MX 2 is a novel regulator of cell cycle in melanoma cells.
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ABSTRACT:
SUBMITTER: Juraleviciute M
PROVIDER: S-EPMC7180100 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Stage VI Xenopus oocytes are suspended at the G2/M transition of meiosis I, and represent an excellent system for the identification and examination of cell cycle regulatory proteins. Essential cell cycle regulators such as MAPK, cyclins and mos have the ability to induce oocyte maturation, causing the resumption of the cell cycle from its arrested state. We have identified the product of a novel Xenopus gene, Speedy or Spy1, which is able to induce rapid maturation of Xenopus oocytes, resulting in the induction of germinal vesicle breakdown (GVBD) and activation of M-phasepromoting factor (MPF). Spy1 activates the MAPK pathway in oocytes, and its ability to induce maturation is dependent upon this pathway. Spy1-induced maturation occurs much more rapidly than maturation induced by other cell cycle regulators including progesterone, mos or Ras, and does not require any of these proteins or hormones, indicating that Spy1-induced maturation proceeds through a novel regulatory pathway. In addition, we have shown that Spy1 physically interacts with cdk2, and prematurely activates cdk2 kinase activity. Spy1 therefore represents a novel cell cycle regulatory protein, inducing maturation through the activation of MAPK and MPF, and also leading to the premature activation of cdk2.
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Project description:We have previously shown that RGC-32 is involved in cell cycle regulation in vitro. To define the in vivo role of RGC-32, we generated RGC-32 knockout mice. These mice developed normally and did not spontaneously develop overt tumors. To assess the effect of RGC-32 deficiency on cell cycle activation in T cells, we determined the proliferative rates of CD4(+) and CD8(+) T cells from the spleens of RGC-32(-/-) mice, as compared to wild-type (WT, RGC-32(+/+)) control mice. After stimulation with anti-CD3/anti-CD28, CD4(+) T cells from RGC-32(-/-) mice displayed a significant increase in [(3)H]-thymidine incorporation when compared to WT mice. In addition, both CD4(+) and CD8(+) T cells from RGC-32(-/-) mice displayed a significant increase in the proportion of proliferating Ki67(+) cells, indicating that in T cells, RGC-32 has an inhibitory effect on cell cycle activation induced by T-cell receptor/CD28 engagement. Furthermore, Akt and FOXO1 phosphorylation induced in stimulated CD4(+) T-cells from RGC-32(-/-) mice were significantly higher, indicating that RGC-32 inhibits cell cycle activation by suppressing FOXO1 activation. We also found that IL-2 mRNA and protein expression were significantly increased in RGC-32(-/-) CD4(+) T cells when compared to RGC-32(+/+) CD4(+) T cells. In addition, the effect of RGC-32 on the cell cycle and IL-2 expression was inhibited by pretreatment of the samples with LY294002, indicating a role for phosphatidylinositol 3-kinase (PI3K). Thus, RGC-32 is involved in controlling the cell cycle of T cells in vivo, and this effect is mediated by IL-2 in a PI3K-dependent fashion.
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Project description:A novel human gene, FN1BP1 (fibronectin 1 binding protein 1), was identified using the human placenta cDNA library. Northern blotting showed a transcript of ∼2.8 kb in human placenta, liver, and skeletal muscle tissues. This mRNA transcript length was similar to the full FN1BP1 sequence obtained previously. We established a conditionally induced stable cell line of Hep3B-Tet-on-FN1BP1 to investigate the preliminary function and mechanism of the secretory FN1BP1 protein. Cell-proliferation and colony-conformation assays demonstrated that FN1BP1 protein suppressed Hep3B cell growth and colonization in vitro. Analysis of Atlas human cDNA expression indicated that after FN1BP1 Dox-inducing expression for 24 h, 19 genes were up-regulated and 22 genes were down-regulated more than two-fold. Most of these gene changes were related to cell-cycle-arrest proteins (p21cip1, p15, and cyclin E1), transcription factors (general transcription factors, zinc finger proteins, transcriptional enhancer factors), SWI/SNF (SWItch/Sucrose NonFermentable) complex units, early-response proteins, and nerve growth or neurotrophic factors. Down-regulated genes were subject to colony-stimulating factors (e.g., GMSFs), and many repair genes were involved in DNA damage (RAD, ERCC, DNA topoisomerase, polymerase, and ligase). Some interesting genes (p21cip1, ID2, GMSF, ERCC5, and RPA1), which changed in the cDNA microarray analysis, were confirmed by semi-qRT-PCR, and similar changes in expression were observed. FCM cell-cycle analysis indicated that FN1BP1 over-expression could result in G1 phase arrest. FN1BP1 might inhibit cell growth and/or colony conformation through G1 phase arrest of the Hep3B cell cycle. These results indicate the potential role of FN1BP1 as a treatment target for hepatocellular carcinoma.
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Project description:Recently, we demonstrated that Pseudomonas aeruginosa Exotoxin T (ExoT) employs two distinct mechanisms to induce potent apoptotic cytotoxicity in a variety of cancer cell lines. We further demonstrated that it can significantly reduce tumour growth in an animal model for melanoma. During these studies, we observed that melanoma cells that were transfected with ExoT failed to undergo mitosis, regardless of whether they eventually succumbed to ExoT-induced apoptosis or survived in ExoT's presence. In this report, we sought to investigate ExoT's antiproliferative activity in melanoma. We delivered ExoT into B16 melanoma cells by bacteria (to show necessity) and by transfection (to show sufficiency). Our data indicate that ExoT exerts a potent antiproliferative function in melanoma cells. We show that ExoT causes cell cycle arrest in G1 interphase in melanoma cells by dampening the G1/S checkpoint proteins. Our data demonstrate that both domains of ExoT; (the ADP-ribosyltransferase (ADPRT) domain and the GTPase activating protein (GAP) domain); contribute to ExoT-induced G1 cell cycle arrest in melanoma. Finally, we show that the ADPRT-induced G1 cell cycle arrest in melanoma cells likely involves the Crk adaptor protein. Our data reveal a novel virulence function for ExoT and further highlight the therapeutic potential of ExoT against cancer.
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Project description:Molecular networks governing responses to targeted therapies in cancer cells are complex dynamic systems that demonstrate nonintuitive behaviors. We applied a novel computational strategy to infer probabilistic causal relationships between network components based on gene expression. We constructed a model comprised of an ensemble of networks using multidimensional data from cell line models of cell-cycle arrest caused by inhibition of MEK1/2. Through simulation of a reverse-engineered Bayesian network model, we generated predictions of G1-S transition. The model identified known components of the cell-cycle machinery, such as CCND1, CCNE2, and CDC25A, as well as revealed novel regulators of G1-S transition, IER2, TRIB1, TRIM27. Experimental validation of model predictions confirmed 10 of 12 predicted genes to have a role in G1-S progression. Further analysis showed that TRIB1 regulated the cyclin D1 promoter via NF?B and AP-1 sites and sensitized cells to TRAIL-induced apoptosis. In clinical specimens of breast cancer, TRIB1 levels correlated with expression of NF?B and its target genes (IL8, CSF2), and TRIB1 copy number and expression were predictive of clinical outcome. Together, our results establish a critical role of TRIB1 in cell cycle and survival that is mediated via the modulation of NF?B signaling. Cancer Res; 77(7); 1575-85. ©2017 AACR.
| S-EPMC5410377 | biostudies-literature
Project description:The inability of targeted BRAF inhibitors to produce long-lasting improvement in the clinical outcome of melanoma highlights a need to identify additional approaches to inhibit melanoma growth. Recent studies have shown that activation of the Wnt/β-catenin pathway decreases tumor growth and cooperates with ERK/MAPK pathway inhibitors to promote apoptosis in melanoma. Therefore, the identification of Wnt/β-catenin regulators may advance the development of new approaches to treat this disease. In order to move towards this goal we performed a large scale small-interfering RNA (siRNA) screen for regulators of β-catenin activated reporter activity in human HT1080 fibrosarcoma cells. Integrating large scale siRNA screen data with phosphoproteomic data and bioinformatics enrichment identified a protein, FAM129B, as a potential regulator of Wnt/β-catenin signaling. Functionally, we demonstrated that siRNA-mediated knockdown of FAM129B in A375 and A2058 melanoma cell lines inhibits WNT3A-mediated activation of a β-catenin-responsive luciferase reporter and inhibits expression of the endogenous Wnt/β-catenin target gene, AXIN2. We also demonstrate that FAM129B knockdown inhibits apoptosis in melanoma cells treated with WNT3A. These experiments support a role for FAM129B in linking Wnt/β-catenin signaling to apoptosis in melanoma.
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Project description:The decision for a cell to self-replicate requires passage from G1 to S phase of the cell cycle and initiation of another round of DNA replication. This commitment is a critical one that is tightly regulated by many parallel pathways. Significantly, these pathways converge to result in activation of the cyclin-dependent kinase, cdk2. It is, therefore, important to understand all the mechanisms regulating cdk2 to determine the molecular basis of cell progression. Here we report the identification and characterization of a novel cell cycle gene, designated Speedy (Spy1). Spy1 is 40% homologous to the Xenopus cell cycle gene, X-Spy1. Similar to its Xenopus counterpart, human Speedy is able to induce oocyte maturation, suggesting similar biological characteristics. Spy1 mRNA is expressed in several human tissues and immortalized cell lines and is only expressed during the G1/S phase of the cell cycle. Overexpression of Spy1 protein demonstrates that Spy1 is nuclear and results in enhanced cell proliferation. In addition, flow cytometry profiles of these cells demonstrate a reduction in G1 population. Changes in cell cycle regulation can be attributed to the ability of Spy1 to bind to and prematurely activate cdk2 independent of cyclin binding. We demonstrate that Spy1-enhanced cell proliferation is dependent on cdk2 activation. Furthermore, abrogation of Spy1 expression, through the use of siRNA, demonstrates that Spy1 is an essential component of cell proliferation pathways. Hence, human Speedy is a novel cell cycle protein capable of promoting cell proliferation through the premature activation of cdk2 at the G1/S phase transition.
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Project description:Activity-guided fractionation of a petroleum ether-soluble extract of the roots of Onosma paniculata, which has been shown to affect the cell cycle and to induce apoptosis in melanoma cells, led to the isolation of several shikonin derivatives, namely, β-hydroxyisovalerylshikonin (1), acetylshikonin (2), dimethylacrylshikonin (3), and a mixture of α-methylbutyrylshikonin and isovalerylshikonin (4+5). All compounds exhibited strong cytotoxicity against eight cancer cell lines and MRC-5 lung fibroblasts, with 3 found to possess the most potent cytotoxicity toward four melanoma cell lines (SBcl2, WM35, WM9, and WM164). Furthermore, 3 and the mixture of 4+5 were found to interfere with cell-cycle progression in these cell lines and led to an increasing number of cells in the subG1 region as well as to caspase-3/7 activation, indicating apoptotic cell death.
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Project description:Smooth muscle cell (SMC) differentiation and proliferation occur simultaneously during embryonic development. The underlying mechanisms especially common factors regulating both processes, however, remain largely unknown. The present study has identified cell division cycle 7 (Cdc7) as one of the factors mediating both the proliferation and SMC differentiation. TGF-? induces Cdc7 expression and phosphorylation in the initial phase of SMC differentiation of pluripotent mesenchymal C3H10T1/2 cells. Cdc7 specific inhibitor or shRNA knockdown suppresses TGF-?-induced expression of SMC early markers including ?-SMA, SM22?, and calponin. Cdc7 overexpression, on the other hand, enhances SMC marker expression. Cdc7 function in inducing SMC differentiation is independent of Dumbbell former 4 or Dbf4, the catalytic subunit of Cdc7 critical for cell proliferation, suggesting that Cdc7 mediates SMC differentiation through a mechanism distinct from cell proliferation. Cdc7 regulates SMC differentiation via activating SMC marker gene transcription. Knockdown of Cdc7 by shRNA inhibits SMC marker gene promoter activities. Mechanistically, Cdc7 interacts with Smad3 to induce SMC differentiation. Smad3 is required for Cdc7 function in inducing SMC promoter activities and marker gene expression. Likewise, Cdc7 enhances Smad3 binding to SMC marker promoter via supporting Smad3 nuclear retention and physically interacting with Smad3. Taken together, our studies have demonstrated a novel role of Cdc7 in SMC differentiation.
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Project description:Epstein-Barr virus (EBV) is implicated in the pathogenesis of multiple human tumours of lymphoid and epithelial origin. The virus infects and immortalizes B cells establishing a persistent latent infection characterized by varying patterns of EBV latent gene expression (latency 0, I, II and III). The CDK1 activator, Response Gene to Complement-32 (RGC-32, C13ORF15), is overexpressed in colon, breast and ovarian cancer tissues and we have detected selective high-level RGC-32 protein expression in EBV-immortalized latency III cells. Significantly, we show that overexpression of RGC-32 in B cells is sufficient to disrupt G2 cell-cycle arrest consistent with activation of CDK1, implicating RGC-32 in the EBV transformation process. Surprisingly, RGC-32 mRNA is expressed at high levels in latency I Burkitt's lymphoma (BL) cells and in some EBV-negative BL cell-lines, although RGC-32 protein expression is not detectable. We show that RGC-32 mRNA expression is elevated in latency I cells due to transcriptional activation by high levels of the differentially expressed RUNX1c transcription factor. We found that proteosomal degradation or blocked cytoplasmic export of the RGC-32 message were not responsible for the lack of RGC-32 protein expression in latency I cells. Significantly, analysis of the ribosomal association of the RGC-32 mRNA in latency I and latency III cells revealed that RGC-32 transcripts were associated with multiple ribosomes in both cell-types implicating post-initiation translational repression mechanisms in the block to RGC-32 protein production in latency I cells. In summary, our results are the first to demonstrate RGC-32 protein upregulation in cells transformed by a human tumour virus and to identify post-initiation translational mechanisms as an expression control point for this key cell-cycle regulator.
| S-EPMC3232240 | biostudies-literature