ABSTRACT
ML-133 is a novel small molecule with potent antiproliferative activity, as shown in cancer cell lines and in a human colon tumor xenograft model. ML-133 reduces the concentration of intracellular labile zinc in HT-29 colon cancer cells, leading to induction of the Krüppel-like factor 4 transcription factor. Krüppel-like factor 4 displaces the positive regulator SP1 from the cyclin D1 promoter, thereby negatively regulating the expression of cyclin D1 and promoting the G(1)-S phase arrest of cell proliferation. The antiproliferative and antitumor activity of ML-133 described in the present study suggests modulation of intracellular zinc homeostasis as a potential strategy for the treatment of several cancer types, and ML-133 represents a promising new class of antitumor agents that deserves further development.
Subject(s)
Antineoplastic Agents/pharmacology , Cell Division/drug effects , Homeostasis/drug effects , Imidazoles/pharmacology , Phenanthrolines/pharmacology , Zinc/metabolism , Base Sequence , Blotting, Western , Cell Cycle , Cell Proliferation/drug effects , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Cyclin D1/genetics , DNA Primers , Electrophoresis, Polyacrylamide Gel , Flow Cytometry , HT29 Cells , Humans , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/biosynthesis , Polymerase Chain Reaction , Promoter Regions, Genetic , Sp1 Transcription Factor/metabolism , Up-Regulation/drug effectsABSTRACT
Substantial actin remodelling occurs prior to mitosis as cells alter their shape in preparation for cytokinesis. In mammalian cells, mitosis is initiated by a heterodimer of cyclin B1 and the cyclin dependent kinase 1 (Cdk1) serine/threonine kinase. In this report. we show that human cyclin B1 binds the actin cross-linking protein Filamin-A (FLNa). The proteins co-immunoprecipitate and co-localize in mitotic human cells. We find that cyclin B1/Cdk1 can phosphorylate FLNa in vitro and reduce its ability to gelate actin. We have also identified serine 1436 as one FLNa residue phosphorylated by cyclin B1/Cdk1 in vitro. Our results suggest a role for cyclin B1/Cdk1 in FLNa-dependent actin remodelling.
Subject(s)
Actins/metabolism , CDC2 Protein Kinase/metabolism , Contractile Proteins/metabolism , Cyclin B/metabolism , Microfilament Proteins/metabolism , Amino Acid Sequence , Contractile Proteins/analysis , Cyclin B/analysis , Cyclin B1 , Filamins , Humans , Microfilament Proteins/analysis , Molecular Sequence Data , Phosphorylation , Protein Interaction Mapping , Serine/metabolism , Two-Hybrid System TechniquesABSTRACT
Some cells undergo apoptosis in response to DNA damage, whereas others do not. To understand the biochemical pathways controlling this differential response, we have studied the intracellular localization of cyclin B1 in cell types sensitive or resistant to apoptosis induced by DNA damage. We found that cyclin B1 protein accumulates in the nucleus of cells that are sensitive to gamma radiation-induced apoptosis (thymocytes, lymphoid cell lines), but remains cytoplasmic in apoptosis-resistant cells (primary and transformed fibroblasts). Treatment of both cell types with leptomycin B, an inhibitor of CRM1-dependent cyclin B1 nuclear export, induces apoptosis. Furthermore, ectopic expression of cyclin B1-5xE, a protein that preferentially localizes to the nucleus, is sufficient to trigger apoptosis. Conversely, expression of cyclin B1-5xA, a predominantly cytoplasmic protein, fails to induce apoptosis. This suggests that nuclear accumulation is necessary for cyclin B1-dependent apoptosis. Our observations are consistent with the idea that localization of cyclin B1 is among the factors determining the cellular decision to undergo apoptosis in response to DNA damage.
Subject(s)
Apoptosis/physiology , Cell Nucleus/chemistry , Cyclin B/physiology , DNA Damage , 3T3 Cells/cytology , 3T3 Cells/radiation effects , 3T3 Cells/ultrastructure , Active Transport, Cell Nucleus/drug effects , Animals , Apoptosis/drug effects , Apoptosis/radiation effects , Burkitt Lymphoma/pathology , COS Cells/cytology , COS Cells/radiation effects , COS Cells/ultrastructure , Chlorocebus aethiops , Cyclin B/analysis , Cyclin B/genetics , Cyclin B1 , Cytoplasm/chemistry , DNA, Neoplasm/radiation effects , Fatty Acids, Unsaturated/pharmacology , Gamma Rays , Humans , Mice , Microscopy, Confocal , Radiation Tolerance , Recombinant Fusion Proteins/physiology , Transfection , Tumor Cells, Cultured/cytology , Tumor Cells, Cultured/radiation effects , Tumor Cells, Cultured/ultrastructureABSTRACT
We have found that EEF1A2, the gene encoding protein elongation factor EEF1A2 (also known as eEF-1 alpha 2), is amplified in 25% of primary ovarian tumors and is highly expressed in approximately 30% of ovarian tumors and established cell lines. We have also demonstrated that EEF1A2 has oncogenic properties: it enhances focus formation, allows anchorage-independent growth and decreases the doubling time of rodent fibroblasts. In addition, EEF1A2 expression made NIH3T3 fibroblasts tumorigenic and increased the growth rate of ES-2 ovarian carcinoma cells xenografted in nude mice. Thus, EEF1A2 and the process of protein elongation are likely to be critical in the development of ovarian cancer.
