title: Mitogenic hormone-induced intracellular message: assay and partial characterization of an activator of DNA replication induced by epidermal growth factor.
abstract: This paper explores the pathway from nuclear quiescence to mitogenesis. It describes an in vitro assay for an activator of DNA replication induced by epidermal growth factor (EGF) in responsive cells. Cytoplasmic extracts from EGF-treated 3T3 cells were found to contain substances that can stimulate DNA synthesis in isolated nuclei from spleen cells of adult frogs. Extracts from untreated resting 3T3 cells lack this activity, and EGF itself is incapable of stimulating DNA synthesis in these cell-free systems. The extract-induced stimulation of incorporation of [3H]dTTP into nuclear DNA is ATP dependent and requires the presence of the four deoxyribonucleoside triphosphates, suggesting the occurrence of replication rather than repair synthesis. This cell-free assay has been used to obtain some initial insights into the mechanism of induction and biochemical characterization of the intermediate in EGF action. Half-maximal induction of the active intracellular substance is achieved at about 0.08 nM EGF, a concentration that correlates well with the concentration required for half-maximal mitogenesis. Studies on the biochemical characteristics of this active substance strongly suggest that the activity is associated with a protein. The activity is nondialyzable and sensitive to trypsin and heat. Sucrose gradient centrifugation of the extract revealed three peaks of activity with molecular weights of 46,000, 110,000, and 270,000 (sedimentation coefficients: 3.7 S, 6.6 S, and 12 S, respectively). These results indicate that receptor-EGF interaction at the cell surface leads to the intracellular generation of protein that are capable of stimulating quiescent nuclei into activity.
mesh_terms: D000818:Animals; D001001:Anura; D002453:Cell Cycle / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects; D002474:Cell-Free System; D003593:Cytoplasm / Q000502:physiology; D004261:DNA Replication / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D008934:Mitogens; D008938:Mitosis / Q000187:drug effects*; D008970:Molecular Weight; D010455:Peptides / Q000494:pharmacology*; D011506:Proteins / Q000494:pharmacology
chemical_list: D008934:Mitogens; D010455:Peptides; D011506:Proteins; D004815:Epidermal Growth Factor