Increase of RhoB in gamma-radiation-induced apoptosis is regulated by c-Jun N-terminal kinase in Jurkat T cells.

The Ras-related small GTP-binding protein RhoB is known to be a pro-apoptotic protein and immediate-early inducible by genotoxic stresses. In addition, JNK activation is known to function in gamma-radiation-induced apoptosis. However, it is unclear how JNK activation and gamma-radiation-dependent RhoB induction are related. Here we verified the relationship between JNK activation and RhoB induction. RhoB induction by gamma-radiation occurred at the transcriptional level and transcriptional activation of RhoB was concomitant with an increase in RhoB protein. gamma-Radiation-induced RhoB expression was markedly attenuated by pretreatment with a JNK-specific inhibitor, SP600125, but not by a p38 MAPK inhibitor, SB203580. Inhibition of JNK caused a decrease in early apoptotic cell death that correlated with RhoB expression. However, PI3K inhibition had no significant effects, indicating that the AKT survival pathway was not involved. The siRNA knockdown of JNK resulted in a decrease in RhoB expression and the siRNA knockdown of RhoB restored cell growth even in the gamma-irradiated cells. These results suggest that RhoB regulation involves the JNK pathway and contributes to the early apoptotic response of Jurkat T cells to gamma-radiation.

Generation of expression vectors for high-throughput functional analysis of target genes in Schizosaccharomyces pombe.

An immediate challenge in the post-genomic era is to assign a biological functions to proteins unraveled by genome analysis. This report is based on studies conducted using Schizosaccharomyces pombe, a simple model organism, and presents various vector systems as tools for high-throughput functional analysis of human genes. We constructed S. pombe expression vectors for efficient cloning of genes via the Gateway system. We modified the pREP and pSLF series vectors, which are widely used for gene expression in S. pombe. The vectors constructed have a uniform backbone of S. pombe autonomously replicating sequence (ARS) elements with different selective markers, namely, urw4 (+) and Saccharomyces cerevisiae LEU2 complementing leul. These vectors contain 3 different strengths of the inducible promoter nmtl, which affect the expression levels of the cloned open reading frames (ORFs). Further, target proteins can be fused with an N-terminal or C-terminal tag such as triple hemagglutinin (3x HA), enhanced green fluorescent protein (EGFP), or Discosoma red fluorescent protein (DsRed). We tested the feasibility of the constructed vectors by using 3 human genes, namely, RAB18, SCC-112, and PTEN. Proper expression of tagged RAB18 was confirmed by western blot analysis. Further, localization of RAB18, SCC112, and PTEN was demonstrated. The constructed vectors can be utilized for high-throughput functional analysis of heterologous genes.

Fission yeast-based screening to identify putative HDAC inhibitors using a telomeric reporter strain.

Transcriptional silencing is regulated by promoter methylation and histone modifications such as methylation and acetylation. We constructed a Schizosaccaromyces pombe reporter strain, KCT120a, to identify modifiers of transcriptional silencing, by inserting the ura4(+) gene into a heterochromatic telomere region. Two compounds inhibited the activity of histone deacetylases, induced acetylation of histone H3 and caused apoptotic cell death in HeLa cells. Expression of gelsolin and p21(waf1/cip1) also increased, as it does in response to HDAC inhibitors such as TSA. Therefore, these compounds appear to be potent inhibitors of HDACs, and hence potential anti-cancer drugs. Our observations suggest that a yeast cell-based assay system for transcriptional silencing may be useful for identifying histone deacetylase inhibitors and other agents affecting chromatin remodeling.