calcimycin and Teratoma

The 78-kDa glucose-regulated protein (GRP78) is ubiquitously expressed in many cell types. Its promoter contains multiple protein-binding sites and functional elements. In this study we examined a high affinity protein-binding site spanning bp -198 to -180 of the rat grp78 promoter, using nuclear extracts from both B-lymphoid and HeLa cells. This region contains a sequence TGACGTGA which, with the exception of one base, is identical to the cAMP-response element (CRE). Site-directed mutagenesis reveals that this sequence functions as a major basal level regulatory element in hamster fibroblast cells and is also necessary to maintain high promoter activity under stress-induced conditions. By gel mobility shift analysis, we detect two specific protein complexes. The major specific complex I, while immunologically distinct from the 42-kDa CRE-binding protein (CREB), binds most strongly to the grp site, but also exhibits affinity for the CRE consensus sequence. As such, complex I may consist of other members of the CREB/activating transcription factor protein family. The minor specific complex II consists of CREB or a protein antigenically related to it. A nonspecific complex III consists of the Ku autoantigen, an abundant 70- to 80-kDa protein complex in HeLa nuclear extracts. By cotransfection experiments, we demonstrate that in F9 teratocarcinoma cells, the grp78 promoter can be transactivated by the phosphorylated CREB or when the CREB-transfected cells are treated with the calcium ionophore A23187. The differential regulation of the grp78 gene by cAMP in specific cell types and tissues is discussed.

Topics: B-Lymphocytes; Base Sequence; Binding Sites; Calcimycin; Cyclic AMP Response Element-Binding Protein; DNA; DNA-Binding Proteins; Endoplasmic Reticulum Chaperone BiP; Gene Expression Regulation; HeLa Cells; HSP70 Heat-Shock Proteins; Humans; Immune Sera; Membrane Proteins; Molecular Sequence Data; Mutagenesis; Phosphorylation; Promoter Regions, Genetic; Regulatory Sequences, Nucleic Acid; Teratoma; Transcriptional Activation; Transfection; Tumor Cells, Cultured

We examined the expression of GRP94 and GRP78 in embryonal carcinoma (EC) cells and during early mouse development. The GRP78 protein is constitutively expressed in the undifferentiated F9 cells whereas the level of GRP94 is below detection limit. These cells, like adult differentiated cells, exhibit enhancement of the GRP94 and GRP78 transcript levels in response to the calcium ionophore A23187, resulting in an increase of GRP94 and GRP78 proteins after 8 h of treatment. The GRP78 promoter has been identified as containing a strong enhancer. To test whether the GRP78 promoter is effective in directing expression of a heterologous gene in F9 cells, CAT fusion genes containing the GRP78 promoter were transfected into F9 cells. In non-induced F9 cells the activity of the GRP78 promoter was about 5- and 10-fold higher than the RSV promoter and the SV40 early promoter, respectively. Treatment of the cells with A23187 further increased the GRP78 promoter activity 3-fold. Since GRP78 is expressed during mouse embryogenesis and the promoter is also active in F9 cells treated with retinoic acid, the GRP78 promoter may be useful for the efficient expression of heterologous genes in undifferentiated and differentiated mouse embryonal cells.

Topics: Animals; Calcimycin; Cell Differentiation; Embryo, Mammalian; Endoplasmic Reticulum Chaperone BiP; Gene Expression Regulation; HSP70 Heat-Shock Proteins; Membrane Proteins; Mice; Promoter Regions, Genetic; RNA; Teratoma; Transcription, Genetic; Transfection; Tumor Cells, Cultured

H6 embryonal carcinoma cells form aggregates of cells in culture medium which contains 2 mM calcium. These aggregates are described as uncompacted, indicating that the individual cells of the aggregate are spherical and are in limited contact with each other. In contrast, compaction of the aggregate, induced by increasing the calcium concentration, results in a tight mass of cells flattened against one another and connected by intercellular junctions. At least 85-97% of the aggregates undergo compaction in 7 mM calcium and are subsequently decompacted if removed to 2 mM calcium. Since calcium ionophore A23187 does not induce compaction, extracellular rather than intracellular calcium seems to be the limiting factor. We have demonstrated that this calcium-induced morphogenetic change is sensitive to inhibition by agents which also prevent the calcium-dependent compaction of the 8-cell mouse embryo. The cytoskeletal-binding drugs tetracaine HCl, colcemid, vinblastine, colchicine, and cytochalasin B each inhibit compaction of H6 aggregates. Interference at surface molecule sites by exposure to the lectins wheat germ agglutinin or concanavalin A or by interruption of glycosylation with exposure to tunicamycin, or by reaction with anti-H6 Fab or anti-F9, also prevent compaction. Since the mouse embryo and embryonal carcinoma cells share certain processes which are involved in initiating and maintaining compaction, these processes and their subsequent roles in differentiation may be examined using embryonal carcinoma cell aggregates.

Topics: Animals; Calcimycin; Calcium; Cell Adhesion; Cell Aggregation; Cell Line; Cytochalasin B; Intercellular Junctions; Lectins; Mice; Microscopy, Electron; Teratoma; Tetracaine