Epigenetic regulation of RhoB loss of expression in lung cancer

<p>Abstract</p> <p>Background</p> <p>RhoB is down-regulated in most lung cancer cell lines and tumor tissues when compared with their normal counterparts. The mechanism of this loss of expression is not yet deciphered.</p> <p>Methods</p> <p>Since no mutation has been reported in the RhoB sequence, we investigated the epigenetic regulation of RhoB expression by analyzing the effect of HDAC inhibitors and methyltransferase inhibitors, by direct sequencing after bisulfite treatment and by methylation specific PCR.</p> <p>Results</p> <p>We first showed that histone deacetylase (HDAC) inhibitors induce a significant RhoB re-expression in lung cancer cell lines whereas only a slight effect was observed with methyl transferase inhibitors. As promoter methylation is the most common epigenetic process in lung cancer, we performed methylation specific PCR and sequence analysis after bisulfite treatment and demonstrated that RhoB was methylated neither in lung cancer cell lines nor in tumor tissues. We also showed that a variable number of tandem repeats sequences in the 5' region of the RhoB gene was involved in HDAC response.</p> <p>Conclusion</p> <p>We thus propose that RhoB regulation of expression occurs mainly by histone deacetylation rather than by promoter hypermethylation and that this process can be modulated by specific 5' sequences within the promoter.</p

Subtype-associated differences in HIV-1 reverse transcription affect the viral replication

Background: The impact of the products of the pol gene, specifically, reverse transcriptase (RT) on HIV-1 replication, evolution, and acquisition of drug resistance has been thoroughly characterized for subtype B. For subtype C, which accounts of almost 60% of HIV cases worldwide, much less is known. It has been reported that subtype C HIV-1 isolates have a lower replication capacity than B; however, the basis of these differences remains unclear. Results: We analyzed the impact of the pol gene products from HIV-1 B and C subtypes on the maturation of HIV virions, accumulation of reverse transcription products, integration of viral DNA, frequency of point mutations in provirus and overall viral replication. Recombinant HIV-1 viruses of B and C subtypes comprising the pol fragments encoding protease, integrase and either the whole RT or a chimeric RT from different isolates of the C and B subtypes, were used for infection of cells expressing CXCR4 or CCR5 co-receptors. The viruses carrying different fragments of pol from the isolates of B and C subtypes did not reveal differences in Gag and GagPol processing and viral RNA incorporation into the virions. However, the presence of the whole RT from subtype C, or the chimeric RT containing either the polymerase or the connection and RNase H domains from C isolates, caused significantly slower viral replication regardless of B or C viral backbone. Subtype C RT carrying viruses displayed lower levels of accumulation of strong-stop cDNA in permeabilized virions during endogenous reverse transcription, and decreased accumulation of both strong-stop and positive strand reverse transcription products in infected cells and in isolated reverse transcription complexes. This decreased accumulation correlated with lower levels of viral DNA integration in cells infected with viruses carrying the whole RT or RT domains from subtype C isolates. The single viral genome assay analysis did not reveal significant differences in the frequency of point mutations between the RT from B or C subtypes. Conclusions: These data suggest that the whole RT as well as distinct polymerase and connection-RNase H domains from subtype C HIV-1 confer a lower level of accumulation of reverse transcripts in the virions and reverse transcription complexes as compared to subtype B, resulting in a lower overall level of virus replication

Salmonella Mitigates Oxidative Stress and Thrives in the Inflamed Gut by Evading Calprotectin-Mediated Manganese Sequestration

Neutrophils hinder bacterial growth by a variety of antimicrobial mechanisms, including the production of reactive oxygen species and the secretion of proteins that sequester nutrients essential to microbes. A major player in this process is calprotectin, a host protein that exerts antimicrobial activity by chelating zinc and manganese. Here we show that the intestinal pathogen Salmonella enterica serovar Typhimurium uses specialized metal transporters to evade calprotectin sequestration of manganese, allowing the bacteria to outcompete commensals and thrive in the inflamed gut. The pathogen's ability to acquire manganese in turn promotes function of SodA and KatN, enzymes that use the metal as a cofactor to detoxify reactive oxygen species. This manganese-dependent SodA activity allows the bacteria to evade neutrophil killing mediated by calprotectin and reactive oxygen species. Thus, manganese acquisition enables S. Typhimurium to overcome host antimicrobial defenses and support its competitive growth in the intestine