Cellular Levels Of Cyclin D1 Research Articles

Bacillus anthracis Edema Toxin Activates Nuclear Glycogen Synthase Kinase 3β

Bacillus anthracis edema toxin (ET) generates high levels of cyclic AMP and impacts a complex network of signaling pathways in targeted cells. In the current study, we sought to identify kinase signaling pathways modulated by ET to better understand how this toxin alters cell physiology. Using a panel of small-molecule inhibitors of mammalian kinases, we found that inhibitors of glycogen synthase kinase 3 beta (GSK-3beta) protected cells from ET-induced changes in the cell cycle. GSK-3beta inhibitors prevented declines in cellular levels of cyclin D1 and c-Jun following treatment of macrophages with ET. Strikingly, cell fractionation experiments and confocal immunofluorescence microscopy revealed that ET activates a compartmentalized pool of GSK-3beta residing in the nuclei, but not in the cytoplasm, of macrophages. To investigate the outcome of this event, we examined the cellular location and activation state of beta-catenin, a critical substrate of GSK-3beta, and found that the protein was inactivated within the nucleus following intoxication with ET. To determine if ET could overcome the effects of stimuli that inactivate GSK-3beta, we examined the impact of the toxin on the Wnt signaling pathway. The results of these experiments revealed that by targeting GSK-3beta residing in the nucleus, ET circumvents the upstream cytoplasmic inactivation of GSK-3beta, which occurs following exposure to Wnt-3A. These findings suggest ET arrests the cell cycle by a mechanism involving activation of GSK-3beta residing in the nucleus, and by using this novel mechanism of intoxication, ET avoids cellular systems that would otherwise reverse the effects of the toxin.

Celecoxib‐induced growth inhibition in SW480 colon cancer cells is associated with activation of protein kinase G

Although it is often assumed that the antitumor effects of nonsteroidal anti-inflammatory drugs (NSAIDs) are due to inhibition of cyclooxgenase (COX) activity, specifically COX-2, there is accumulating evidence that COX-2 independent mechanisms can also play an important role. Studies with sulindac sulfone (Aptosyn) and related derivatives have revealed a novel pathway of tumor growth inhibition and apoptosis mediated by activation of the guanosine 3',5' monophosphate (cGMP)-dependent enzyme protein kinase G (PKG). The present study indicates that concentrations of the NSAIDs celecoxib, indomethacin, and meclofenamic acid that inhibit growth of SW480 human colon cancer cells inhibit subcellular cGMP-phosphodiesterase (PDE) enzymatic activity and in intact cells induce a two- to threefold increase in intracellular levels of cGMP. This is associated with phosphorylation of the protein VASP, a marker of PKG activation, activation of JNK1 and a decrease in cellular levels of cyclin D1; effects seen with other agents that cause activation of PKG in these cells. On the other hand even a high concentration of the COX-2 specific inhibitor rofecoxib (500 microM) did not inhibit growth of SW480 cells. Nor did rofecoxib inhibit cGMP-PDE activity or cause other changes related to PKG activation in these cells. Since activation of the PKG pathways by celecoxib, indomethacin, and meclofenamic acid in this cell culture system required high concentrations of these compounds, it remains to be determined whether activation of this pathway contributes to the in vivo antitumor effects of specific NSAIDs.

Phosphorylation of p68 RNA Helicase Plays a Role in Platelet-derived Growth Factor-induced Cell Proliferation by Up-regulating Cyclin D1 and c-Myc Expression

p68 RNA helicase is a protypical member of DEAD box family RNA helicase. The protein plays an important role in the cell developmental program and organ maturation. We demonstrated previously that, in response to growth factor platelet-derived growth factor (PDGF)-BB stimulation, p68 is phosphorylated at Tyr(593), and the phosphorylation of p68 promotes epithelial-mesenchymal transition via promoting beta-catenin nuclear translocation (Yang, L., Lin, C., and Liu, Z. R. (2006) Cell 127, 139-155). We show here that the tyrosine phosphorylation of p68 also mediates the effects of PDGF in stimulating cell proliferation. The phosphorylated p68 (referred to as phospho-p68) promotes cell proliferation by activating the transcription of cyclin D1 and c-Myc genes. We show that the ATPase/helicase activities of p68 are required for the activation of cyclin D1 transcription. The phospho-p68 participates in the complex assembled at the cyclin D1 and c-Myc promoters, which strongly suggests a direct role in transcriptional regulation. Furthermore, our data demonstrated that the phosphorylation of p68 at Tyr(593) plays a role in mediating the autocrine loop effects of PDGF, suggesting an important role for p68 phosphorylation in cell proliferation.

Protein Kinase C β Enhances Growth and Expression of Cyclin D1 in Human Breast Cancer Cells

Although alterations in the expressions of protein kinase C (PKC) have been implicated in breast carcinogenesis, the roles of specific isoforms in this process remain elusive. In the present study, we examined the specific roles of PKCbeta1 and beta2 in growth control in human breast cancer cell lines. The PKCbeta-specific inhibitor LY379196 significantly inhibited growth of the breast cancer cell lines MCF-7, MDA-MB-231, and BT474, but not the normal mammary epithelial cell line MCF-10F. Treatment of MCF-7 cells with LY379196 caused an increase in the fraction of cells in the G(1) phase of the cell cycle. To explore the roles of PKCbeta1 and beta2, we used cDNA expression vectors that encode wild-type and constitutively activated or dominant negative mutants of these two proteins. When compared with vector controls, derivatives of MCF-7 cells that stably overexpress wild-type PKCbeta1 or PKCbeta2 displayed a slight increase in growth rate; derivatives that stably express the constitutively active mutants of PKCbeta1 or PKCbeta2 displayed a marked increase in growth rate; and derivatives that stably express a dominant negative mutant of PKCbeta1 or beta2 displayed inhibition of growth. The derivatives of MCF-7 cells that stably express the constitutively activated mutants of PKCbeta1 or beta2 were more resistant to growth inhibition by LY379196 than the vector control MCF-7 cells. Immunoblot analysis indicated that MCF-7 cells that stably overexpress wild-type or constitutively activated mutants of PKCbeta1 or beta2 had higher cellular levels of cyclin D1 than vector control cells, whereas cells that express a dominant negative mutant had decreased levels of cyclin D1. The derivatives that stably express the constitutively activated mutants of PKCbeta1 or beta2 also displayed increased cyclin D1 promoter activity in transient transfection luciferase reporter assays, and this induction of activity requires activator protein 1. Constitutively activated PKCbeta1 and beta2 also enhanced the transcription of c-fos in transient transfection luciferase reporter assays. Thus, PKCbeta1 and beta2 may play important positive roles in the growth of at least a subset of human breast cancers. Therefore, inhibitors of these isoforms may be useful in breast cancer chemoprevention or therapy.

Eukaryotic Translation Initiation Factor 4E Is a Cellular Target for Toxicity and Death Due to Exposure to Cadmium Chloride

Whether translation initiation factor 4E (eIF4E), the mRNA cap binding and rate-limiting factor required for translation, is a target for cytotoxicity and cell death induced by cadmium, a human carcinogen, was investigated. Exposure of human cell lines, HCT15, PLC/PR/5, HeLa, and Chang, to cadmium chloride resulted in cytotoxicity and cell death, and this was associated with a significant decrease in eIF4E protein levels. Similarly, specific silencing of the expression of the eIF4E gene, caused by a small interfering RNA, resulted in significant cytotoxicity and cell death. On the other hand, overexpression of the eIF4E gene was protective against the cadmium-induced cytotoxicity and cell death. Further studies revealed the absence of alterations in the eIF4E mRNA level in the cadmium-treated cells despite their decreased eIF4E protein level. In addition, exposure of cells to cadmium resulted in enhanced ubiquitination of eIF4E protein while inhibitors of proteasome activity reversed the cadmium-induced decrease of eIF4E protein. Exposure of cells to cadmium, as well as the specific silencing of eIF4E gene, also resulted in decreased cellular levels of cyclin D1, a critical cell cycle and growth regulating gene, suggesting that the observed inhibition of cyclin D1 gene expression in the cadmium-treated cells is most likely due to decreased cellular level of eIF4E. Taken together, our results demonstrate that the exposure of cells to cadmium chloride resulted in cytotoxicity and cell death due to enhanced ubiquitination and consequent proteolysis of eIF4E protein, which in turn diminished cellular levels of critical genes such as cyclin D1.

Nuclear Association of Cyclin D1 in Human Fibroblasts: Tight Binding to Nuclear Structures and Modulation by Protein Kinase Inhibitors

The association of cyclin D1 with nuclear structures was investigated in normal human fibroblasts by using hypotonic detergent extraction procedures, immunofluorescence quantitation with flow cytometry, and Western blot analysis. About 20% of the total cellular levels of cyclin D1 was found to be tightly bound to nuclear structures, being the complex formation resistant to DNase I treatment and to high salt extraction. Maximal levels of the insoluble form of the protein were found in the middle to late G1 phase of the cell cycle. Cell fractionation and immunoprecipitation techniques after in vivo 32P-labeling showed that both soluble and nuclear-bound forms of cyclin D1 were phosphorylated. Both fractions were reactive to an anti-phosphotyrosine antibody, while only the latter was detectable with an anti-phosphoserine antibody. Treatment with the protein kinase inhibitor staurosporine, which induces a cell cycle arrest in early G1 phase, strongly reduced cyclin D1 phosphorylation. Concomitantly, the ratio of nuclear-bound/total cyclin D1 levels was reduced by about 60%, compared with the control value. The protein kinase A specific inhibitor isoquinoline-sulfonamide (H-89) induced a similar reduction in the ratio, with no significant modification in the total amount of protein. In contrast, both calphostin C and bisindolylmaleimide, specific inhibitors of protein kinase C, consistently increased by 30–50% the ratio of nuclear-bound/total amount of the cyclin protein. These results suggest that, during the G1 phase, formation of an insoluble complex of cyclin D1 occurs at nuclear matrix structures and that this association is mediated by a protein kinase A-dependent pathway.