Month: August 2019

In accordance with this function, Necdin overexpression shows growth inhibitory properties in NIH3T3 and SaOS cell lines. However, it is also expressed in myogenic precursors that have a high proliferating potential. Necdin is a p53 target gene and physically interacts with the p53 protein product suggesting a functional relationship. Furthermore, the expression of Necdin can protect cells from apoptosis in different models, including p53-induced apoptosis. Therefore we hypothesize that during carcinogenesis, and depending on the cellular context, Necdin possesses opposing functions and may act as a tumor suppressor based on its similarity with pRb proteins, or as an oncogene through its capacity to inhibit apoptosis and p53-dependent tumor suppressive cell fates. Results reported here support this dual functionality for Necdin. We show that despite the growth suppressive functions of Necdin, it was possible to derive growing cell populations expressing constitutively high levels of Necdin. These high levels of Necdin interfered with p53 activity and contributed to an ineffective growth arrest in response to stress. Overall, we provide evidence suggesting that upregulation of Necdin expression could provide advantages for p53 wild type cells during early carcinogenesis through its ability to decrease signaling from p53 pathways. Interestingly, we found higher Necdin expression to be associated with low malignancy potential ovarian tumors, where p53 mutations are rare, compared to high grade invasive ovarian cancers. Among all candidates identified, the gene encoding Necdin was selected for further study. Microarray analysis showed an upregulation of mRNA up to five-fold. In addition, a second probe set was associated with the Necdin gene and also revealed a 3.6-fold upregulation, although with a P value of 0.04. To further validate the microarray data, Necdin expression was analyzed on an extended set of six NIH3T3 sub-clones and nine independent PyLT-expressing NIH3T3 stable clones not included in our initial analysis. The higher expression levels of Nectin observed when PyLT is expressed, as determined by Northern blot analysis, correlated well with the data derived from microarray analyses. Moreover, a nonradioactive Dig-labeled probe gave only one specific band around the expected size of 1.6 kb, confirming the identity of the lower band in Figure 1 as Necdin. Some clones with variable levels of PyLT expression were also used to confirm that the variation measured at the RNA level was reproduced at the protein level for Necdin. Furthermore, when we derived a new heterogeneous Torin 1 population of NIH3T3 cells expressing PyLT, we again observed an upregulation of Necdin expression compared to a vectortransfected population control. Necdin variation could be seen as early as 72 hrs posttransfection of PyLT. These results show that elevated Necdin expression levels were a reproducible and MK-1775 955365-80-7 constant phenotype in PyLT-expressing NIH3T3 cells and not caused by a clonogenic effect, thus suggesting that Necdin may be involved in some of PyLT oncogenic functions. Necdin interacts with p53 and possibly modulates its activity, which raises the possibility that PyLT exerts its inhibitory effect on p53 through Necdin induction. Nutlin-3 is a small molecule antagonist of MDM2, which prevents the interaction between MDM2 and p53, thus promoting the accumulation of p53 in cells. It has been recently shown that nutlin-3 can efficiently induce cell cycle arrest or apoptosis in different cancer cell lines with functional p53. To assess the response induced in our model, the NIH3T3 cell line was treated with nutlin-3 and proliferation was followed by flow cytometry.

We did not detect apparent cell death as evaluated by the sub-G1 content. When PyLT-expressing NIH3T3 cells were treated with the same dose of nutlin-3, we observed an important delay in growth arrest without a significant elevation in the amount of cell death. To confirm that growth arrest obtained in our model was actually dependent on p53, we used a dominant-negative p53 peptide, GSE22, delivered by lentivirus. As revealed by immunostaining, high infection efficiencies were reached with lentiviruses since almost all cells showed expression of GSE22, which resulted in an accumulation of nonfunctional p53 in the nucleus. Inactivation of p53 by GSE22 expression conferred almost complete resistance to nutlin-3 thereby showing the p53dependence of nutlin-3 induced cell cycle arrest in NIH3T3 cells. These results show that PyLT expression clearly protects against a p53-dependent growth arrest, which supports previous reports on the inhibitory activity of the viral protein on p53. Genes regulated by PyLT were identified in a mouse fibroblast cell culture model. Considering that PyLT has antiapoptotic activities, that it maintains strong homologies in essential domains to the transforming oncogenes SV40LT and E1A, and that its expression in transgenic mice leads to tumors development, it was hypothesized that these PyLT structure-function properties could provide clues to early steps during the transformation process. Since NIH3T3 cells were already immortalized mostly through the biallelic deletion of the INK4 locus, PyLT-mediated immortalization was not a selection criteria in our model and we Paclitaxel Microtubule inhibitor considered candidate genes as possibly immortalization-independent. Our microarray analysis identified a list of potential transformation-associated candidate genes that corroborates the existing literature and point out the importance of viral proteins as tools to Z-VAD-FMK identify events related to cancer progression. For example, Transgelin, an actin-binding protein downregulated in our study, is also downregulated in virally transformed human cells and in human breast, colon and lung cancers. Alternatively, DNA methyltransferase 1, which contributes to the maintenance of tumor suppressors silencing in colon cancer progression and in tumorigenic cell lines, is also upregulated by PyLT expression. Importantly, Dmnt1 is recognized as a significant event during the carcinogenesis process in models related to polyomavirus T antigen expression including the prostate cancer mouse model expressing SV40LT, and transformation of cell lines by SV40LT or the human polyomavirus BKV. Interestingly our main candidate gene, Necdin, was also upregulated in a mouse prostate cancer progression model based on SV40LT expression. Initial observations for Necdin expression in human cancer suggested a tumor suppressor function due to its lack of expression in brain tumor cell lines, its decrease in melanomas, and in bladder cancer cell lines and tumors. Conversely, more recent studies revealed loss of imprinting and upregulation of Necdin in pancreatic cancer. As a whole, Necdin function in cancer remains poorly defined and warrants further investigation. One way to identify closely interacting proteins is to monitor their mRNA expression levels since they are often co-regulated. Since the group of genes shown in Table S2 most closely correlates with PyLT expression at the transcriptional level, these genes represent good candidates for functional associations. One particularly promising member of this group is Necdin, whose gene product has Rb-like growth regulatory activities and has been shown to interact with p53 and viral oncogenes such as SV40LT and E1A.

Whereas Akt phosphorylation has been reported to be downregulated by enzastaurin, likely through an indirect mechanism as Akt is not a direct target of the drug. CUDC-907 However, enzastaurin has also been reported to have little effect on Akt phosphorylation in glioma cells. In the UM cells studied here, Akt phosphorylation was only affected in Mel285 cells by enzastaurin. Interestingly, Nutlin-3 although both Akt and Erk1/2 phosphorylation were decreased by enzastaurin, Mel285 cells, like other GNAQ wild type cells, were less sensitive to enzastaurin in comparison to GNAQ mutated cells where only Erk1/2 phosphorylation was affected. In agreement with sensitivity to enzastaurin, inhibition of Erk1/2 phosphorylation was accompanied by increased p27Kip1 accumulation and decreased expression of cyclin D1, Bcl-2 and survivin in GNAQ mutant cells whereas only survivin was downregulated in Mel285 cells. Furthermore, inhibition of Erk1/2 phosphorylation by MEK1/2 inhibitors increased sensitivity of GNAQ wild type cells to enzastaurin and was associated with similar alterations in the expression of p27Kip1, cyclin D1, Bcl-2 and/or survivin to GNAQ mutant cells treated with enzastaurin. Our findings suggest that the suppression of Erk1/2 phosphorylation may be the major contributor to the increased sensitivity of GNAQ mutant UM cells to the antiproliferative action of enzastaurin through altering the expression of p27, ccyclin D1, Bcl-2 and survivn. These observations further support the oncogenic role for GNAQ mutations via activation of MAPK. The signaling pathways downstream of GNAQ are multifold and include activation of the PKC family members. Our results indicate that UM cell lines have varying expression and phosphorylation patterns of PKC isoforms, independent of GNAQ mutational status. The effects of enzastaurin on the expression and phosphorylation of PKC isoforms in UM cells are complex. Additional studies are needed to determine whether GNAQ mutational status influences the effects of enzastaurin on various PKC isoforms and the potential therapeutic ramifications of these effects. Nonetheless, some PKC isoforms were downregulated by enzastaurin in UM cell carrying GNAQ mutations. In particular, the expression and phosphorylation of PKCh, PKCe, and PKCb were reduced by enzastaurin in GNAQ mutated cells. Our functional studies revealed that these PKC isoforms are indeed more critical for growth of UM cells harboring GNAQ mutations than those with wild type GNAQ. Together, our findings suggest that enzastaurin may exert increased antiproliferative action through inhibiting these PKC isoforms in GNAQ mutated UM cells. Inhibition of these isoforms may play a role in enzastaurininduced inhibition of Erk1/2 phosphorylation, since activation of PKCe and PKCbII have been shown to trigger several major signaling pathways including MAPK. In addition, the inhibition of PKCbII by enzastaurin or small interfering RNA decreased Erk1/2 phosphorylation in metastatic hepatocellular carcinoma cells. It is noteworthy that although enzastaurin had little effect in general on the expression and/or phosphorylation of PKC isoforms in GNAQ wild type C918 cells, it did decrease the expression of PKCe and PKCb phosphorylation in another GNAQ wild type cell line Ocm1. However, enzastaurin did not significantly alter Erk1/2 phosphorylation in both cell lines, suggesting other PKC isoforms and/or PKC independent mechanisms for Erk1/2 activation in Ocm1 cells. Complicating this interpretation, Ocm1 cells have been shown to carry the common V600E BRAF mutation that constitutively activates the MAPK pathway. Furthermore, PKCa and PKCd have been reported to activate Erk1/2 in mouse melanoma. Both PKCa and PKCd are expressed in Ocm1 cells.

In mutated cell lines, there was an estimated decrease of 21% in viability for each unit increase in enzastaurin concentration. Based upon the estimated equations, the EC50 values are 9.15 mM and 2.85 mM for wild type and mutated cell lines, respectively. There is little evidence for differences in response in enzastaurin concentrations of 1 mM or less. At concentrations of 2 mM or above, there was a statistically significant reduction in the viability of mutated cell lines compared with wild type. To better understand the differential responses of UM cells based on GNAQ mutational status, we investigated cell cycle progression alterations with drug exposure. Enzastaurin treatment for 48 hours significantly increased the G1 population while decreasing the S population in all three cell lines harboring GNAQ mutations. In agreement with these findings, enzastaurin significantly decreased BrdU incorporation in mutant cell lines. These results suggest that enzastaurin induced G1 arrest in the cell lines harboring mutations. In comparison, the G1 population of the wild type cell lines was either unaltered or decreased by enzastaurin. A significant increase in the G2/M population was observed in Ocm1 and Mel285 cells. A mild increase in the S population and a significant increase in BrdU uptake were observed in Ocm3 cells treated with 5 and 10 mM enzastaurin. As enzastaurin is known to induce apoptosis in many types of cancer cells, we next examined whether enzastaurin induced apoptosis of UM cells using Annexin V-FITC staining. Treatment with 4 mM enzastaurin for 72 hours induced a slight increase in apoptosis in mutant cell line 92.1 but not in the wild type cell line C918. Because enzastaurin is highly bound by serum protein, we tested if reduced serum concentrations would increase its apoptotic effects. In the presence of 1% serum, treatment with 5 mM enzastaurin for 72 hours induced substantial apoptosis in the cell lines Mel202, 92.1 and Omm1.3 harboring GNAQ mutations, and in the wild type cell lines Ocm1, but failed to do so in cell line C918 which is wild type for GNAQ. An increase in cleaved caspase-3 fragments was also observed in enzastaurin-treated Mel202, 92.1 and Omm1.3 mutant cells and Ocm1 wild type cells, but not C918 cells. These findings suggest that UM cells carrying GNAQ mutations and some GNAQ wild type/BRAF mutant cells are more sensitive to the apoptotic activity of enzastaurin and that enzastaurin exerted increased antiproliferative effect on GNAQ mutant UM cells through ICG-001 induction of G1 arrest and apoptosis. GNAQ mutations at codon 209 have been recently found in nearly 50% of UM patients. These mutations can lead to Niraparib activation of a number of cell signaling pathways. In the present study, we demonstrate for the first time that UM cell lines harboring GNAQ mutations are more sensitive to the antiproliferative effects of the PKC inhibitor enzastaurin than those possessing wild type GNAQ. Enzastaurin inhibits proliferation of mutant UM cells through induction of G1 cell cycle arrest and apoptosis. We have further characterized signaling and molecular mechanisms underlying differential responses of GNAQ wild type and mutant cells to enzastaurin. The PI3K/Akt and MAPK pathways are frequently activated in malignant tumors. Erk1/2 activation is commonly found in UM, independent of GNAQ, RAS, and BRAF mutational status, and are crucial for UM development. GNAQ mutations have been reported to be oncogenic through activating the Erk1/2 pathway in UM cells. In the current study, we show that enzastaurin reduced Erk1/2 phosphrylation in all three GNAQ mutant UM cell lines and in one wild type cell line.

Palmitoylation is carried out by the DHHC-family of palmitoyltransferases. The most commonly used inhibitor of protein palmitoylation is 2bromopalmitate. However, this compound is active only at relatively high concentrations of 100 mM as a broad-spectrum inhibitor that also affects myristoylation. Other identified lipidic inhibitors were shown to exhibit only low mM activity. However, recent insight into the palmitoylation cycle of the cell has led to the development of promising inhibitors of acyl-protein thioesterase 1, which hydrolyzes the palmitoyl-ester bond. Here we report the design and application of three FRETbiosensors that can detect membrane anchorage of N-myristoylated proteins in mammalian cells. These biosensors exploit nanoclustering-induced FRET making them therefore in addition uniquely suitable for the detection of novel nanocluster modulators. Such modulators may represent a novel class of pharmacological compounds that attenuate the action of membrane anchored signaling molecules. We demonstrate that these biosensors report on the inhibition of NMTs and Met-APs and can potentially be employed in cell-based high-throughput screening. In conclusion, Yes-NANOPS is suitable for screening of chemical compound libraries and should have similar potential also for genetic screening applications. In summary, our cytometric assay merges the benefits of imaging-based high content screening and plate reader based cellular assays. The Emax value rapidly integrates essential features of the subcellular localization that is commonly obtained by cell imaging. On the other hand, the assay can be carried out at a rate comparable to that of conventional plate reader based assays. Most importantly, our assay has the unique potential for the discovery of nanoclustering modulators of myristoylated proteins, which may provide a new approach for their pharmacological Tasocitinib modulation. The importance of nanoclustering has been demonstrated for Ras signaling and by analogy, we expect that inhibition of nanoclustering of myristoylated proteins will critically affect their signaling activity, too. Our previous data showed that heterotrimeric G protein alpha subunits from the Gaq and Gai/o subfamily laterally segregate into distinct membrane nanodomains. This may suggest that with the help of our FRET-biosensors inhibitors against specific nanoclusters can be developed. Our assay is flexible and can be adapted to other cell lines, provided that they allow for sufficiently high expression of the biosensor to determine the Emax parameter. It is even conceivable to implement the biosensors in protozoan pathogens, e.g. in order to understand the mechanism of action of membrane organization disrupting compounds. These features, the discovery of novel nanocluster inhibitors and the potential for a cellular high-throughput assay, clearly distinguish our assay from existing formats. The standard assay for N-myristoylation is radioactive and albeit successful even in the high-throughput setting, not really optimal towards that goal. Only recently two complementary non-radioactive in vitro assays have been published. The first detects fluorometrically the released CoA-SH and is thus generally sensitive to hydrolyzing compounds in the screening context. In the second assay a click-chemistry amenable myristate-analogue is SAR131675 utilized and detected by an ELISA-assay like procedure in both cellular and tissue samples.