Characterization of MEQC and functional studies of glypican and p23

Abstract

MEQC (mvc embryonic quail cardiomyocytes) is a permanent cell line derived from cardiac tumors produced by infection of 3-day quail with the MC29 myelocytoma virus, which contains the v-myc proto-oncogene. This cell line can be induced to differentiate as evidenced by expression of muscle specific markers upon co-culture with NIH 3T3 fibroblasts. When MEQC are treated with low concentrations of BrdU before co-culture, they can no longer be induced to express phenotypic markers. In the current study, I have isolated BrdU-sensitive transcripts from MEQC cells by subtractive hybridization and investigated their distribution in developing chick embryos. In all, 29 transcripts were isolated, 14 of which could be identified by sequence comparison with the Genbank data base. Complete sequences were obtained for two of the remaining transcripts, pX19 and pX27. pX19 encodes a protein of 23 kDa that contains an amphipathic alpha-helix previously described only in plant seed embryos. By in situ hybridization pX19 was identified mainly in hemopoietic tissues; it was also found in cardiac cushion mesenchyme by PCR. The clone pX27 was identified as the avian homologue of mammalian glypican core proteins and was localized in early stages of development to the cephalic regions of the neural folds, rostral paraxial mesoderm, and newly formed somites. Later, glypican transcripts were found in the apical epidermal ridge of the limb buds, mantle zone of the telencephalon, and endocardial cushions of the atrioventricular canal and aortopulmonary outflow tract. An antibody raised against the glypican core protein was localized to the cell membrane in MEQC cells. Furthermore, upon withdraw of serum from cultures of MEQC expression of glypican transcripts was enhanced and the cells tended to clump. Administration of a glypican antisense oligonucleotide prevented cell clumping and blocked the migration of endocardial endothelial cells over collegen gel. Taken together, these results suggest that MEQC express transcripts unique to either myocardium or endocardium and provide a useful system from which transcripts expressed during development can be isolated.