atrial-natriuretic-factor and Teratoma

We assessed the temporal transcriptional activity profiles of the genes for type-B natriuretic factor, BNF, the isoform ANF, and other cardiac muscle proteins in differentiating cultures derived from multipotential mouse cell lines. P19 embryonal carcinoma cells and D3 embryonic stem cells were induced for in vitro cardiac myogenesis; RNA was isolated at regular intervals throughout the differentiation programs, and mRNAs were detected by reverse transcriptase mediated polymerase chain reactions. The transcriptional activation profiles of the ANF and BNF gene were similar, but there were quantitative differences that were best assayed by use of competitive internal DNA standards. The levels of induced BNF transcripts were highest in the P19 developmental system reaching approximately 10% of adult mouse ventricular muscle levels; those for ANF were lower, but also readily detected. The cell lines may be used to define the regulatory control elements for natriuretic factor gene expression, in stably transfected cell lines, during cardiac muscle growth.

Topics: Animals; Atrial Natriuretic Factor; Base Sequence; Cell Line; DNA Primers; DNA, Complementary; Gene Expression Regulation; Growth Inhibitors; Heart; Interleukin-6; Introns; Leukemia Inhibitory Factor; Lymphokines; Mice; Molecular Sequence Data; Myocardium; Natriuretic Peptide, Brain; Polymerase Chain Reaction; Recombinant Proteins; RNA, Messenger; Stem Cells; Teratoma; Transcription, Genetic; Transfection; Tumor Cells, Cultured

We examined the transcriptional activity profile of the gene for atrial natriuretic factor (ANF) in mouse embryonal carcinoma P19 cells which had been induced for in vitro cardiac myogenesis. Differentiation was assessed visually, by the degree of spontaneous beating activity, and by the appearance of striated muscle structures detected by immunofluorescence with a myosin heavy chain antibody. Northern blot analysis of RNA isolated at regular intervals throughout the differentiation program revealed abundant cardiac alpha-actin transcripts beginning at Day 6, reaching maximum levels during Days 7 to 8 and declining to low levels by Days 12 to 15. Throughout this period, the transcriptional profile of the ANF gene was similar to that of alpha-actin but at lower levels; thus, in vivo stages of abundant ANF and structural muscle gene transcription were not reached and these gene expression states appear to be uncoupled. Using the more sensitive assay of reverse transcriptase-mediated polymerase chain reactions, we observed the presence of ANF transcripts even in small samples of muscle-induced P19 cells and not in neuron-induced or undifferentiated P19 cells. Induced ANF transcript levels reached about 5-10% that found in adult atrium muscle tissue. ANF gene activity was further corroborated by nuclear transcriptional run-on assays. The P19 stem cell model system will be of value in the study of early events during cardiac muscle commitment and differentiation.

Topics: Actins; Animals; Atrial Natriuretic Factor; Base Sequence; Blotting, Northern; Cell Differentiation; Dimethyl Sulfoxide; Fluorescent Antibody Technique; Gene Expression; Genes; Mice; Molecular Sequence Data; Myocardium; Polymerase Chain Reaction; RNA, Messenger; Teratoma; Time Factors; Transcription, Genetic; Tumor Cells, Cultured