Thomas E. Webb

Effects of 5-azacytidine on polyribosomes and on the control of tyrosine transaminase activity in rat liver

Abstract 1. 1. The effects of the nucleoside analog 5-azacytidine on general protein biosynthesis in rat liver, and on the induction and inactivation of hepatic tyrosine transaminase and tryptophan pyrrolase were investigated. 2. 2. 5-Azacytidine caused a breakdown of hepatic polyribosomes to inactive monomers and dimers, and inhibited the incorporation of amino acids into soluble liver proteins; these effects are attributed in part to incorporation of the analog into RNA. 3. 3. The hormonal induction of tryptophan pyrrolase, but not that of tyrosine transaminase, was inhibited by 5-azacytidine. 4. 4. 5-Azacytidine prevented the inactivation of tyrosine transaminase, but not that of tryptophan pyrrolase, which normally occurs following hormonal induction. 5. 5. The data support the hypothesis that the synthesis of tyrosine transaminase is controlled, at least in part, at the translational level of protein biosynthesis. The results also support the theory that the inactivation of tyrosine transaminase following its hormonal induction is dependent on the synthesis of a degradative enzyme.

Hydrocortisone-mediated changes in the concentration of tyrosine transaminase in rat liver: an immunochemical study.

Abstract By standard immunochemical techniques, with a specific antiserum against purified hepatic tyrosine transaminase, it has been shown that the degradation of this enzyme in the livers of adrenalectomized rats is totally eliminated between one and four hours after the administration of an optimum inducing dose of hydrocortisone (i.e. during the initial phase of the induction cycle when the rate of enzyme synthesis increases 4- to 5-fold over the basal value). An immunochemical analysis of the second phase of the induction cycle (i.e. the fall in enzyme activity which begins approximately 5.5 hours after the initiation of induction) indicates that the rate of enzyme synthesis returns to the basal level during this period; the drop to the basal rate occurs within seven, hours after the administration of the corticosteroid. Immunochemical measurements were also employed to study the nature of the effects of several inhibitors of protein biosynthesis on the induced enzyme. The results indicate that the maintenance of the tyrosine transaminase activity at or near the induced level by 8-azaguanine, 5-azacytidine or cycloheximide results from a failure to re-initiate the degradation of the enzyme.

Posttranscriptional control in the steroid-mediated induction of hepatic tyrosine transaminase.

The purine analog azaguanine does not inhibit the initial induction of hepatic tyrosine transaminase by hydrocortisone. However, the continued induced synthesis of tyrosine transaminase, elicited by repeated doses of hydro-cortisone, is inhibited approximately 64 percent in the presence of the analog after 7 to 8 hours and appears to be almost completely inhibited by 9 to 10 hours; this suggests that the induction cycle involves the activation and renewal of a pool of preexisting messenger RNA.

Concurrent changes in the concentration of monomeric ribosomes and the rate of ribosome synthesis in rat liver

Abstract (1) The size of the ribosomal monomer pool and the rate of ribosome synthesis has been studied as a function of the time of rat liver regeneration. (2) The pool of free ribosomal monomers rapidly decreases to a minimal value of 40 per cent that of normal liver 12–19 h after partial hepatectomy, during which period the number of cells preparing for division is known to be maximal. The size of the monomer pool then gradually returns to normal as the regenerative process proceeds, attaining a value typical of resting liver at approx. 80 h post-operative. (3) Based on 2 and 4 h labelling in vivo there is a linear and inverse relationship between the rate of incorporation of label from [6- 14 C]orotic acid into the RNA of cytoplasmic ribosomes and the size of the monomer pool. (4) Inhibitors of protein biosynthesis which promote the conversion of polyribosomes to non-functional monomers inhibit ribosome synthesis to a greater extent than those which maintain the integrity of the polyribosome. (5) The correspondence between the rate of transport of newly synthesized ribosomes to the cytoplasm and the size of the non-functional monomer pool, suggests that the two parameters are directly, or indirectly coupled.

Ribosome formation in rat liver: Evidence for post-transcriptional control

Abstract An investigation of the amount of label incorporated from [6- 14 C]orotic acid into the nuclear and cytoplasmic ribosomal RNA of non-proliferating rat liver, during a 1-h treatment with several inhibitors of protein biosynthesis, gave the following results: 1. 1. The incorporation into nuclear 45-S, 28-S and 18-S RNA of liver was increased during a 1-h treatment with anisomycin and cycloheximide by 50%, and with puromycin by 25%; 8-azaguanine showed little effect. 2. 2. The appearance of label in newly synthesized ribosomes increased approx. 25% after cycloheximide or anisomycin treatment; in contrast, this parameter was reduced to 25% that of the controls after puromycin treatment, and to 50% that of the controls after 8-azaguanine treatment. This differential effect of the inhibitors on ribosome transport was also observed in the slow growing Hepatoma 7800. 3. 3. The size of the ribosomal subunit (60 S and 40 S) pools were not significantly altered by the inhibitors, even though puromycin and 8-azaguanine caused extensive conversion of the polyribosomes to monomers and dimers. The results suggest that the differential effect of the inhibitors on ribosome formation and polyribosome structure are related. They further suggest that this feed-back control, which may involve monomeric ribosomes, acts at a post-transcriptional step. There appears to be a pool of ribosomal proteins in non-proliferating rat liver and in the slow growing hepatoma which is sufficient to support ribosome synthesis for at least 1 h.

Polyribosome breakdown in rat liver following administration of 8-azaguanine

Abstract A gradual breakdown in vivo of the polyribosomes in the post-mitochondrial supernatant of normal and regenerating rat liver to monomers and dimers is observed up to 5 h after a single injection (110 mg/kg) of 8-azaguanine. There follows a recovery phase, lasting in excess of 12 h, during which the size distribution returns to normal. During the recovery phase the size distribution is similar to that of neoplastic liver. Low doses of actinomycin D partially inhibits the 8-azaguanine-induced breakdown of the polyribosomes. [14C]Orotic acid was incorporated into the ribosomes of the monomer, dimer and polyribosome pools at equal rates during both the breakdown and recovery phases, suggesting that the pools were interconvertable. The data are consistent with the substitution of 8-azaguanine for guanine in an RNA component(s) which has a relatively short half-life (cf. ribosomal RNA) and which normally contributes to the integrity of the polyribosome.

Liver regeneration in the hypophysectomized rat: Influence of corticosteroids on the size distribution and synthesis of ribosomal components

Abstract The increased incorporation of label from orotic-6- 14 C acid into ribosomal RNA and changes in the size distribution of ribosomal components, normally occurring during the early phases of liver regeneration in the intact rat, are also observed in the regenerating liver of the hypophysectomized rat. Neither parameter in the resting and regenerating liver was adversely affected by the administration of pharmacological doses of corticosteroids to the intact rat. However, identical treatment of the hypophysectomized rat resulted in a marked inhibition of the incorporation of label from orotic-6- 14 C acid into ribosomal RNA and partial breakdown of the polyribosomes in the regenerating liver within 20 hours of partial hepatectomy. The phenomena appear to be peculiar to proliferating tissue, since pharmacological doses of hydrocortisone neither caused polyribosome breakdown nor inhibited orotic-6- 14 C acid incorporation in the resting liver of the hypophysectomized rat.