Different from Caco-2 cells, FET cells do not show varying macroH2A1 levels under regular culture conditions, making them a suitable tool for assessing the effects of macroH2A1 depletion in a cell line with a naturally high and stable expression of www.selleckchem.com/products/crenolanib-cp-868596.html macroH2A1. This approach allowed us to assess the effects of varying macroH2A1 levels in a second independent cell-line model complementing the Caco-2 differentiation experiment. RNA from knockdown and control experiments was analyzed using the same PCR arrays as in the above Caco-2 differentiation experiment (Figure 5). Expression changes were calculated comparing knockdown to control cells. Overall, we observed more subtle expression changes than in the previous experiment.
This was expected, as we compared two sets of cancer cells that were both proliferating, whereas previously, we compared cells with two distinct phenotypes, colon cancer cells on one hand and cells resembling enterocytes without tumorigenic potential on the other. Knockdown of macroH2A1.1 was very specific and did not lead to major changes in macroH2A1.2 levels, yet knockdown of macroH2A1.2 involved a decrease in macroH2A1.1 levels (Figure 6A). Figure 6 Knockdown of macroH2A1 isoforms is associated with a phenotype enhancing proliferation and metastasis. A: Transient knockdown of macroH2A1.1 and macroH2A1.2 is determined by qPCR (left) and Western blot analysis (right). B: Effects of macroH2A1.1 knockdown … FET cells with reduced macroH2A1.
1 levels showed a phenotype consistent with enhanced proliferation and DNA replication (up-regulation of HERC5, BRCA2, CCND2, HUS1, NBN, and CITED2), favoring survival (up-regulation of apoptotic inhibitor gene SERPINB2, down-regulation of CDKN1A), as well as a relief of gene silencing (up-regulation of GADD45A) (Figure 6B). Transcriptional repressors (ID1, TXB3) and markers of cell cycle arrest and growth inhibition (CDKN1A, CDKN1C, CDKN2C, and MAP2K6) were suppressed. Surprisingly, CDKN2B, classically described as a cell cycle inhibitor, was up-regulated following macroH2A1.1 knockdown, whereas we had expected a down-regulation. Interestingly, these data paralleled recent findings in chronic lymphocytic leukemia and small lymphocytic lymphoma showing specific overexpression of p15 (CDKN2B) along with up-regulation of CCND2 in the proliferation centers of these tumors.
18 Notably, we observe an up-regulation of the gene for telomerase (TERT), mirroring the results of the differentiation experiment, as well as other genes with known oncogenic potential GSK-3 (BMI1, EGR1, ETS1, HERC5). Especially interesting is the up-regulation of several genes that have been shown to be involved in migration and metastasis in various other cancer types (ALDH1A3, CDK5R1, FN1, PLAU, SERPINE1, SPARC) (see Supplemental Table S318�C43 at http://ajp.amjpathol.org). Results of the macroH2A1.2 knockdown revealed a similar phenotype (Figure 6C).