Indeed, the promoter reporter constructs induced by FL Mkl1 were also strongly induced by mutB1 Mkl1, but not by SAP Mkl1. In contrast, the promoter construct for Acta2, a gene from the SRF dependent SAP independent gene set was strongly induced by SAP Mkl1 but not by mutB1 Mkl1, as and Nox4, for which some activity was lost by shortening the promoters, selleck chemicals the 200 bp proximal promoters of all other genes tested were induced equally well as the longer con structs. Thus, we conclude that there are many genes that are regulated similarly as tenascin C requiring the SAP domain of Mkl1 to induce transcription from their proximal promoter. The different HC11 cell strains proliferate at different rates and show distinct migration behaviors Next, we tested whether the differential gene expression seen in the different HC11 strains overexpressing either FL.

mutB1 or SAP Mkl1 constructs have functional consequences on their behavior. Since most of the SAP dependent transcripts Inhibitors,Modulators,Libraries are proposed to have a function in cancer, we decided to analyze two main functions im portant for cancer progression proliferation and cell mi gration. An approximately equal overexpression of the different Mkl1 protein variants in the HC11 cell lines was confirmed by Western blot analysis. An HC11 cell strain stably transfected with an empty vector expressing only endogenous Mkl1 was also included in these studies. The proliferation rates of the HC11 strains were ana lyzed Inhibitors,Modulators,Libraries using a 5 bromo 2 deoxyuridine incorp oration assay. The incorporated BrdU was measured immediately after plating as well as at 24, 48, 72 and 96 h.

Compared to empty vector. FL or mutB1 transfected Inhibitors,Modulators,Libraries HC11 strains, there was a significant decrease in BrdU uptake into newly synthesized DNA in HC11 expected for a typical SRF Mkl1 target gene. All promoters that revealed SAP dependency were shortened to 200 bp upstream of the TSS to test whether this was sufficient to relay the Mkl1 response, as it has been seen previously Inhibitors,Modulators,Libraries for tenascin C. With the exception of Krt5 SAP cells over Inhibitors,Modulators,Libraries the entire time period tested. To investigate cell motility, we used a transfilter migration assay. Similarly to the effect on cellular proliferation, the expression of SAP Mkl1 significantly inhibited HC11 cell migration by 2. 7 fold compared to endogenous or full length Mkl1 expression, and more than 3. 5 fold compared to mutB1 Mkl1 expression.

Thus, overexpression of FL Mkl1 protein in HC11 cells did not affect their behavior. However, overexpres sion of SAP Mkl1 led to a significant reduction in the proliferative and migratory ability of HC11 epithelial cells, either through a dominant negative effect of SAP Mkl1 on SRF mediated action and or a positive impact of the SAP dependent Mkl1 target genes on these functions important antiangiogenic for cancer progression.