Glucocorticoid-induced changes in liver: inhibition of nuclear Ca2+, Mg(2+)-dependent endonuclease activity in response to dexamethasone administration.

The endogenous Ca2+, Mg(2+)-dependent endonuclease activity in nuclei from livers of rats receiving daily injections of the synthetic glucocorticoid dexamethasone was examined with respect to the production of both single and double strand breaks in chromatin DNA. The ability to form single strand breaks was measured by means of a nick translation assay and double strand breaks by following the appearance of nucleosomal ladders. A fall in the activity causing double strand breaks to approximately 50 per cent of the control value was apparent at 12 h after the first injection of the steroid. A fall of 25-30 per cent was also observed in the nicking activity but this was not apparent until 24 h after the first steroid injection. Both endonuclease activities remained at these lower levels for the remainder of the period of treatment. Nuclear extracts from dexamethasone-treated rats also showed a reduced ability to produce nucleosomal ladders when incubated with rat muscle nuclei, indicating that the inhibition observed in intact nuclei from treated animals was independent of any changes in chromatin structure. On the other hand the nick translation activity of the two extracts was the same when calf thymus DNA was used as the substrate suggesting that steroid-induced alterations in chromatin structure may be a critical factor in the reduced level of this activity observed in intact nuclei.

H1 histone sub-type distribution and DNA topoisomerase activity in skeletal muscle of tumour-bearing rats.

In an investigation of possible causes of the transcriptional defect which has been observed in muscles atrophying in response to tumour growth, the amounts of histone H1 subtypes and the activities of DNA topoisomerase I and II, factors which can affect the structural organisation of chromatin, were studied in nuclei of skeletal muscle of rats bearing a Walker 256 carcinoma. The H1 histones were separated by SDS/PAGE into three fractions, H1.1, H1.2 and H1(0). The level of H1.2 but not that of H1.1 or H1(0) was increased in the tumour-bearing animal. Tumour growth did not affect the activity of either DNA topoisomerase I or II.

Glucocorticoid-induced changes in liver: effect of dexamethasone administration on DNA topoisomerase I and II activities and distribution of histone H1 subtypes.

The activities of DNA topoisomerase I and II and the relative proportions of the histone H1 subtypes were investigated in rat liver which was undergoing hypertrophy and exhibiting increased transcriptional activity following the administration of dexamethasone. There was a rise in the level of activity of DNA topoisomerase I and a slight fall in that of DNA topoisomerase II. The relative proportions of the H1 subtypes were altered due to a preferential increase in H1.1. The results are discussed in relation to the effect of glucocorticoids on the transcription and replication of hepatic DNA.

Altered levels of histone H1(0) and DNA topoisomerase activity in the liver of the tumour-bearing rat.

Walker 256 carcinoma cells were injected subcutaneously into the dorsal region of rats. After 9 days of tumour growth increases were observed in liver mass and DNA content and in the incorporation of [3H]-thymidine into liver DNA. These changes were accompanied by a fall in the H1(0) fraction of liver histones and rises in both DNA topoisomerase I and II activities.

Protein and enzyme content of plasma membranes derived from Walker 256 carcinoma cells grown as ascitic or solid tumours.

A preliminary investigation of enzymes functioning at the cell surface indicates that when Walker 256 carcinoma cells growing ascitically are induced to grow as a solid tumour there is a marked increase in the activities of Na+, K(+)-ATPase, Mg(2+)-ATPase, 5'-nucleotidase, alkaline phosphatase and phosphodiesterase I; 5'-nucleotidase and alkaline phosphatase being particularly affected. These enzyme changes occur in the absence of any major alteration in the protein composition of the plasma membrane.

Actin and actin-binding proteins in plasma membranes derived from Walker 256 ascites or solid tumour cells.

Plasma membranes from Walker 256 carcinoma cells grown ascitically or as a solid tumour were examined with respect to actin content, [3H]cytochalasin B-binding and the binding of 125I-labelled G-actin to membrane proteins separated by SDS-PAGE. Differences were observed both in cytochalasin B-binding to membrane actin and affinity of 125I-labelled G-actin for specific membrane proteins.

Glucocorticoid-mediated muscle atrophy: alterations in transcriptional activity of skeletal muscle nuclei.

Glucocorticoid-induced muscle atrophy is associated with a decrease in the level of protein synthesis and a loss of RNA. This paper reports the behaviour of RNA polymerase I- and RNA polymerase II-directed transcription (EC 2.7.7.6) in nuclei isolated from skeletal muscles of rats given a catabolic dose of dexamethasone acetate (5 mg per Kg body weight) over a period of 4 days. Both activities were altered by the dexamethasone treatment. In the case of RNA polymerase I-mediated transcription there was a loss of template-engaged enzymes indicating the existence of an inhibition of initiation of transcription while the rate of elongation of bound enzymes was unaltered. The number of RNA polymerase II-chromatin bound enzymes was increased, but the mean polynucleotide elongation rate was reduced. The possibility that glucocorticoids may impair the elongation stage of transcription in skeletal muscle by increasing the frequency of premature termination of transcripts is discussed. No evidence was obtained for any increase in ribonuclease activity in muscle nuclei of dexamethasone-treated animals.

Increased liver alanine in the tumour-bearing host. Altered levels of some enzymes involved in alanine metabolism.

A 2-fold increase in hepatic alanine concentration was observed in rats bearing a Walker 256 carcinoma growing sub-cutaneously. Decreases were observed in the activities of both cytosolic and mitochondrial isozyme forms of L-alanine-2-oxoglutarate aminotransferase. Activities of two enzymes involved in a secondary pathway of haem synthesis involving alanine, L-alanine-4,5-dioxovalerate aminotransferase and the NADP-requiring isozyme form of 4-oxo-5-hydroxyvalerate dehydrogenase were also reduced but there was no change in liver porphyrin concentration. L-alanine-glyoxalate aminotransferase activity was unaffected. The results are discussed in relation to the utilisation of alanine as a gluconeogenic substrate in the tumor-bearing host.

Response of skeletal muscle RNA polymerases I and II to tumour growth.

The muscle wasting which occurs in animals bearing a transplantable tumour is accompanied by a decrease in the level of protein synthesis and a loss in RNA. This paper examines the behaviour of RNA polymerases I and II (EC 2.7.7.6) in nuclei isolated from skeletal muscle of rats bearing a Walker 256 carcinoma. Marked decreases were observed in template-engaged RNA polymerase I and II activities and in free RNA polymerase I activity. Free RNA polymerase II activity was unaltered. When assays were carried out at high (NH4)2SO4 concentration or in the presence of heparin the diminished RNA polymerase I activity was still apparent, but heparin and high ionic strength overcame the inhibition of RNA polymerase II. Loss of RNA polymerase I activity was associated with a decrease in the number of template-engaged enzyme molecules and in the polynucleotide elongation rate. The number of template-engaged RNA polymerase II molecules was unaltered by tumour growth, but the polynucleotide elongation rate was significantly reduced. No evidence was obtained for any alteration in ribonuclease activity in nuclei or whole muscles of tumour-bearing rats. These results demonstrate an effect of the tumor on transcription in skeletal muscle of its host.

A differential effect of the Walker 256 carcinoma on the tyrosine protein kinase activity of liver plasma membrane domains.

Protein kinase activity was investigated in plasma membrane fractions enriched in either sinusoidal or lateral domains prepared from liver of normal rats and rats bearing a Walker 256 carcinoma. Membrane proteins were phosphorylated on tyrosine, threonine and serine residues in both fractions. Phosphorylation of tyrosine residues was increased in sinusoidal preparations from tumour-bearing rats but not in lateral preparations. An increase in histone phosphorylation by sinusoidal preparations from tumour-bearing rats was found to be due to serine protein kinase activity.

Plasma membrane associated actin in liver of normal and tumour-bearing rats.

A study was made of the association of actin with different plasma membrane fractions from liver of normal rats and from the enlarged liver of rats bearing a Walker 256 carcinoma where a decrease in the state of polymerisation of cytoplasmic actin has been previously observed. As estimated by the DNAase I inhibition assay, actin constituted approx. 7% and 3%, respectively, of the protein of membrane fractions enriched in lateral or bile-canalicular domains, but only trace amounts were found in the sinusoidal fraction. [3H]Cytochalasin B binding indicated the presence of 20 and 13 pmol of high-affinity binding sites per mg protein in lateral and bile-canalicular fractions, but none in the sinusoidal. Kd for cytochalasin B binding was of the order of 1 nM for lateral and bile-canalicular fractions. Polypeptide profiles obtained by SDS/urea/polyacrylamide gel electrophoresis of non-ionic detergent-insoluble residues differed for all three fractions although some proteins, including actin, occurred as major components of both bile-canalicular and lateral regions. Tumour growth had no effect on the actin content, high-affinity cytochalasin B binding or polypeptide profiles of the three membrane fractions.

Effect of tumour growth on hepatic neutral ribonuclease and its inhibitor and on RNA polymerase activity of liver nuclei.

In rats bearing a subcutaneously implanted Walker 256 carcinoma an early rise in liver DNA content was followed by a two-fold increase in RNA content between the 6th and 10th day of tumour growth. Total hepatic neutral ribonuclease and its inhibitor were unaffected by tumour growth. No alteration in RNA polymerase I and II activities of liver nuclei was observed except for a 47% increase in RNA polymerase I on the 8th day of tumour growth.

Alterations in hepatic 5'-nucleotidase in the tumor-bearing rat.

The effect of the growth of the Walker 256 carcinoma on the level of 5'-nucleotidase and alkaline phosphatase in the whole liver and in an isolated hepatocyte membrane preparation of its host was investigated. Alkaline phosphatase activities of whole liver and plasma membrane were increased approximately 5-fold by tumor growth. A 50% decrease in whole liver 5'-nucleotidase activity was observed in tumor-bearing rats while the 5'-nucleotidase activity per milligram membrane protein was unaltered. Tumor growth would therefore appear to affect a pool of 5'-nucleotidase which is not associated with the plasma membrane.

Actin depolymerising activity in tumour-bearing rats.

The actin depolymerising activity in plasma, liver and gastrocnemius muscle of normal and tumour-bearing rats and in Walker 256 carcinoma grown in the solid and ascitic form was measured. Similar levels of actin depolymerising activity were observed in solid and ascitic forms of the tumour. No alteration in actin depolymerising activity was found to accompany the increased levels of unpolymerised actin found in the plasma and tissues of the tumour-bearing rat. It was concluded that changes in the state of polymerisation of actin in the tissues of the tumour-bearing host could not be attributed to an effect of the tumour on actin depolymerising activity.

Actin polymerisation in Walker 256 carcinoma cells from solid or ascitic tumours.

Use was made of the differential DNase I assay to estimate the relative amounts of polymerised and unpolymerised actin in Walker 256 carcinoma cells from solid or ascitic tumours. The concentration of actin per unit DNA and the relative amount of actin present in a polymerised form were both greater in ascitic tumour cells than in cells from solid tumours.

Actin synthesis and polymerization in the liver of fed and fasted rats bearing a Walker 256 carcinoma.

The effect of tumor growth on the amount, state of polymerization, and synthesis of liver actin was investigated in fed and fasted rats bearing a Walker 256 carcinoma. The increase in liver size in the tumor-bearing animal was accompanied by a rise in the total amount of actin and protein, although the amounts present per g liver fell in both cases. Soluble actin increased in both concentration and total amount in the tumor-bearing animals. While the ratio of total actin to total protein in liver was unaltered by tumor growth, the ratio of soluble actin to total actin was increased. The incorporation of [3H]leucine into liver actin relative to that into liver protein, in vivo, was not affected by tumor growth, but the radioactivity incorporated into soluble actin relative to total actin in the livers of the tumor-bearing rats was increased. Liver polysome preparations from tumor-bearing rats showed an increased ability to synthesize actin and total protein, whereas polysomes from skeletal muscle of tumor-bearing rats exhibited a decreased synthesis of actin and total protein. These results suggest that, in the liver of the tumor-bearing rat, while there is an increase in actin synthesis in parallel with a net increase in protein synthesis, there is a decrease in the polymerization of action. In livers of both control and tumor-bearing rats, the consumption of a meal was accompanied by a decrease in soluble actin relative to total actin and an increase in the synthesis of action relative to total protein.

RNA polymerase and ribonuclease activities in skeletal muscle of prednisolone-treated rats.

Nuclei isolated from gastrocnemius muscles of rats given prednisolone acetate i.p. showed decreased levels of Mg2l-dependent RNA polymerase activity while the Mn2+-dependent polymerase activity showed no significant change from control levels. Both acid and alkaline ribonuclease activities in gastrocnemius and liver of prednisolone-treated rats were lower than levels in untreated rats. Although evidence was obtained for the existence of an inhibitor of ribonuclease activity in skeletal muscle, the effect of the glucocorticoid on ribonuclease activity was not due to increased levels of any such inhibitor.