cyclin-d1 and Carcinoma--Embryonal

Lysophosphatidic acid (LPA) is a lipid growth factor that induces proliferation of fibroblasts by activating the cAMP response element binding protein (CREB). Here, we further investigated whether LPA induces proliferation of P19 cells, a line of pluripotent embryonic carcinoma cells. 5'-Bromo-2-deoxyuridine incorporation and cell viability assays showed that LPA stimulated proliferation of P19 cells. Immunoblot experiments with P19 cells revealed that the mitogen activated protein kinases, including p-ERK, p38, pAKT, glycogen synthase kinase 3β, and CREB were phosphorylated by treatment with 10 μM LPA. LPA-induced phosphorylation of CREB was efficiently blocked by U0126 and H89, inhibitors of the MAP kinases ERK1/2 and mitogen- and stress-activated protein kinase 1, respectively. Involvement of cyclin D1 in LPA-induced P19 cell proliferation was verified by immunoblot analysis in combination with pharmacological inhibitor treatment. Furthermore, LPA up-regulated CRE-harboring cyclin D1 promoter activity, suggesting that CREB and cyclin D1 play significant roles in LPA-induced proliferation of P19 embryonic carcinoma cells.

Topics: Animals; Carcinoma, Embryonal; Cell Line, Tumor; Cell Proliferation; Cyclic AMP Response Element-Binding Protein; Cyclin D1; Lysophospholipids; MAP Kinase Signaling System; Mice; Mice, Inbred C3H; p38 Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 90-kDa; Up-Regulation

Our previous studies have shown that microRNA-383 (miR-383) expression is downregulated in the testes of infertile men with maturation arrest (MA). However, the underlying mechanisms of miR-383 involved in the pathogenesis of MA remain unknown. In this study, we showed that downregulation of miR-383 was associated with hyperactive proliferation of germ cells in patients with mixed patterns of MA. Overexpression of miR-383 in NT2 (testicular embryonal carcinoma) cells resulted in suppression of proliferation, G1-phase arrest and induction of apoptosis, whereas silencing of miR-383 reversed these effects. The effects of miR-383 were mediated through targeting a tumor suppressor, interferon regulatory factor-1 (IRF1), and miR-383 was negatively correlated with IRF1 protein expression in vivo. miR-383 inhibited IRF1 by affecting its mRNA stability, which subsequently reduced the levels of the targets of IRF1, namely cyclin D1, CDK2 and p21. Downregulation of IRF1 or cyclin D1, but not that of CDK2, enhanced miR-383-mediated effects, whereas silencing of p21 partially inhibited the effects of miR-383. Moreover, miR-383 downregulated CDK4 by increasing proteasome-dependent degradation of CDK4, which in turn resulted in an inhibition of phosphorylated retinoblastoma protein (pRb) phosphorylation. These results suggest that miR-383 functions as a negative regulator of proliferation by targeting IRF1, in part, through inactivation of the pRb pathway. Abnormal testicular miR-383 expression may potentiate the connections between male infertility and testicular germ cell tumor.

Topics: Animals; Apoptosis; Carcinoma, Embryonal; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Embryonal Carcinoma Stem Cells; G1 Phase; Humans; Infertility, Male; Interferon Regulatory Factor-1; Male; Mice; MicroRNAs; Phosphorylation; Retinoblastoma Protein; Testicular Neoplasms

We studied the ability of F9 teratocarcinoma cells to arrest in G1/S and G2/M checkpoints after gamma-irradiation. Wild-type p53 protein was rapidly accumulated in F9 cells after gamma-irradiation, however, this was followed not by a G1/S arrest but by a short and reversible delay of the cell cycle in G2/M. In order to elucidate the reasons of the lack of G1/S arrest in F9 cells, we investigated the expression of p53 downstream target Cdk inhibitor p21WAF1/CIP1. In spite of p53-dependent activation of p21WAF1/CIP1 gene promoter and p21WAF1/CIP1 mRNA accumulation upon irradiation, the p21WAF1/CIP1 protein was not detected by either immunoblot or immunofluorescence techniques. However, the cells treated with a specific proteasome inhibitor lactacystin revealed the p21WAF1/CIP1 protein both in non-irradiated and irradiated cells. Therefore we suggest that p21WAF1/CIP1 protein is degraded by a proteasome-dependent mechanism in F9 cells and the lack of G1/S arrest after gamma-irradiation is due to this degradation. We also examined the expression and activity of cell cycle regulatory proteins: G1- and G2-cyclins and cyclin-dependent kinases. In the absence of functional p21WAF1/CIP1 inhibitor, the activity of G1 cyclin/Cdk complexes was insufficiently inhibited to cause a G1 arrest, whereas a decrease of cdc2 and cyclin B1-associated kinase activities was enough to contribute to a reversible G2 arrest following gamma-irradiation. After gamma-irradiation, the majority of F9 cells undergo apoptosis implying that wt-p53 likely triggers pro-apoptotic gene expression in DNA damaged cells. Elimination of defected cells might ensure maintenance of genome integrity in the remaining cell population.

Topics: Acetylcysteine; Animals; Apoptosis; Carcinoma, Embryonal; CDC2-CDC28 Kinases; Cell Cycle; Cyclin B; Cyclin B1; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA Damage; G1 Phase; Gamma Rays; Mice; Multienzyme Complexes; Nocodazole; Promoter Regions, Genetic; Proteasome Endopeptidase Complex; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; S Phase; Tumor Cells, Cultured; Tumor Suppressor Protein p53

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.

Topics: Carcinoma, Embryonal; Cell Cycle; Cell Differentiation; Cyclin D1; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Protein Processing, Post-Translational; Tretinoin; Tumor Cells, Cultured; Ubiquitins

The growth rate of malignant F9 embryonal carcinoma cells slows considerably following all-trans-retinoic acid-induced differentiation into benign parietal endoderm. To determine the mechanism of this process, we examined the expression of cyclins D1, D2, and D3 and the activity of their associated kinases. Cyclin D1 and D3 mRNA levels decreased during complete differentiation induced by all-trans-retinoic acid and dibutyryl cAMP, while the levels of cyclin D2 and the cyclin-dependent kinase (Cdk) inhibitor p27 mRNAs increased. Ultimately, terminally differentiated cells possessed 50% of the Cdk4-associated kinase activity observed in undifferentiated cells. Since numerous genes are differentially regulated during parietal endoderm differentiation, it is difficult to determine whether retinoic acid affects cell cycle gene expression directly or if these changes are caused by differentiation. We found that the retinoid X receptor (RXR)-selective agonists LG100153 and LG100268 significantly inhibited F9 cell growth without causing overt terminal differentiation as assessed by anchorage-independent growth and differentiation-associated gene expression. As seen in cells induced to differentiate by the RAR agonist all-trans-retinoic acid, RXR activation led to an increase in the number of cells in G1 phase. RXR agonists also sharply induced the levels of the Cdk regulatory subunits, cyclin D2 and D3. However, Cdk4-dependent kinase activity was reduced by RXR-selective retinoid treatment. These observations suggest that some retinoids can directly inhibit proliferation and regulate Cdk4-dependent kinase activity without inducing terminal differentiation.

Topics: Alitretinoin; Animals; Antineoplastic Agents; Benzoates; Biomarkers; Carcinoma, Embryonal; Cell Adhesion; Cell Differentiation; Cell Division; Cyclin D1; Cyclin D2; Cyclin D3; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Cyclins; Endoderm; Gene Expression Regulation, Neoplastic; Nicotinic Acids; Proto-Oncogene Proteins; Receptors, Retinoic Acid; Retinoid X Receptors; RNA, Messenger; Stem Cells; Tetrahydronaphthalenes; Transcription Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured