Immunofluorescence analysis of the time-course of extinction, reexpression, and activation of albumin production in rat hepatoma-mouse fibroblast heterokaryons and hybrids.

Abstract

A combination of a sensitive immunocytochemical stain for intracellular albumin and Hoechst 33258 dye for identification of parental nuclei was used to investigate the time-course of extinction, reexpression and activation of albumin production in fusion products of 1s (hyperdiploid), or 2s (hypertetradiploid) rat hepatoma [Fao and 2s-F] cells with mouse fibroblasts (L cells or embryonic EM cells) [mouse hepatoma BWI-J cells were also used]. In all combinations the initial event was the extinction of albumin production. Extinction occurred immediately after fusion when the mouse fibroblast was a normal embryonic (senescent ?) cell. In the case of an L cell, rat albumin was synthesized and secreted during the first 12 h after fusion; no production of mouse albumin occurred. Albumin production ceased. After fusion (8-12 d), young hybrid colonies resumed the synthesis of rat albumin (reexpression), and several days later the production of mouse albumin began (activation). The patterns of reexpression and activation indicated that chromosome loss was not necessary for either event to occur, and that the cells active in the synthesis of mouse albumin were a subpopulation of those cells already engaged in the production of rat albumin. Apparently extinction was mediated by diffusible factor(s) from the L-cell parent that acted in the hepatoma nucleus to prevent the formation of new albumin mRNA; reexpression and activation were gene dosage-dependent but extinction was not; and previously active genes were more rapidly expressed than previously silent ones.