Shape change and Nitric Oxide (NO) Modulation of Core Pluripotent TF Expression

我)。我们之前的研究表明，当人类结直肠癌细胞从水平单层培养转变为垂直静止悬浮培养，并在层流剪切应力下转变为球形细胞时，细胞内NO水平会增加（Laguinge等人，Cancer Res. 64:2643- 8,2004）。目前，我们已经发现，当CRC细胞继续保持单层培养时，它们开始聚集并垂直生长，要么聚集起来，要么脱离并形成球体，这取决于细胞系。随着这些单层培养的继续，NANOG基因表达增加。因此，我们假设细胞内NO水平与NANOG表达有关，并且实际上可能调节NANOG表达。我们的初步研究结果部分支持了这一假设，因为在克隆A细胞中，细胞内NO水平的增加与NANOG表达的增加有关，而与CX-1无关。此外，NOS抑制剂ng - monommethyl - l- arginine acetate （L-NMMA）在两种结直肠癌细胞系中均抑制细胞内NO，并降低克隆A的NANOG基因表达，而CX-1则没有。相反，在短期单层培养中，将细胞暴露于生理或药物浓度的外源性NO供体中会增加CRC中NANOG的表达。在cGMP供体和拮抗剂的初步实验中，没有明确显示NO对NANOG表达的影响，这表明NO通过一些中间蛋白的亚硝基化/硝化作用影响NANOG的表达。我们目前的假设是NO引起染色质重塑，从而促进NANOG基因的表达。此外，我们还假设在人类癌症中重新表达的基因是NANOGP8，这是15号染色体上的一个逆转录基因，其开放阅读框与12号染色体上的亲本野生型nanog8相同，只有一个氨基酸替代。亲本基因被启动子甲基化沉默，而有证据表明逆转录基因的表达受组蛋白乙酰化调节。最近来自另外两组的数据表明，NO通过s -亚硝基化抑制HDAC活性，我们假设乙酰化是激活逆转录基因的方法。二)。人类结直肠癌细胞在血清存在下单层培养形成致密培养，包括圆形细胞数量增加，附着减少但并非没有。在CX-1和克隆A的单层培养过程中，细胞之间的距离逐渐增加，最终从扩散的、平坦的、水平的结构转变为更圆的、垂直的结构。使用高密度单层培养作为研究NANOG基因表达的手段，CX-1和克隆a在NANOG表达上存在明显差异，因为在血清存在和附着于底物的情况下继续培养。CX-1以周期性的方式上调基因表达，似乎有一个3天的周期，而克隆a似乎在培养期结束时显著增加NANOG的表达。这些结果与早期基因表达微阵列数据（未发表的数据）一致，表明NANOG与上皮间充质转化（EMT）和去分化相关的基因一起上调。由于EMT被认为对支持癌症的扩散（一个被称为转移的过程）很重要，因此很可能观察到的基因表达变化与形成的球体的基因变化一起过渡到垂直生长阶段是导致转移的细胞形状和细胞功能之间相互作用的关键标志。这些结果被克隆A和CX-1稳定的含有绿色荧光蛋白（GFP）的NANOG启动子构建体的转染物证实。扁平细胞呈GFP阴性，而圆形或球形细胞呈GFP阳性，表明NANOG启动子被激活。综上所述，这些结果表明NANOG基因表达对细胞形状敏感，随着细胞脱离并过渡到悬浮培养，NANOG基因表达增加。当我们注意到在单层培养中延长培养时间和增加CRC细胞密度会导致细胞形状的变化时，我们试图确定细胞内NO浓度是否也会增加。使用对细胞内NO水平敏感的荧光染料，我们发现密集的单层培养增加了细胞内NO，但不同细胞系的细胞内NO浓度不同。克隆A细胞的细胞内NO水平高于CX-1细胞，且CX-1细胞的细胞内NO基础水平较高。在高浓度启动的单层培养中，随着细胞内NO的增加，收集总RNA和蛋白质，并探测转录因子和csc相关膜蛋白。细胞高密度培养48小时后，CD44、CD133和ALDH1A1蛋白浓度未升高。而在CX-1和克隆A中，Nanog的表达在蛋白表达和转录水平上均有增加。我们之前的研究表明，NO生成的一般抑制剂ng - monom甲基- l-精氨酸乙酸酯（L-NMMA）不仅可以降低细胞内NO，还可以通过影响诱导型（iNOS或NOS2）和内皮型（eNOS或NOS3）一氧化氮合酶来影响细胞存活。本研究表明，克隆A和CX-1在暴露于5M L-NMMA后48小时，细胞内NO显著降低。对照组不进行治疗。结果证实，L-NMMA可显著且仅适度降低NO的表达，但可显著降低NANOG的表达。我们也开始评估外源性NO对单层培养结直肠癌中NANOG表达的影响。用稀释的二乙胺一氧化氮（DEANO）培养结直肠癌24 - 72小时。24小时后收集细胞，用qRT-PCR分析NANOG和诱导NO合成酶（iNOS或NOS2）的总RNA。药理学浓度大于100 M的DEANO诱导两种结直肠癌细胞系的形状发生变化，并与底物分离。相比之下，克隆A在DEANO浓度高达100 M时NANOG表达没有显著变化，而CX-1在100 M DEANO时NANOG表达增加了6倍。cGMP供体8-Bromo-cGMP或cGMP抑制剂1H-[1,2,4]- oxadiazolo -[4,3- A]-quinoxalin-1-one （ODQ）的初步实验表明，它们都不会影响CX-1或克隆A细胞的NANOG，但还需要进一步的实验。这支持了最近的报道，即HDAC2的s -亚硝基化通过增加组蛋白乙酰化导致染色质重塑。三世。形状变化可能通过不涉及hdac NO或s -亚硝基化的途径影响NANOG的表达。然而，我们的重点将是1)确定NANOGP8或NANOG本身是否被激活，2)确定克隆A和CX-1之间的差异是否与HDAC活性的差异有关。

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.