8, resulting in a dominant negative effect to affect the proper expression levels of a variety of genes and plant development.