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

A549 and BEAS-2B cells were stably transfected with a firefly luciferase reporter gene controlled by the RhoB promoter

<p><b>Copyright information:</b></p><p>Taken from "Epigenetic regulation of RhoB loss of expression in lung cancer"</p><p>http://www.biomedcentral.com/1471-2407/7/220</p><p>BMC Cancer 2007;7():220-220.</p><p>Published online 30 Nov 2007</p><p>PMCID:PMC2222678.</p><p></p> Cells were treated with 5 AzaC (10 μM) for 3 days, TSA (1 μM) for 1 day, combination of both or not treated (cont.). Luciferase activity was normalized with protein quantity

RhoB expression was analyzed in various normal and cancer cell lines by RT-PCR

<p><b>Copyright information:</b></p><p>Taken from "Epigenetic regulation of RhoB loss of expression in lung cancer"</p><p>http://www.biomedcentral.com/1471-2407/7/220</p><p>BMC Cancer 2007;7():220-220.</p><p>Published online 30 Nov 2007</p><p>PMCID:PMC2222678.</p><p></p> Expression was also analyzed in normal (N) and tumor (T) tissues obtained from patient operated from lung cancer. B. MSP analysis was performed on DNA extracted from the same cell lines and from normal and tumor tissues. U and M designed PCR product obtained with nmethylated or ethylated specific primers. C. Scheme of the 5' promoter region. The 5' region of was identified previously [18]. We used a CpG island searcher [29] that screens for CpG islands that meet the following criteria: CG percentage > 55%; observed CpG/expected CpG > 0.65; length > 500 bp. This rich-CpG islands region is , and all CpG are represented by . D. An example of RhoB sequence after bisulfite sequencing is shown with CpG island in square. Comparison between normal and tumor tissue is shown