i.) Abstract We have previously shown that human CRC cells increase intracellular NO levels as they transition from horizontal monolayer cultures to vertical stationary suspension cultures and to cells in spheroids subjected to laminar flow shear stress (Laguinge et al., Cancer Res. 64:2643-8, 2004). Currently, we have found that as cultures of CRC cells continue in monolayer they begin to crowd and grow vertically either by rounding up or by detachment and forming spheroids depending on the cell line. As these monolayer cultures are continued, NANOG gene expression increases. As a result, we postulated that intracellular levels of NO would be associated with NANOG expression and may, in fact, regulate NANOG expression. Our preliminary findings partially support the postulate since intracellular NO levels increase in association with increased expression of NANOG in Clone A cells but not CX-1. In addition, the NOS inhibitor NG-Monomethyl-L-arginine acetate (L-NMMA) inhibits intracellular NO in both CRC lines and decreases NANOG gene expression in Clone A but not CX-1. In contrast, the exposure of cells in monolayer to exogenous NO donors at physiologic to pharmacologic concentrations increases NANOG expression in CRC in short term monolayer culture. Preliminary experiments with cGMP donors and antagonists do not clearly show an effect on NANOG expression which suggests that NO affects NANOG expression through nitrosylation/nitration of some intermediary protein. Our current postulate is that NO causes chromatin remodeling that facilitates expression of the NANOG gene. In addition, we also hypothesize that the gene that is re-expressed in human cancers is NANOGP8 which is a retrogene on chromosome 15 whose open reading frame is identical save for one amino acid substitution to the parent wild type NANOG on chromosome 12. The parent gene is silenced by promoter methylation while there is evidence that the retrogene expression is regulated by histone acetylation. Recent data from two other groups indicate that NO inhibits HDAC activity by S-nitrosylation and we would postulate that acetylation is the method by which the retrogene is activated. ii.) Progress Human CRC cells cultured in monolayers in the presence of serum form dense cultures that include increasing numbers of rounded cells with decreased but not absent attachment. During culture of CX-1 and Clone A in monolayer cultures, cells gradually increase their closeness to each other and eventually transition from a spreading, flat, horizontal architecture to one that is more rounded and vertically oriented. Using high density monolayer cultures as a means of studying the expression of the NANOG gene, there are distinct differences between CX-1 and Clone A in the expression of NANOG as the cultures continue in the presence of serum and attachment to a substrate. CX-1 upregulates gene expression in periodic fashion that appears to have a 3-day cycle while Clone A appears to increase NANOG expression dramatically at the end of the culture period. These results are in line with earlier gene expression microarray data (Unpublished data) that suggested NANOG was upregulated along with genes associated with epithelial:mesenchymal transition (EMT) and dedifferentiation were increased. Since EMT is considered to be important for supporting the spread of cancer (a process known as metastasis), it is quite likely that the gene expression changes observed with the transition to a vertical growth phase combined with the gene changes in formed spheroids are critical signs of the interplay between cell shape and cell function that leads to metastasis. These results were confirmed with stable transfectants of Clone A and CX-1 containing a NANOG promoter construct with Green Fluorescent Protein (GFP). Flat cells are GFP negative while cells that are rounded or in spheres are positive for GFP and indicate activation of the NANOG promoter. Taken together, these results indicate that the gene expression of NANOG is sensitive to cell shape with an increase in the gene expression of NANOG as cells detach and transition to suspension culture. Assessment of NO and NANOG expression in Monolayer 2-D and Static 3-D Cultures When we noticed that extending the time of culture and increasing the density of CRC cells in monolayer cultures led to changes in the shape of cells, we then sought to determine if the intracellular concentration of NO also increased. Using a fluorescent dye that is sensitive to intracellular levels of NO we found that densely populated monolayer cultures increased cellular NO but that different cell lines had different concentrations of intracellular NO. Clone A cells increased intracellular NO levels more than CX-1 cells did and CX-1 cells had higher basal levels of intracellular NO. As intracellular NO increases in monolayer cultures that were initiated at high concentration, total RNA and protein was collected and probed for transcription factors and CSC-associated membrane proteins. As cells were cultured in High Density for up to 48 Hr, the protein concentration of CD44, CD133, and ALDH1A1 did not increase. However, there was an increase in Nanog expression in both CX-1 and Clone A in protein expression and transcript level. Modulation of NO levels Alters NANOG Expression We have previously shown that a general inhibitor of NO production, NG-Monomethyl-L-arginine acetate (L-NMMA), not only decreases intracellular NO but affects cell survival through effects on inducible (iNOS or NOS2) and endothelial (eNOS or NOS3) nitric oxide synthase. Here we show that Clone A and CX-1 in monolayer culture decrease their intracellular NO significantly 48 hr after exposure to 5M L-NMMA. Controls were left untreated. The results confirm that L-NMMA decreases NO significantly and only modestly but significantly decreases NANOG expression. We have also begun to assess the effects of exogenous NO on NANOG expression in CRC cultured in monolayer. CRC were cultured with dilutions of diethylamine nitric oxide (DEANO) for 24 - 72 Hr. Cells were harvested at 24 Hr and total RNA analyzed by qRT-PCR for NANOG and inducible NO synthase (iNOS or NOS2). Pharmacologic concentrations of of DEANO above 100 M induced shape change in both CRC lines with detachment from the substrate. In contrast, Clone A did not have a significant change in NANOG expression with DEANO concentrations of up to 100 M. However, CX-1 demonstrated a 6-fold increase in NANOG expression at 100 M DEANO. Preliminary experiments with the cGMP donor 8-Bromo-cGMP or the cGMP inhibitor 1H-[1,2,4]-Oxadiazolo-[4,3-a]-quinoxalin-1-one (ODQ) suggest that neither affects NANOG in CX-1 or Clone A cells, although further experiments are needed. This supports recent reports that S-nitrosylation of HDAC2 leads to chromatin remodeling through increased histone acetylation. iii. Future Directions Shape change may affect NANOG expression through pathways that do not involve NO or S-nitrosylation of HDAcs. However, our focus will be on 1) identifying whether NANOGP8 or NANOG itself are activated and 2) identifying whether the differences between Clone A and CX-1 are related to differences in HDAC activity.
