Month: January 2018

To understand the changes in adhesion and motility, we used confocal R428 fluorescence microscopy to monitor the localization and expression of the adherens junction proteins E-cadherin, b-catenin and Zo-1 in matched samples of parental, LGR5 knockdown or LGR5 overexpressing LIM1899 cells. Fixed cells were incubated with the appropriate INCB28060 antibodies and fluorescent secondary antibodies, and co-stained with rhodamine-phalloidin to visualize actin. Neither the levels nor distribution of E-cadherin changed significantly with up-or down-regulation of LGR5, however there was a consistent recruitment of b-catenin to the cell junctions in LIM1899 overexpressing LGR5. This was surprising as the LIM1899 cell line carries an activating b-catenin mutation resulting in a predominantly cytosolic bcatenin, with some weak association to the membranes but rarely seen at the cell-cell junctions; this distribution is insensitive to wnt signalling stimulation or inhibition. The tight-junction molecule Zo-1 was also increased at the cell-cell contacts in LIM1899 cells overexpressing LGR5. The relative amounts of these proteins in cells under- or over-expressing LGR5 were also assessed by immunoblotting, confirming little change in total b-catenin levels, marginal increase in Ecadherin, and significant increase of Zo-1. Overall, these results are consistent with an enhancement of cell-cell adhesion in cells overexpressing LGR5. Given the effects of LGR5 modulation on cell migration, we hypothesized that LGR5 levels might affect, directly or indirectly, the expression or localization of adhesion molecules. CD44, CD133 and CD166 are adhesion molecules expressed on intestinal stem cells and colorectal cancer stem cells and therefore can be expected to have overlapping expression patterns to LGR5. As these molecules have been used extensively as stem cell markers, we also wanted to assess whether changes in LGR5 expression resulted in altered patterns of expression for these markers. CD133 is not expressed on LIM1899, as assessed by flow cytometry, while CD44 and CD166 are expressed at high levels. Only CD44 surface expression is weakly enhanced by upregulation of LGR5 in these cells, but there is no appreciable change in total cellular CD44 as assessed by immunoblotting. Confocal microscopy revealed that the cell surface distribution of CD44 is subtly altered in LGR5 knockdown cells. In parental LIM1899 cells and in cells overexpressing LGR5, CD44 associates with actin rings as assessed by morphology and colocalization with actin. When LGR5 expression is reduced or abolished by inhibitory RNAs, CD44 is more uniformly distributed on the cell surface and is missing selectively from the focal actin rings. This phenomenon was observed consistently with knockdown of LGR5, either by siRNA or shRNA, in both Lim1899 and LIM1215 cells. The distribution of CD44 in LIM 1899 cells overexpressing LGR5 was unaltered.

Furthermore, the phenomenon of amplification in 3q appears to be quite different from that in 5p. For instance, the level of gain or amplification was lower than in 5p and it did not include the entire 3q arm in all the cell lines; instead, only certain sparse regions were found altered recurrently. In fact, the average log2 ratio of MRRs at 5p was almost 2 fold times higher than those located at 3q. The full arm or several regions of 3q were found to be amplified, similarly to the findings in previous reports. This could explain the lower proportion of deregulated genes found in 3q compared with that in 5p. However, even in those cell lines where most of 3q was gained, the proportion of deregulated genes did not rise or increased modestly. In addition, in 3q, the proportion of LY2835219 moa downregulated genes was higher than the proportion of upregulated genes, particularly in 3q26, where 8 of 11 deregulated genes were downregulated, even some of them were recurrently gained. Furthermore, similarly to 5p, deregulated genes seemed to be grouped in GDC-0199 clusters in 3q26�C29. These findings indicate that an increase in the copy number does not necessarily mean that genes located in those regions will be upregulated. It suggests that, in those entirely amplified regions, epigenetic mechanisms could be involved in gene repression. On the other hand, the increased frequency of downregulated genes with the number of amplified SNPs in the subset of genes located in MRRs, which seems to be not entirely amplified, supports that partial gene amplification may be a mechanism of gene silencing. This idea has been proposed theoretically. The 3q26 region has been previously identified as gained or amplified in biopsies or cell lines derived from CC by using CGH or FISH. Recognized tumor genes, such as EVI1 and MDS1, and genes associated previously with CC are located in this region. However, it has not been demonstrated that these genes were upregulated, particularly in the same samples where the CN alterations were found. In this study, EVI1, TERC, PIK3CA, and LAMP3 were neither found CN altered recurrently nor upregulated in all the cell lines studied. TERC was found gained in CaLo, CaSki, and HeLa but upregulated only in HeLa. However, conclusions with these negative results from the microarrays may be too risky without the validation with different methodologies, like qPCR and qRT-PCR. Interestingly, the gene encoding for tumor necrosis factor superfamily member 10, a protein that induces apoptosis in transformed and tumor cells, was found to be downregulated in the 4 cell lines, even though the gene was recurrently gained. This gene is located in the same region as 2 other downregulated and 2 upregulated genes, which have not been previously associated with CC. However, the protein encoded by ECT2 is a transforming protein that is a nuclear guanine nucleotide exchange factor and regulates RhoB-mediated cell death after DNA damage in cervical cell lines.