isopropyl-thiogalactoside and Leukemia--Myeloid--Acute

The MyD118/Gadd45/CR6 gene family (also termed Gadd45beta/alpha/gamma) has been identified as genes which are rapidly induced by genotoxic agents, during terminal differentiation, as well as by apoptotic cytokines. In recent years, evidence has emerged that the proteins encoded by these genes play pivotal roles in negative growth control, including growth suppression and apoptotic cell death. However, under what physiological condition these proteins mediate either cell cycle arrest or apoptosis, and the molecular nature of apoptotic pathways involved are currently unclear. Thus, to further explore the effects of these genes on cell growth and cell viability, either in the presence or absence of extrinsic stress, we have established M1 myeloblastic leukemia and H1299 lung carcinoma cell lines, where high level ectopic expression of MyD118, Gadd45, or CR6 can be induced by isopropyl beta-D-thiogalactopyranoside (IPTG). By taking advantage of these cell lines, it was observed that in the absence of genotoxic stress, inducible expression of MyD118, Gadd45 and/or CR6 resulted in retardation of cellular proliferation and accumulation of cells in the G1 phase of the cell cycle. Ectopic expression of these proteins also was found to sensitize the cells to apoptosis induced by genotoxic agents such as UV, MMS, gamma-irradiation and VP16. Finally, evidence has been obtained that in the absence of stress, ectopic expression of MyD118/Gadd45/CR6 is insufficient to activate the MTKl/JNK/p38 stress cascade, and that enhancement of genotoxic stress induced apoptosis by these proteins may involve apoptotic pathways other than the JNK/p38 pathways.

Topics: Antigens, Differentiation; Apoptosis; Blotting, Northern; Blotting, Western; Carrier Proteins; Cell Cycle; DNA Damage; DNA Repair; Etoposide; Flow Cytometry; GADD45 Proteins; Gene Expression Regulation; Genetic Vectors; Humans; Intracellular Signaling Peptides and Proteins; Isopropyl Thiogalactoside; Leukemia, Myeloid, Acute; Lung Neoplasms; Mitogen-Activated Protein Kinases; Nucleic Acid Synthesis Inhibitors; Oxidative Stress; Protein Biosynthesis; Proteins; Transfection; Tumor Cells, Cultured

CHOP (GADD153) is a member of the C/EBP family and a stress-induced protein. To investigate the role of CHOP in cellular growth, we expressed CHOP conditionally in M1 myeloblastic leukemia cells that do not express p53 protein. More than 60% of M1 cells died through apoptosis 72 h after CHOP induction. Site-directed mutagenesis revealed that this process requires leucine zipper domain but neither intact basic region nor trans-activation domain. CHOP-mediated apoptosis accompanied downregulation of bcl-2 mRNA and overexpression of Bcl-2 delayed the process. Our results indicate that CHOP can induce apoptosis in a p53-independent manner.

Topics: Animals; Apoptosis; CCAAT-Enhancer-Binding Proteins; Cell Line; Cloning, Molecular; Cricetinae; DNA Damage; DNA-Binding Proteins; Genes, bcl-2; Isopropyl Thiogalactoside; Kinetics; Leukemia, Myeloid, Acute; Mice; Mutagenesis, Site-Directed; Nuclear Proteins; Proto-Oncogene Proteins c-bcl-2; Recombinant Proteins; RNA, Messenger; Transcription Factor CHOP; Transcription Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured; Tumor Suppressor Protein p53