cyclin-d1 and lactacystin

The detection of cell cycle proteins in Alzheimer's disease (AD) brains may represent an early event leading to neurodegeneration. To identify cell cycle modifiers with anti-Abeta properties, we assessed the effect of Differentiation-Inducing Factor-1 (DIF-1), a unique, small-molecule from Dictyostelium discoideum, on the proteolysis of the amyloid beta-protein precursor (APP) in a variety of different cell types. We show that DIF-1 slows cell cycle progression through G0/G1 that correlates with a reduction in cyclin D1 protein levels. Western blot analysis of DIF-treated cells and conditioned medium revealed decreases in the levels of secreted APP, mature APP, and C-terminal fragments. Assessment of conditioned media by sandwich ELISA showed reduced levels of Abeta40 and Abeta42, also demonstrating that treatment with DIF-1 effectively decreases the ratio of Abeta42 to Abeta40. In addition, DIF-1 significantly diminished APP phosphorylation at residue T668. Interestingly, site-directed mutagenesis of APP residue Thr668 to alanine or glutamic acid abolished the effect of DIF-1 on APP proteolysis and restored secreted levels of Abeta. Finally, DIF-1 prevented the accumulation of APP C-terminal fragments induced by the proteasome inhibitor lactacystin, and calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN). Our findings suggest that DIF-1 affects G0/G1-associated amyloidogenic processing of APP by a gamma-secretase-, proteasome- and calpain-insensitive pathway, and that this effect requires the presence of residue Thr668.

Topics: Acetylcysteine; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Benzazepines; Cell Line; Cell Line, Tumor; CHO Cells; Cricetinae; Cricetulus; Cyclin D1; Fibroblasts; Glioma; Hexanones; Humans; Hydrocarbons, Chlorinated; Indoles; Leupeptins; Mice; Peptide Fragments; Proteasome Inhibitors; Purines; Recombinant Fusion Proteins; Roscovitine; Threonine

In response to DNA damage, eukaryotic cells initiate a complex signalling pathway, termed the DNA damage response (DDR), which coordinates cell cycle arrest with DNA repair. Studies have shown that oncogene-induced senescence, which provides a barrier to tumour development, involves activation of the DDR. Using a genome-wide RNA interference (RNAi) screen, we have identified 17 factors required for oncogenic BRAF to induce senescence in primary fibroblasts and melanocytes. One of these factors is an F-box protein, FBXO31, a candidate tumour suppressor encoded in 16q24.3, a region in which there is loss of heterozygosity in breast, ovarian, hepatocellular and prostate cancers. Here we study the cellular role of FBXO31, identify its target substrate and determine the basis for its growth inhibitory activity. We show that ectopic expression of FBXO31 acts through a proteasome-directed pathway to mediate the degradation of cyclin D1, an important regulator of progression from G1 to S phase, resulting in arrest in G1. Cyclin D1 degradation results from a direct interaction with FBXO31 and is dependent on the F-box motif of FBXO31 and phosphorylation of cyclin D1 at Thr 286, which is known to be required for cyclin D1 proteolysis. The involvement of the DDR in oncogene-induced senescence prompted us to investigate the role of FBXO31 in DNA repair. We find that DNA damage induced by gamma-irradiation results in increased FBXO31 levels, which requires phosphorylation of FBXO31 by the DDR-initiating kinase ATM. RNAi-mediated knockdown of FBXO31 prevents cells from undergoing efficient arrest in G1 after gamma-irradiation and markedly increases sensitivity to DNA damage. Finally, we show that a variety of DNA damaging agents all result in a large increase in FBXO31 levels, indicating that induction of FBXO31 is a general response to genotoxic stress. Our results reveal FBXO31 as a regulator of the G1/S transition that is specifically required for DNA damage-induced growth arrest.

Topics: Acetylcysteine; Ataxia Telangiectasia Mutated Proteins; Cell Cycle Proteins; Cell Line, Tumor; Cyclin D1; Cysteine Proteinase Inhibitors; DNA Damage; DNA-Binding Proteins; F-Box Proteins; G1 Phase; Humans; Melanoma; Proteasome Endopeptidase Complex; Protein Serine-Threonine Kinases; Transcriptional Activation; Tumor Suppressor Proteins; Ubiquitination

Cyclin D1 is an important regulator of G1-S phase cell cycle transition and has been shown to be important for breast cancer development. GSK3beta phosphorylates cyclin D1 on Thr-286, resulting in enhanced ubiquitylation, nuclear export and degradation of the cyclin in the cytoplasm. Recent findings suggest that the development of small-molecule cyclin D1 ablative agents is of clinical relevance. We have previously shown that the histone deacetylase inhibitor trichostatin A (TSA) induces the rapid ubiquitin-dependent degradation of cyclin D1 in MCF-7 breast cancer cells prior to repression of cyclin D1 gene (CCND1) transcription. TSA treatment also resulted in accumulation of polyubiquitylated GFP-cyclin D1 species and reduced levels of the recombinant protein within the nucleus.. Here we provide further evidence for TSA-induced ubiquitin-dependent degradation of cyclin D1 and demonstrate that GSK3beta-mediated nuclear export facilitates this activity. Our observations suggest that TSA treatment results in enhanced cyclin D1 degradation via the GSK3beta/CRM1-dependent nuclear export/26S proteasomal degradation pathway in MCF-7 cells.. We have demonstrated that rapid TSA-induced cyclin D1 degradation in MCF-7 cells requires GSK3beta-mediated Thr-286 phosphorylation and the ubiquitin-dependent 26S proteasome pathway. Drug induced cyclin D1 repression contributes to the inhibition of breast cancer cell proliferation and can sensitize cells to CDK and Akt inhibitors. In addition, anti-cyclin D1 therapy may be highly specific for treating human breast cancer. The development of potent and effective cyclin D1 ablative agents is therefore of clinical relevance. Our findings suggest that HDAC inhibitors may have therapeutic potential as small-molecule cyclin D1 ablative agents.

Topics: Acetylcysteine; Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Cyclin D1; Cytoplasm; Enzyme Inhibitors; Exportin 1 Protein; Fatty Acids, Unsaturated; Female; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Karyopherins; Leupeptins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Receptors, Cytoplasmic and Nuclear; Recombinant Fusion Proteins; RNA Interference; Transfection; Ubiquitin

Cyclin D1 is a proto-oncogene that is overexpressed in many cancers including breast and prostate. It plays a role in cell proliferation through activation of cyclin-dependent kinases. Curcumin, a diferuloylmethane, is a chemopreventive agent known to inhibit the proliferation of several breast and prostate cancer cell lines. It is possible that the effect of curcumin is mediated through the regulation of cyclin D1. In the present report we show that inhibition of the proliferation of various prostate, breast and squamous cell carcinoma cell lines by curcumin correlated with the down-regulation of the expression of cyclin D1 protein. In comparison, the down-regulation by curcumin of cyclin D2 and cyclin D3 was found only in selective cell lines. The suppression of cyclin D1 by curcumin led to inhibition of CDK4-mediated phosphorylation of retinoblastoma protein. We found that curcumin-induced down-regulation of cyclin D1 was inhibited by lactacystin, an inhibitor of 26S proteosome, suggesting that curcumin represses cyclin D1 expression by promoting proteolysis. We found that curcumin also down-regulated mRNA expression, thus suggesting transcriptional regulation. Curcumin also inhibited the activity of the cyclin D1 promoter-dependent reporter gene expression. Overall our results suggest that curcumin down-regulates cyclin D1 expression through activation of both transcriptional and post-transcriptional mechanisms, and this may contribute to the antiproliferative effects of curcumin against various cell types.

Topics: Acetylcysteine; Base Sequence; Cell Division; Curcumin; Cyclin D1; Cyclin D2; Cyclin D3; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Cyclins; DNA Primers; Down-Regulation; Humans; Phosphorylation; Promoter Regions, Genetic; Proto-Oncogene Mas; Proto-Oncogene Proteins; Retinoblastoma Protein; RNA, Messenger; Tumor Cells, Cultured

Indomethacin-induced G(1) arrest and apoptosis of human colorectal cancer (CRC) cells is associated with a dose-dependent decrease in beta-catenin protein levels. Beta-catenin plays a pivotal role in the WNT signalling pathway and its expression is frequently dysregulated at early stages of colorectal carcinogenesis. The objective of this study was to investigate the effect of indomethacin on catenin expression and downstream WNT signalling events in human CRC cells. Beta-catenin, gamma-catenin and T-cell factor (TCF) target gene (cyclin D1, c-MYC and PPARdelta) expression was studied following indomethacin treatment of SW480 and HCT116 cells. Cyclin D1 was used as a model TCF target gene for analysis of beta-catenin-TCF-4 DNA binding and trans-activation. Indomethacin treatment was associated with a specific decrease in beta-catenin (but not gamma-catenin) expression. Resulting TCF target gene expression was gene specific (cyclin D1, decreased; c-MYC, increased; PPARdelta, no significant change). Cyclin D1 promoter analysis revealed that indomethacin disrupted formation of a beta-catenin-TCF-4-DNA complex. Indomethacin-induced G(1) arrest and apoptosis is associated with specific beta-catenin down-regulation in human CRC cells in vitro. Differential expression of TCF target genes following indomethacin treatment implies complex effects on multiple genes which play an important role in colorectal carcinogenesis.

Topics: Acetylcysteine; beta Catenin; Blotting, Western; Colorectal Neoplasms; Cyclin D1; Cytoskeletal Proteins; Desmoplakins; DNA; Dose-Response Relationship, Drug; Down-Regulation; Electrophoretic Mobility Shift Assay; gamma Catenin; Gene Expression Regulation, Neoplastic; Humans; Indomethacin; Interleukin-2; Isoenzymes; Nitrobenzenes; Promoter Regions, Genetic; Protein Kinase C; Protein Kinase C beta; Proto-Oncogene Proteins c-myc; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Sulfonamides; Trans-Activators; Transcription Factors; Tumor Cells, Cultured

A bicyclic hexapeptide, RA-VII or O-methyl deoxybouvardin, isolated from Rubia cordifolia, is known to inhibit protein biosynthesis in vitro and in vivo. We here demonstrate that the treatment of human colon cancer DLD-1 cells with RA-VII induces cell growth inhibition associated with a partial G1 arrest and a rapid decrease (below 2 h) in the level of cyclin D1 protein. Since cycloheximide, another protein synthesis inhibitor, neither decreased the amount of cyclin D1 in the cells nor arrested cells in G1 phase, it is unlikely that this RA-VII-induced reduction of cyclin D1 was fully dependent on its direct inhibitory effect of protein synthesis. Northern blot analysis revealed that RA-VII did not affect the level of cyclin D1 mRNA. Meanwhile, pre-treatment of cells with lactacystin, a proteasome inhibitor, abolished the RA-VII-induced decrease in cyclin D1. Moreover, RA-VII still decreased cyclin D1 protein in the presence of cycloheximide. These results indicate that the RA-VII-induced cyclin D1 decrease depends on cyclin D1 degradation via the ubiquitin-proteasome pathway and does not require additional protein synthesis. RA-VII might actively proceed the degradation process of cyclin D1 via the ubiquitin-proteasome pathway in DLD-1 cells.

Topics: Acetylcysteine; Antineoplastic Agents, Phytogenic; Blotting, Northern; Blotting, Western; Cell Cycle; Cyclin D1; Cycloheximide; Cysteine Proteinase Inhibitors; Formazans; Humans; Peptides, Cyclic; Protein Synthesis Inhibitors; RNA, Messenger; Tetrazolium Salts; Tumor Cells, Cultured

Quinidine inhibits proliferation and promotes cellular differentiation in human breast tumor epithelial cells. Previously we showed quinidine arrested MCF-7 cells in G(1) phase of the cell cycle and led to a G(1) to G(0) transition followed by apoptotic cell death. The present experiments demonstrated that MCF-7, MCF-7ras, T47D, MDA-MB-231, and MDA-MB-435 cells transiently differentiate before undergoing apoptosis in response to quinidine. The cells accumulated lipid droplets, and the cytokeratin 18 cytoskeleton was reorganized. Hyperacetylated histone H4 appeared within 2 h of the addition of quinidine to the medium, and levels were maximal by 24 h. Quinidine-treated MCF-7 cells showed elevated p21(WAF1), hypophosphorylation and suppression of retinoblastoma protein, and down-regulation of cyclin D1, similar to the cell cycle response observed with cells induced to differentiate by histone deacetylase inhibitors, trichostatin A, and trapoxin. Quinidine did not show evidence for direct inhibition of histone deacetylase enzymatic activity in vitro. HDAC1 was undetectable in MCF-7 cells 30 min after addition of quinidine to the growth medium. The proteasome inhibitors MG-132 and lactacystin completely protected HDAC1 from the action of quinidine. We conclude that quinidine is a breast tumor cell differentiating agent that causes the loss of HDAC1 via a proteasomal sensitive mechanism.

Topics: Acetylation; Acetylcysteine; Animals; Anti-Bacterial Agents; Breast Neoplasms; Cell Cycle; Cell Differentiation; Cell Division; Chickens; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Cytoskeleton; Down-Regulation; Enzyme Inhibitors; Female; G1 Phase; Histone Deacetylase 1; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Immunoblotting; Keratins; Leupeptins; Multienzyme Complexes; Peptides; Phosphorylation; Proteasome Endopeptidase Complex; Quinidine; Retinoblastoma Protein; Time Factors; Tumor Cells, Cultured

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

Hypothemycin was originally isolated as an antifungal metabolite of Hypomyces trichothecoides. Here we report that treatment on v-K-ras-transformed NIH3T3 cells (DT cells) with hypothemycin caused drastic decrease in amount of cyclin D1 protein with concomitant prolongation of G1 phase in their cell cycle. Analysis using hypothemycin-resistant mutant of Schizosaccharomyces pombe (S. pombe) was carried out to show that S. pombe rhp6+ (homologue of Saccharomyces cerevisiae RAD6) and mammalian ubiquitin-conjugating enzyme 2 (ubc2) are the targets of hypothemycin or its downstream molecules in ubiquitin-conjugation process. Furthermore, in the presence of lactacystin, a specific inhibitor for proteasome, hypothemycin greatly enhanced the accumulation of multi-ubiquitinated form of cyclin D1 in DT cells. Therefore, it is indicated that hypothemycin facilitates ubiquitinating process of cyclin D1. In terms of malignant phenotype, hypothemycin inhibited anchorage-independent growth and reverted the morphology of DT cells. On the contrary, their morphology still remained transformed in the additional presence of lactacystin. Our results suggest that cyclin D1 is a key molecule working downstream in ras-signaling and that the transformation can be inhibited by the compound which can activate ubiquitin-proteasome pathway including degradation of cyclin D1.

Topics: 3T3 Cells; Acetylcysteine; Animals; Antineoplastic Agents; Blotting, Western; Cell Line, Transformed; Cell Transformation, Neoplastic; Cyclin D1; Cysteine Endopeptidases; DNA, Complementary; Down-Regulation; G1 Phase; Mice; Mitosporic Fungi; Multienzyme Complexes; Proteasome Endopeptidase Complex; Repressor Proteins; Schizosaccharomyces; Ubiquitins; Zearalenone

Cyclin D1, a critical positive regulator of G1 progression, has been implicated in the pathogenesis of certain cancers. Regulation of cyclin D1 occurs at the transcriptional and posttranscriptional level. Here we present evidence that cyclin D1 levels are regulated at the posttranscriptional level by the Ca2+-activated protease calpain. Serum starvation of NIH 3T3 cells resulted in rapid loss of cyclin D1 protein that was completely reversible by calpain inhibitors. Actinomycin D and lovastatin induced rapid loss of cyclin D1 in prostate and breast cancer cells that was reversible by calpain inhibitors and not by phenylmethylsulfonyl fluoride, caspase inhibitors, or lactacystin, a specific inhibitor of the 26 S proteasome. Treatment of intact NIH 3T3, prostate, and breast cancer cells with a calpain inhibitor dramatically increased the half-life of cyclin D1 protein. Addition of purified calpain to PC-3-M lysates resulted in Ca2+-dependent cyclin D1 degradation. Transient expression of the calpain inhibitor calpastatin increased cyclin D1 protein in serum-starved NIH 3T3 cells. Cyclins A, E, and B1 have been reported to be regulated by proteasome-associated proteolysis. The data presented here implicate calpain in cyclin D1 posttranslational regulation.

Topics: 3T3 Cells; Acetylcysteine; Animals; Calpain; Cyclin B; Cyclin B1; Cyclin D1; Cysteine Proteinase Inhibitors; Dactinomycin; Half-Life; Humans; Male; Mice; Prostatic Neoplasms; Protein Synthesis Inhibitors; Tumor Cells, Cultured