Inhibition of Egr-1 by a dominant-negative mutant, or siRNA-mediated knockdown, significantly decreased Pazopanib purchase the expression of the caspase-8 inhibitor protein, c-FLIP, especially its short isoform (c-FLIPS) and Egr-1 expression associated with high c-FLIP expression in a number of cancer cell lines. The reduction in c-FLIP expression was only partial; however, this experiment probably underestimated the effect of Egr-1 on c-FLIP expression because the maximum transfection efficiency of DN-Egr-1 that we could achieve was 50% in HCT15 cells. Nonetheless, we cannot exclude the contribution of other Egr-1 regulated genes to TRAIL sensitivity. The 5�� region of the c-FLIP gene contains an Egr-1 binding site.

Given that the Egr-1 binding site is a rare promoter element, and that the mouse c-FLIP promoter also contains an Egr-1 binding site (data not shown), it may be a bona fide site and thus indicate a direct regulation of c-FLIP by Egr-1; however, only experimental evidence can confirm it. The c-FLIP promoter also contains a number of AP-1 binding sites and c-Jun is known to be activated by DR4 and DR5. However, inhibition of c-Jun with a dominant-negative construct failed to alter TRAIL sensitivity (data not shown), indicating that c-Jun does not have a major role in regulating c-FLIP expression. Inhibition of Egr-1 affected c-FLIPS expression more than of c-FLIPL probably because of a differential degradation of c-FLIP isoforms. C-FLIPS has been shown to be more prone to ubiquitylation and degradation than c-FLIPL.

Lysines 192 and 195 are principal ubiquitin acceptors in c-FLIPS but not in c-FLIPL because a 19 amino acid tail, which is specific to c-FLIPS and adjacent to the two target lysines, is required for correct positioning and subsequent ubiquitylation of the target lysines (Poukkula et al, 2005). The considerable level of basal Egr-1 expression in colon carcinoma cells can maintain high c-FLIP expression levels, in particular c-FLIPs, and can thus reduce TRAIL sensitivity. Furthermore, upon DR4/DR5 stimulation Egr-1 becomes induced, which may further inc
Sir, The study by Gonzalez de Castro et al (2012) published in British Journal of Cancer compared the sensitivity and specificity of three different methods, the COBAS KRAS mutation kit by Roche (Basel, Switzerland), the Therascreen KRAS kit by Qiagen (Hilden, Germany) and direct Sanger sequencing of PCR product (PCR/sequencing), to detect KRAS mutations in formalin-fixed paraffin-embedded tissues from colorectal carcinoma (CRC) patients. The study clearly demonstrated Entinostat the good reproducibility of the COBAS test. However, we feel that some conclusions might be misleading.