Decrease In Cyclin D1 Protein Research Articles

Salicylate suppresses the oncogenic hyaluronan network in metastatic breast cancer cells

The oncogenic role of hyaluronan in several aspects of tumor biology has been well established. Recent studies by us and others suggest that inhibition of hyaluronan synthesis could represent an emerging therapeutic approach with significant clinical relevance in controlling different breast cancer subtypes, including triple-negative breast cancer. Epidemiological and preclinical studies have revealed the therapeutic potential of aspirin (acetyl salicylate), a classical anti-inflammatory drug, in patients with cancer. However, the underlying molecular mechanisms remain unknown. The present study demonstrates that salicylate, a break down product of aspirin in vivo, alters the organization of hyaluronan matrices by affecting the expression levels of hyaluronan synthesizing (HAS1, 2, 3) and degrading (HYAL-1, -2) enzymes, and that of hyaluronan receptor CD44. In particular, salicylate was found to potently activate AMPK, a kinase known to inhibit HAS2 activity, and caused a dose-dependent decrease of cell associated (intracellular and membrane-bound) as well as secreted hyaluronan, followed by the down-regulation of HAS2 and the induction of HYAL-2 and CD44 in metastatic breast cancer cells. These salicylate-mediated effects were associated with the redistribution of CD44 and actin cytoskeleton that resulted in a less motile cell phenotype. Interestingly, salicylate inhibited metastatic breast cancer cell proliferation and growth by inducing cell growth arrest without signs of apoptosis as evidenced by the substantial decrease of cyclin D1 protein and the absence of cleaved caspase-3, respectively. Collectively, our study offers a possible direction for the development of new matrix-based targeted treatments of metastatic breast cancer subtypes via inhibition of hyaluronan, a pro-angiogenic, pro-inflammatory and tumor promoting glycosaminoglycan.

Combined Inhibition of mTOR and CDK4/6 Is Required for Optimal Blockade of E2F Function and Long-term Growth Inhibition in Estrogen Receptor-positive Breast Cancer.

The cyclin dependent kinase (CDK)-retinoblastoma (RB)-E2F pathway plays a critical role in the control of cell cycle in estrogen receptor-positive (ER+) breast cancer. Small-molecule inhibitors of CDK4/6 have shown promise in this tumor type in combination with hormonal therapies, reflecting the particular dependence of this subtype of cancer on cyclin D1 and E2F transcription factors. mTOR inhibitors have also shown potential in clinical trials in this disease setting. Recent data have suggested cooperation between the PI3K/mTOR pathway and CDK4/6 inhibition in preventing early adaptation and eliciting growth arrest, but the mechanisms of the interplay between these pathways have not been fully elucidated. Here we show that profound and durable inhibition of ER+ breast cancer growth is likely to require multiple hits on E2F-mediated transcription. We demonstrate that inhibition of mTORC1/2 does not affect ER function directly, but does cause a decrease in cyclin D1 protein, RB phosphorylation, and E2F-mediated transcription. Combination of an mTORC1/2 inhibitor with a CDK4/6 inhibitor results in more profound effects on E2F-dependent transcription, which translates into more durable growth arrest and a delay in the onset of resistance. Combined inhibition of mTORC1/2, CDK4/6, and ER delivers even more profound and durable regressions in breast cancer cell lines and xenografts. Furthermore, we show that CDK4/6 inhibitor-resistant cell lines reactivate the CDK-RB-E2F pathway, but remain sensitive to mTORC1/2 inhibition, suggesting that mTORC1/2 inhibitors may represent an option for patients that have relapsed on CDK4/6 therapy. Mol Cancer Ther; 17(5); 908-20. ©2018 AACR.

Geranylgeraniol suppresses the viability of human DU145 prostate carcinoma cells and the level of HMG CoA reductase

The rate-limiting enzyme of the mevalonate pathway, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, provides essential intermediates for the prenylation of nuclear lamins and Ras and dolichol-mediated glycosylation of growth factor receptors. The diterpene geranylgeraniol downregulates the level of HMG CoA reductase and suppresses the growth of human liver, lung, ovary, pancreas, colon, stomach, and blood tumors. We evaluated the growth-suppressive activity of geranylgeraniol in human prostate carcinoma cells. Geranylgeraniol induced dose-dependent suppression of the viability of human DU145 prostate carcinoma cells (IC50=80±18 µmol/L, n=5) following 72-h incubations in 96-well plates. Cell cycle was arrested at the G1 phase with a concomitant decrease in cyclin D1 protein. Geranylgeraniol-induced apoptosis was detected by flow cytometric analysis, fluorescence microscopy following acridine orange and ethidium bromide dual staining, and caspase-3 activation. Geranylgeraniol-induced viability suppression was accompanied by concentration-dependent decrease in the level of HMG CoA reductase protein. As a nonsterol molecule that downregulates HMG CoA reductase in the presence of sterols, geranylgeraniol may have potential in the chemoprevention and/or therapy of human prostate cancer.

The all- trans retinoic acid (atRA)-regulated gene Calmin ( Clmn) regulates cell cycle exit and neurite outgrowth in murine neuroblastoma (Neuro2a) cells

The vitamin A metabolite all- trans retinoic acid (atRA) functions in nervous system development and regulates cell proliferation and differentiation. Neuroblastoma cells (SH-SY5Y and Neuro2a or N2A) exposed to atRA undergo growth inhibition and neuronal differentiation, both of which are preceded by an increase in Clmn mRNA. Treatment of N2A cells with atRA produces a reduction in phosphohistone 3 immunostaining and BrdU incorporation, both indicators of a reduction in cell proliferation. These effects are nearly eliminated in atRA-treated sh Clmn knockdown cells. Loss of Clmn in the mouse N2A cell line also results in a significant reduction of atRA-mediated neurite outgrowth, a response that can be rescued by reintroduction of the Clmn sequence. In contrast, ectopic overexpression of Clmn produces an increase in the cyclin dependent kinase inhibitor, p21 Cip1, a decrease in cyclin D1 protein and an increase in hypophosphorylated Rb, showing that Clmn participates in G 1/S arrest. Clmn overexpression alone is sufficient to inhibit N2A cell proliferation, whereas both Clmn and atRA must be present to induce neurite outgrowth. This study shows that the atRA-responsive gene Clmn promotes exit from the cell cycle, a requisite event for neuronal differentiation.

3’UTR mediated regulation of the cyclin D1 proto-oncogene

In mantle cell lymphoma (MCL), over-expression of cyclin D1 is the hallmark of malignant transformation and results from it’s juxtaposition to the immunoglobulin heavy chain enhancer. In addition, genomic deletions or point mutations leading to premature truncation of the cyclin D1 3’UTR have been reported in a several MCL patients as well as in cell lines isolated from various tumors types. We demonstrate that the expression of cyclin D1 with or without the 3’UTR has different phenotypic consequences in stably transduced fibroblasts, with the hyper-proliferative phenotype of cyclin D1 closely linked to the deletion of its 3’UTR. In our study, the loss of the cyclin D1 3’UTR led to a significant upregulation of the protein. However, the loss of AU-rich elements (AREs) from the cyclin D1 UTR results in a significant decrease in cyclin D1 protein and UTR-tagged reporter expression. In contrast, the levels of cyclin D1 protein can be significantly reduced by microRNAs of the miR15/16 family and the miR17-92 cluster that directly target the cyclin D1 3’UTR. Most importantly, these microRNAs regulated the levels of the endogenous cyclin D1 protein encoded by an mRNA with a full 3’UTR but not with 3’ UTR deletions. Taken together, our data highlight the regulatory role of the cyclin D1 3’UTR in the expression and phenotype of cyclin D1 and suggest that in MCL and solid tumors with cyclin D1 3’UTR mutations, the loss of microRNA target sites, rather than ARE elements contribute to the pathogenic over-expression of the cyclin D1 protein.

P21 is required for atRA‐mediated growth inhibition of MEPM cells, which involves RAR

All-trans retinoic acid (atRA), a metabolite of vitamin A, is essential for embryonic development. Thus the spatial and temporal dispersal of RA must be tightly controlled. Previous studies show that excessive atRA led to growth inhibition and p21 accumulation in mouse embryonic palatal mesenchymal (MEPM) cells. We reported here the identification of p21 as a required mediator during atRA-induced growth inhibition. atRA caused a G1 arrest in the cell cycle with an increase in the proportion of cells in G0/G1 and a decrease in the proportion of cells in S phase. In addition to a marked effect on cell cycling, atRA also triggered DNA fragmentation, reflected by an increase of the fraction of cells in the sub-G(1) population. Western blot analysis revealed that atRA treatment led to an increase in p21 level and a decrease in cyclin D1 protein and Rb phosphorylation. Using luciferase assay with reporter gene regulated by p21 promoter, we showed that atRA increased the reporter activity in a dose-dependent manner; and p21 siRNA blocked the growth inhibition by atRA, suggesting that p21 is required for atRA-mediated growth inhibition. Moreover, the induction of p21 by atRA was partially attenuated when RAR was silenced with specific siRNA. atRA stimulated RARE-driven reporter gene activity dose-dependently. Using chromatin immunoprecipitation, we demonstrated that RAR protein could bind to the p21 promoter. Taken together, our results indicate p21 is responsible for atRA-induced growth inhibition of MEPM cells and RAR plays a role during this process.

Characterization of a Novel Primary Mammary Tumor Cell Line Reveals that Cyclin D1 Is Regulated by the Type I Insulin-Like Growth Factor Receptor

The importance of type I insulin-like growth factor receptor (IGF-IR) overexpression in mammary tumorigenesis was recently shown in two separate transgenic models. One of these models, the MTB-IGFIR transgenics, was generated in our lab to overexpress IGF-IR in mammary epithelial cells in a doxycycline (Dox)-inducible manner. To complement this transgenic model, primary cells that retained Dox-inducible expression of IGF-IR were isolated from a transgenic mammary tumor. This cell line, RM11A, expressed high levels of IGF-IR, phosphorylated Akt, and phosphorylated extracellular signal-regulated kinase 1/2 in the presence of Dox. IGF-IR overexpression provided the primary tumor cells with a survival advantage in serum-free media and seemed to induce ligand-independent activation of the IGF-IR because RM11A cells cultured in the presence of Dox were largely nonresponsive to exogenous IGFs. IGF-IR overexpression also augmented the growth of RM11A cells in vivo because injection of these cells into mammary glands of wild-type mice produced palpable tumors in 15.8 +/- 3.4 days when the mice were administered Dox, compared with 57.8 +/- 6.3 days in the absence of Dox. DNA microarray analysis revealed a number of genes regulated by IGF-IR, one of which was cyclin D1. Suppression of IGF-IR expression in vitro or in vivo was associated with a decrease in cyclin D1 protein, suggesting that at least some of the proliferative actions of IGF-IR are mediated through cyclin D1. Therefore, this article characterizes the first primary murine mammary tumor cell line with inducible IGF-IR expression. These cells provide a powerful in vitro/in vivo model to examine the function of IGF-IR in mammary tumorigenesis.

Onopordum cynarocephalum induces apoptosis and protects against 1,2 dimethylhydrazine-induced colon cancer

The potential chemopreventive properties of the crude extract of Onopordum cynarocephalum were evaluated. Growth inhibition was investigated in FHs74Int human normal intestinal cells and ModeK mouse normal intestinal cell line and in two human colon cancer cells HCT-116 (p53+/+) and HT-29 (p53+/-). The extract was not cytotoxic to FHs74Int cells at concentrations 2-fold higher than the IC50 of HCT-116 cells. The extract inhibited dose-dependently the growth of HCT-116 cells (IC50=0.18 mg/ml) to a greater extent than HT-29 cells (IC50=1.8 mg/ml). The p53 wild-type HCT-116 cells were more sensitive than p53 mutant HT-29 cells to the pro-apoptotic effects of the plant extract; five times lower concentrations were needed to induce apoptosis in HCT-116 cells. Apoptosis induction by the extract was associated with an increase in the expression of pro-apoptotic proteins such as p53 and Bax, and a significant inhibition of the anti-apoptotic protein Bcl-2. Significant decrease in cyclin D1 protein and increase in p21 protein was observed in extract-treated HCT-116 cells. In vivo, the crude extract injected intra-peritoneally reduced the number of tumors by 64% (p<0.0001) and decreased the mean size of aberrant crypt foci in the DMH model of colon cancer. These data collectively suggest that O. cynarocephalum has potential anti-colon cancer effects.

Iron chelation regulates cyclin D1 expression via the proteasome: a link to iron deficiency–mediated growth suppression

AbstractIron (Fe) plays an important role in proliferation, and Fe deficiency results in G1/S arrest. Despite this, the precise role of Fe in cell-cycle control remains unclear. Cyclin D1 plays a critical function in G1 progression by interacting with cyclin-dependent kinases. Previously, we examined the effect of Fe depletion on the expression of cell-cycle control molecules and identified a marked decrease in cyclin D1 protein, although the mechanism involved was unknown. In this study, we showed that cyclin D1 was regulated posttranscriptionally by Fe depletion. Iron chelation of cells in culture using desferrioxamine (DFO) or 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311) decreased cyclin D1 protein levels after 14 hours and was rescued by the addition of Fe. Cyclin D1 half-life in control cells was 80 ± 15 minutes (n = 5), while in chelator-treated cells it was significantly (P < .008) decreased to 38 ± 3 minutes (n = 5). Proteasomal inhibitors rescued the Fe chelator–mediated decrease in cyclin D1 protein, suggesting the role of the proteasome. In Fe-replete cells, cyclin D1 was degraded in an ubiquitin-dependent manner, while Fe depletion induced a ubiquitin-independent pathway. This is the first report linking Fe depletion–mediated growth suppression at G1/S to a mechanism inducing cyclin D1 proteolysis.

Double blockade of cell cycle progression by coptisine in vascular smooth muscle cells

Coptisine, an isoquinoline alkaloid isolated from rhizome of Coptis japonica, inhibits proliferation of vascular smooth muscle cells (VSMCs). The aim of this study was to evaluate the action of coptisine, along with berberine (a structurally similar isoquinoline alkaloid), on progression of the cell cycle in VSMCs. Coptisine displayed antiproliferative action against VSMCs by blocking the cell cycle at G 1 and G 2/M phases. The G 1 block was shown by inhibition of [ 3H]thymidine incorporation into VSMCs at coptisine concentrations higher than 15 μM. The mechanism underlying the G 1 arrest involved a decrease in cyclin D1 protein, although cyclin E, A, and B were not affected by coptisine treatment. The selective reduction in cyclin D1 protein was mainly attributable to accelerated proteolysis via proteasome-dependent pathway, since it was inhibited by a proteasome inhibitor, N-carbobenzoxy- l-leucinyl- l-leucinyl- l-norleucinal (MG132) and further the mRNA level of cyclin D1, protein synthesis, and mitogen-activated protein kinase (MAPK) activity remained unaltered. The mechanism underlying the G 2/M arrest involved partial inhibition of tubulin polymerization, which was apparent at coptisine concentration of 3 μM. Berberine arrested the cell cycle at G 1 phase via a mechanism identical with coptisine, but did not cause block at G 2/M phase. The results demonstrate that a small difference in the structure between isoquinoline alkaloids produces a big difference in activity, and that coptisine has a unique double action in arresting the cell cycle of VSMCs.

Mantle Cell Lymphoma: SAHA Blocks Akt/mTOR Pathway and Reduces Cyclin D1 Protein Levels by Affecting Translation.

Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase (HDAC) inhibitor. We have found that SAHA has profound antiproliferative activities against mantle cell lymphoma (MCL) cell lines (ED50 ≅ 1 μM SAHA for 3 days), and this was associated with a rapid decrease of Cyclin D1 protein as seen by western blot. For example, eight hours treatment with SAHA (5 μM), reduced the Cyclin D1 levels by 80 % in three of 3 MCL cell lines (Jeko1, SP49, SP53). In contrast, other HDAC inhibitors including valproic acid and trichostatin A, were unable to reduce protein levels of Cyclin D1 as rapidly or as completely as SAHA in these MCL cell lines. Also, a myelogenous leukemia cell line, K562 expressed low levels of Cyclin D1 and exposure of these cells to SAHA (8 hrs) had almost no effect on protein levels of Cyclin D1, although SAHA had profound antiproliferative effect on these cells. A hallmark of MCL cells is the t(11; 14)(q13; q32) chromosomal change which rearranges the enhancer region of the immunoglobulin heavy chain to the regulatory region of the cyclin D1 gene inducing overexpression of Cyclin D1 mRNA, leading to high expression of Cyclin D1 protein. Levels of Cyclin D1 mRNA (Northern blot) remained elevated in these cell lines during an 8 hr exposure to SAHA (5 μM). Also, stability of Cyclin D1 protein in these MCL lines as measured by pulse-chase assay and cyclohexamide treatment minimally changed during the 8 hours of exposure to SAHA in the MCL cell lines. Taken together, we speculated that translation of the Cyclin D1 may be slowed after treatment of the cells with SAHA. Since translation of Cyclin D1 protein is regulated by eIF4E, the binding activity of eIF4E to the cap site of mRNA was analyzed by immunoprecipitation using 7 methyl-GTP sepharose beads. Binding activity decreased rapidly after the treatment with SAHA. Since hypophosphorylated eIF4EBP binds eIF4E and blocks the function of eIF4E, activity of eIF4E is regulated by eIF4EBP and its phosphorylation status. Furthermore, Akt phosphorylates and activates mTOR which then phosphorylates eIF4EBP and prevents eIF4EBP from binding to eIF4E. We found that eIF4EBP, mTOR, and Akt proteins are markedly phosphorylated resulting in an activated Akt/mTOR pathway in the MCL cell lines. Levels of phospho-eIF4EBP, phosph-mTOR and phospho-Akt decreased rapidly after treatment with SAHA. Also, PI3K inhibitor, LY294002, transiently decreased the level of Cyclin D1 protein. In summary, our data suggest that 1) Akt/mTOR pathway is activated in MCL cells; 2) Translation of Cyclin D1 is dependent on this pathway in these cells; 3) Blockade of this pathway reduces the protein levels of Cyclin D1 rapidly by decreasing its translation; 4) SAHA blocks this pathway through a mechanism other than HDAC inhibition. This discovery may encourage a reappraisal of the mechanism of action of this interesting therapeutic compound and also focus attention to developing additional inhibitors of this pathway in cancers associated with dysregulated Cyclin D1.

P53 represses cyclin D1 transcription through down regulation of Bcl-3 and inducing increased association of the p52 NF-kappaB subunit with histone deacetylase 1.

The p53 and NF-kappaB transcription factor families are important, multifunctional regulators of the cellular response to stress. Here we have investigated the regulatory mechanisms controlling p53-dependent cell cycle arrest and cross talk with NF-kappaB. Upon induction of p53 in H1299 or U-2 OS cells, we observed specific repression of cyclin D1 promoter activity, correlating with a decrease in cyclin D1 protein and mRNA levels. This repression was dependent on the proximal NF-kappaB binding site of the cyclin D1 promoter, which has been shown to bind the p52 NF-kappaB subunit. p53 inhibited the expression of Bcl-3 protein, a member of the IkappaB family that functions as a transcriptional coactivator for p52 NF-kappaB and also reduced p52/Bcl-3 complex levels. Concomitant with this, p53 induced a significant increase in the association of p52 and histone deacetylase 1 (HDAC1). Importantly, p53-mediated suppression of the cyclin D1 promoter was reversed by coexpression of Bcl-3 and inhibition of p52 or deacetylase activity. p53 therefore induces a transcriptional switch in which p52/Bcl-3 activator complexes are replaced by p52/HDAC1 repressor complexes, resulting in active repression of cyclin D1 transcription. These results reveal a unique mechanism by which p53 regulates NF-kappaB function and cell cycle progression.

Dictyostelium Differentiation-inducing Factor-3 Activates Glycogen Synthase Kinase-3β and Degrades Cyclin D1 in Mammalian Cells

In search of chemical substances applicable for the treatment of cancer and other proliferative disorders, we studied the signal transduction of Dictyostelium differentiation-inducing factors (DIFs) in mammalian cells mainly using HeLa cells. Although DIF-1 and DIF-3 both strongly inhibited cell proliferation by inducing G(0)/G(1) arrest, DIF-3 was more effective than DIF-1. DIF-3 suppressed cyclin D1 expression at both mRNA and protein levels, whereas the overexpression of cyclin D1 overrode DIF-3-induced cell cycle arrest. The DIF-3-induced decrease in the amount of cyclin D1 protein preceded the reduction in the level of cyclin D1 mRNA. The decrease in cyclin D1 protein seemed to be caused by accelerated proteolysis, since it was abrogated by N-acetyl-Leu-Leu-norleucinal, a proteasome inhibitor. DIF-3-induced degradation of cyclin D1 was also prevented by treatment with lithium chloride, an inhibitor of glycogen synthase kinase-3beta (GSK-3beta), suggesting that DIF-3 induced cyclin D1 proteolysis through the activation of GSK-3beta. Indeed, DIF-3 dephosphorylated Ser(9) and phosphorylated tyrosine on GSK-3beta, and it stimulated GSK-3beta activity in an in vitro kinase assay. Moreover, DIF-3 was revealed to induce the nuclear translocation of GSK-3beta by immunofluorescent microscopy and immunoblotting of subcellular protein fractions. These results suggested that DIF-3 activates GSK-3beta to accelerate the proteolysis of cyclin D1 and that this mechanism is involved in the DIF-3-induced G(0)/G(1) arrest in mammalian cells.

Overexpression of retinoic acid receptors alpha and gamma into neoplastic epidermal cells causes retinoic acid-induced growth arrest and apoptosis.

Retinoids are essential for normal epidermal differentiation and are used for the prevention and treatment of numerous skin disorders and cancers in humans. In previous studies, we have shown that retinoic acid receptors (RARs) -alpha and -gamma are down-regulated during skin tumor progression. The transduction of v-ras(Ha) into primary mouse keratinocytes is sufficient to reduce both RARalpha and RARgamma protein levels as well as inhibit their transactivation functions. Our primary objective is to investigate the roles that RARalpha and RARgamma play in keratinocyte tumor cell proliferation. Through retroviral gene transduction, we overexpressed RARalpha or RARgamma into neoplastic mouse epidermal cells with down-regulated endogenous RAR proteins. Following all-trans retinoic acid (RA) treatment, RARalpha- and RARgamma-transduced cell lines exhibit a progressive, dose-dependent growth inhibition relative to the control LXSN cell lines. Further characterization of RAR-transduced cells following RA treatment reveals that both RARalpha and RARgamma cause a decrease in S-phase population, while only RARalpha causes a simultaneous G(0)/G(1) block as evidenced by reduced [(3)H]-thymidine incorporation and flow cytometric analysis of DNA content. Following RA treatment, both receptors cause an early, transient increase in the cyclin-dependent kinase inhibitor (CDKI) p21 proteins, while only RARalpha causes a simultaneous sharp, brief increase in the CDKI p16 protein. A later decrease in cyclin D(1) protein is also evident in RARalpha- and RARgamma-transduced cells. Chromatin condensation and PARP cleavage are observed in both RARalpha- and RARgamma-transduced cells indicating an RA-induced apoptosis that may be caspase dependent. Furthermore, both receptors cause a late upregulation and apparent cleavage of the squamous differentiation marker protein kinase C (PKC)-eta. These results suggest that RARalpha and RARgamma enhance growth suppression and apoptosis of neoplastic epidermal keratinocytes. This growth inhibitory effect of both retinoid receptors in neoplastic keratinocytes may be achieved through distinct as well as overlapping mechanisms of cell cycle control.

Retinoic Acid Promotes Ubiquitination and Proteolysis of Cyclin D1 during Induced Tumor Cell Differentiation

Mechanisms by which differentiation programs engage the cell cycle are poorly understood. This study demonstrates that retinoids promote ubiquitination and degradation of cyclin D1 during retinoid-induced differentiation of human embryonal carcinoma cells. In response to all-trans-retinoic acid (RA) treatment, the human embryonal carcinoma cell line NT2/D1 exhibits a progressive decline in cyclin D1 expression beginning when the cells are committed to differentiate, but before onset of terminal neuronal differentiation. The decrease in cyclin D1 protein is tightly associated with the accumulation of hypophosphorylated forms of the retinoblastoma protein and G(1) arrest. In contrast, retinoic acid receptor gamma-deficient NT2/D1-R1 cells do not growth-arrest or accumulate in G(1) and have persistent cyclin D1 overexpression despite RA treatment. Notably, stable transfection of retinoic acid receptor gamma restores RA-mediated growth suppression and differentiation to NT2/D1-R1 cells and restores the decline of cyclin D1. The proteasome inhibitor LLnL blocks this RA-mediated decline in cyclin D1. RA treatment markedly accelerates ubiquitination of wild-type cyclin D1, but not a cyclin D1 (T286A) mutant. Transient expression of cyclin D1 (T286A) in NT2/D1 cells blocks RA-mediated transcriptional decline of a differentiation-sensitive reporter plasmid and represses induction of immunophenotypic neuronal markers. Taken together, these findings strongly implicate RA-mediated degradation of cyclin D1 as a means of coupling induced differentiation and cell cycle control of human embryonal carcinoma cells.

Sustained activation of extracellular-signal-regulated kinase 1 (ERK1) is required for the continued expression of cyclin D1 in G1 phase.

In Chinese hamster embryo fibroblasts (IIC9 cells), platelet-derived growth factor (PDGF) stimulated mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAP kinase/ERK) activity, but not that of c-jun N-terminal kinase (JNK), and induced G1 phase progression. ERK1 activation was biphasic and was sustained throughout the G1 phase of the cell cycle. PDGF induced cyclin D1 protein and mRNA levels in a time-dependent manner. Inhibition of PDGF-induced ERK1 activity by the addition of a selective inhibitor of MEK1 (MAP kinase kinase/ERK kinase 1) activation, PD98059, or transfection with a dominant-negative ERK1 (dnERK-) was correlated with growth arrest. In contrast, growth was unaffected by expression of dominant-negative JNK (dnJNK-). Interestingly, addition of PD98059 or dnERK-, but not dnJNK-, resulted in a dramatic decrease in cyclin D1 protein and mRNA levels, concomitant with a decrease in cyclin D1-cyclin-dependent kinase activity. To investigate the importance of sustained ERK1 activation, ERK1 activity was blocked by the addition of PD98059 throughout G1. Addition of PD98059 up to 4 h after PDGF treatment decreased ERK1 activity to the levels found in growth-arrested IIC9 cells. Loss of cyclin D1 mRNA and protein expression was observed within 1 h after inhibition of the second sustained phase of ERK1 activity. Disruption of sustained ERK1 activity also resulted in G1 growth arrest. These data provide evidence for a role for sustained ERK activity in controlling G1 progression through positive regulation of the continued expression of cyclin D1, a protein known to positively regulate G1 progression.