isopropyl-thiogalactoside and Fibrosarcoma

Induction of a cyclin-dependent kinase inhibitor p21Waf1/ Cip1/Sdi1 is an integral part of cell growth arrest associated with senescence and damage response. p21 overexpression from an inducible promoter resulted in senescence-like growth arrest in a human fibrosarcoma cell line. After release from p21-induced growth arrest, cells re-entered the cell cycle but displayed growth retardation, cell death and decreased clonogenicity. The failure to form colonies was associated with abnormal mitosis and endoreduplication in the recovering cells and was correlated with the induced level of p21 and the duration of p21 induction. p21 induction was found to inhibit the expression of multiple proteins involved in the execution and control of mitosis. p21-induced depletion of the cellular pools of mitosis-control proteins was followed by asynchronous resynthesis of such proteins after release from p21, which explains the observed mitotic abnormalities. Genetic destabilization in cells recovering from p21-induced growth arrest may conceivably play a role in carcinogenesis and tumor progression.

Topics: Autoantigens; Calcium-Binding Proteins; Carrier Proteins; CDC2 Protein Kinase; Cell Cycle Proteins; Cell Division; Centromere Protein A; Chromosomal Proteins, Non-Histone; Cyclin B; Cyclin B1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA; Dose-Response Relationship, Drug; Fibrosarcoma; Fluorescent Dyes; Fungal Proteins; Humans; Isopropyl Thiogalactoside; Microfilament Proteins; Mitosis; Nuclear Proteins; Organic Chemicals; Polo-Like Kinase 1; Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Tumor Cells, Cultured

MEK kinases (MEKKs) are serine-threonine kinases that regulate sequential protein phosphorylation pathways involving mitogen-activated protein kinases (MAPKs), including members of the Jun kinase (JNK) family. MEKK1 is a 196 kDa protein that when cleaved by caspase-3-like proteases generates an active COOH-terminal kinase domain. Expression of the MEKK1 kinase domain is sufficient to induce apoptosis. Mutation of MEKK1 to prevent its proteolytic cleavage protects cells from MEKK1-mediated cell death even though the JNK pathway is still activated, indicating that JNK activation is not sufficient to induce cell death. The inducible acute expression at modest levels of the activated MEKK1 kinase domain can be used to potentiate the apoptotic response to low dose ultraviolet irradiation and cisplatin. Similarly, in L929 fibrosarcoma cells inducible acute expression of the kinase domain of MEKK1 markedly increased the cell death response to tumor necrosis factor alpha (TNF alpha). The findings demonstrate that acute expression of an active form of MEKK1 can potentiate the cell death response to external stress stimuli. Manipulation of MEKK1 proteolysis and its regulation of signal pathways involved in apoptosis has significant potential for anticancer therapies when used in combination with therapeutic agents at doses that alone have little or modest effects on cell viability.

Topics: 3T3 Cells; Animals; Apoptosis; Cell Line, Transformed; Cisplatin; Enzyme Activation; Enzyme Induction; Fibrosarcoma; Humans; Isopropyl Thiogalactoside; JNK Mitogen-Activated Protein Kinases; Kidney; L Cells; MAP Kinase Kinase 4; MAP Kinase Kinase Kinase 1; Mice; Mitogen-Activated Protein Kinase Kinases; Phosphorylation; Protein Kinases; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Recombinant Fusion Proteins; Signal Transduction; Stress, Physiological; Transfection; Tumor Cells, Cultured; Ultraviolet Rays