pactamycin and Liver-Neoplasms

Vertebrate fibrinogen consists of two sets of three nonidentical polypeptides that are synthesized in the liver. The subunits of fibrinogen have been synthesized in a cell-free, membrane-free translation system and compared with (alpha), polypeptides of fibrinogen purified from rat plasma and (b) subunits synthesized and secreted by hepatoma cells grown in culture. Rat hepatoma monolayers were grown with or without tunicamycin to prevent or allow glycosylation of the B beta and gamma subunits, respectively. Sodium dodecyl sulfate polyacrylamide gel analysis indicated that each of the polypeptides translated in vitro from mRNA is larger than its corresponding nonglycosylated fibrinogen chain. The primary translation A alpha, B beta, and gamma chains are larger than their authentic nonglycosylated counterparts by 600, 1100, and 3000 daltons, respectively. Furthermore, the preA alpha and preB beta translation products are thrombin sensitive. These results strongly imply that signal peptides exist on each of the primary translation products of fibrinogen.

Topics: Animals; Base Sequence; Cell-Free System; Cells, Cultured; Fibrinogen; Gene Expression Regulation; Liver Neoplasms; Liver Neoplasms, Experimental; Pactamycin; Polyribosomes; Protein Biosynthesis; Protein Precursors; Rats; RNA, Messenger; Thrombin; Tunicamycin

Topics: Animals; Antimetabolites; Carcinoma, Hepatocellular; Cytotoxicity, Immunologic; Dactinomycin; Depression, Chemical; Guinea Pigs; Liver Neoplasms; Macrophages; Mitomycins; Neoplasms, Experimental; Pactamycin; Puromycin; Puromycin Aminonucleoside

The modes of action of insulin and of inhibitors of protein synthesis on the degradation of labeled cellular proteins have been studied in cultured hepatoma (HTC) cells. Protein breakdown is accelerated upon the deprivation of serum (normally present in the culture medium), and this enhancement is inhibited by either insulin or cycloheximide. An exception is a limited class of rapidly turning over cellular proteins, the degradation of which is not influenced by insulin or cycloheximide. Alternative hypotheses to explain the relationship of protein synthesis to the regulation of protein breakdown, viz., control by the levels of precursors of protein synthesis, regulation by the state of the ribosome cycle, or requirement for a product of protein synthesis, have been examined. Protein breakdown was not influenced by amino acid deprivation, and measurements of valyl-tRNA levels in HTC cells subjected to various experimental conditions showed no correlation between the levels of charged tRNAVal and the rates of protein degradation. Three different inhibitors of protein synthesis (puromycin, pactamycin, and cycloheximide) suppressed enhanced protein breakdown in a similar fashion. A direct relationship was found between the respective potencies of these drugs to inhibit protein synthesis and to block enhanced protein breakdown. When cycloheximide and insulin were added following a prior incubation of HTC cells in a serum-free medium, protein breakdown was maximally suppressed within 15-30 min. Actinomycin D inhibited protein breakdown only after a time lag of about 90 min. It is suggested that the regulation of protein breakdown in hepatoma cells requires the continuous formation of a product of protein synthesis, in a manner analogous to the mode of the control of this process in bacteria.

Topics: Carcinoma, Hepatocellular; Cell Line; Cycloheximide; Dactinomycin; Insulin; Liver Neoplasms; Neoplasm Proteins; Neoplasms, Experimental; Pactamycin; Protein Biosynthesis; Puromycin; RNA, Transfer