Zhi-mu saponin inhibits alpha-fetoprotein gene expression in developing rat liver

1. A saponin isolated from the Chinese herb zhi-mu (Anemarrhena asphodeloides Bunge) modifies alpha-fetoprotein production when injected into newborn rats. 2. The serum level of AFP was determined quantitatively by immunorocket electrophoresis. 3. AFP serum levels were reduced to 60% of the control by zhi-mu saponin (ZMS). 4. The lower AFP level in drug treated rat serum is not due to a change in the pattern of serum AFP variants. 5. AFP mRNA levels in ZMS-treated rat livers, measured by RNA dot hybridization, decreased to about 50% of control levels after 4 days treatment. 6. Results from tritium labeled dexamethasone competition assays suggest that ZMS may act on AFP gene expression through glucocorticoid receptor mediated action.     

Changes in expression of albumin and alpha-fetoprotein genes during rat liver development and neoplasia.

Albumin mRNA was isolated and purified from rat liver polysomes by a combination of immunoprecipitation of specific polysomes, poly(U)-Sepharose 4B chromatography, and fractionation of the resulting poly(A)-containing RNA on a sucrose gradient. alpha-Fetoprotein (AFP) mRNA was isolated from Morris hepatoma 7777 by a similar procedure. The purity of the mRNA preparations was determined by analytical gel electrophoresis under denaturing conditions, analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the polypeptides synthesized in a wheat germ cell-free system, and the kinetics of hybridization to cDNA transcribed from albumin mRNA and AFP mRNA. The albumin mRNA possessed a chain length of approximately 2265 nucleotides and the AFP mRNA possesed a length of approximately 2235 nucleotides when examined under stringent denaturing conditions on agarose gels containing 10 mM methylmercury hydroxide. Analysis of poly(A) content by a hybridization assay with [3H]poly(U) revealed the presence in albumin mRNA of a poly(A) region containing approximately 100 adenosine residues. The AFP mRNA preparation was found to contain an average poly(A) tract of approximately 190 bases. Thus, albumin mRNA appears to contain approximately 330 untranslated nucleotides, and AFP mRNA appears to contain a similar number (approximately 285) of noncoding, nonpoly(A) bases. The purified albumin and AFP mRNA's were used as templates for synthesis of full-length cDNA hybridization probes. Both of the probes selectively hybridized to their templates with kinetics expected for single RNA species the sizes of albumin and AFP mRNA. ROt analysis was used to quantitate albumin and AFP mRNA sequences during normal liver postnatal development and liver oncogenesis. The number of polysomal AFP mRNA molecules per liver was found to drastically decrease during the first weeks of postnatal life, concomitant with a decline in the AFP synthetic capacity of the livers and in the serum concentrations of AFP. During this period, the concentration of albumin mRNA molecules per cell in the liver remained at high, approximately constant levels. In Morris hepatoma 7777, the concentration of AFP-specifying sequences was at least 10(3)-fold higher than that found in normal adult liver, whereas the content of albumin nRNA was four- to five-fold lower. These changes in concentration of albumin and AFP mRNA sequences closely correlated with a parallel variation in the specific protein synthetic capacity of the tissues.     

The expression of alpha-fetoprotein and albumin genes in rat liver during chemical carcinogenesis

The effect of the hepatocarcinogen 3′-methyl-4-dimethylaminoazobenzene on α-fetoprotein (AFP) and albumin gene expression in rat liver was studied. Serum concentrations of AFP and albumin were measured. Amounts of AFP mRNA and albumin mRNA in rat livers were determined by hybridization of total cytoplasmic RNAs to their cDNAs. Dramatic increases in serum AFP concentrations coincided with increases in AFP biosynthesis and amount of AFP mRNA in livers of carcinogen-treated rats. In contrast, no or little change in albumin mRNA concentration was found in livers of rats treated with 3′-methyl-4-dimethylaminoazobenzene. Concomitantly, there was little change in liver albumin biosynthesis or serum albumin concentrations during hepatocarcinogenesis.     

Developmental and steroid hormonal regulation of insulin-like growth factor II expression

We have investigated the influence of steroid hormones on insulin-like growth factor II (IGF-II) expression. Hepatic IGF-II mRNA decreased gradually during postnatal development, reaching adult levels at 3 weeks of age. Treatment of 1-day-old rats for 4 days with 10 micrograms/day of the glucocorticoid dexamethasone (DEX) reduced IGF-II mRNA levels 10-fold in liver and inhibited body weight gain. Estradiol and testosterone did not affect IGF-II expression. A dose-response relationship between IGF-II mRNA levels and the different amounts of DEX injected was seen. IGF-II levels remained low after withdrawal of DEX, indicating an irreversible effect. Albumin expression was increased in newborn rat livers after DEX treatment. Our results suggest that glucocorticoids play an important role in the regulation of IGF-II expression. The mechanism for glucocorticoid-induced reduction of IGF-II mRNA is still unclear; however, our findings indicate that DEX inhibits IGF-II by causing premature differentiation of the liver.     

Changes induced in rat liver polysomal polyadenylated RNAs by depletion of circulating glucocorticoids.

The effects of adrenalectomy on the complexity and the relative abundances of rat liver polyadenylated mRNAs have been investigated. The qualitative and quantitative changes induced by adrenalectomy have been measured by hybridisation of polysomal polyadenylated RNAs from the livers of normal and adrenalectomised rats with total cDNAs, fractionated cDNAs, cDNA representing RNAs specific to normal liver, total unique-sequence DNA and unique-sequence DNA complementary to normal liver polysomal RNA. These analyses indicated that, by 14 days after adrenalectomy, the equivalent of about 7000 sequences of average length 2000 nucleotides can no longer be detected in liver polysomes. Many other sequences are decreased in abundance as compared to normal liver, but some abundant sequences become more abundant. Administration of a glucocorticoid hormone (dexamethasone) very rapidly reverses these changes.     

Differential expression of albumin and alpha-fetoprotein genes in fetal tissues of mouse and rat

We have carried out a comparative analysis of the expression of the albumin and alpha-fetoprotein (AFP) genes in yolk sac and liver at different stages of fetal and postnatal life, in rat and mouse. Albumin and AFP mRNA levels were examined in these tissues by R0t analysis of RNA excess-cDNA hybridization data and/or by Dot blot hybridization. In addition, size analysis of the mRNA sequences were performed by electrophoretic fractionation on agarose gels containing methylmercury hydroxide and hybridization to radioactive cloned rat and mouse albumin and AFP cDNA probes. In the mouse, substantial amounts of albumin mRNA molecules were found in the yolk sac at different stages of development, while minimal levels of albumin mRNA sequences were detected in the rat yolk sac. The mouse yolk sac albumin mRNA molecules were found to be associated with the polysomes and to be functional in cell-free translation systems. In the rat, a reciprocal relationship appears to exist between the concentrations of the two mRNAs in yolk sac and embryonic liver. In contrast, in the mouse a parallel increase in both albumin and AFP mRNA levels was found in these tissues during fetal development. These results suggest that the expression of the albumin and AFP genes may be subjected to different regulatory events in these two members of the Muridae family.     

Induction of heme oxygenase in rat liver. Increase of the specific mRNA by treatment with various chemicals and immunological identity of the enzymes in various tissues as well as the induced enzymes

A specific antibody was prepared against rat liver heme oxygenase which had been induced by bromobenzene treatment. Immunochemical studies with this antibody (IgG) revealed that heme oxygenases from livers of rats treated with hemin, Cd2+, Co2+, or bromobenzene from rat spleen and also from kidney of Sn2+-treated rats were all immunochemically identical. Cell-free synthesis of heme oxygenase was performed in a rabbit reticulocyte lysate system using polysomes isolated from livers of rats treated with either hemin, Cd2+, or bromobenzene, and it was found that translatable mRNA specific for heme oxygenase was actually increased in the liver of rats treated with any of those inducers. Also, the ability of liver polysomes to direct cell-free synthesis of heme oxygenase was apparently proportional to the activity of heme oxygenase in the liver from which polysomes were prepared. The heme oxygenase protein synthesized either in vivo or in vitro showed a molecular weight of 31,000 when examined by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and fluorography. This value is essentially identical with the molecular weight of heme oxygenase purified from rat liver and indicates that a precursor form of heme oxygenase may not be involved in the heme oxygenase synthesis.     

The effect of dexamethasone treatment on the expression of the regulatory genes of ketogenesis in intestine and liver of suckling rats

The influence of the injection of dexamethasone on ketogenesis in 12 day old suckling rats was studied in intestine and liver by determining mRNA levels and enzyme activity of the two genes responsible for regulation of ketogenesis: carnitine palmitoyl transferase I (CPT 1) and mitochondrial HMG-CoA synthase. Dexamethasone produced a 2 fold increase in mRNA and activity of CPT I in intestine, but led to a decrease in mitt HMG-CoA synthase. In liver the mRNA levels and activity of both CPT I and mitt HMG-CoA synthase decreased. Comparison of these values with the ketogenic rate of both tissues following dexamethasone treatment suggests that mitt HMG-CoA synthase could be the main gene responsible for the regulation of ketogenesis in suckling rats. The changes produced in serum ketone bodies by dexamethasone, with a profile that is more similar to the ketogenic rate in the liver than that in the intestine, indicate that liver contributes more to ketone body synthesis in suckling rats. Two day treatment with dexamethasone produced no change in mRNA or activity levels for CPT I in liver or intestine. While mRNA levels for mitt HMG-CoA synthase changed little, the enzyme activity is decreased in both tissues.     

alpha-Fetoprotein and albumin mRNA levels in liver regeneration and carcinogenesis

Using a titration procedure, we measured the proportion of alpha-fetoprotein (AFP) and albumin mRNA in normal, regenerating, and preneoplastic rat livers. AFP mRNA constitutes approximately 0.006% of the polysomal polyadenylated RNA of normal livers and this proportion increases only slightly before the onset of DNA synthesis in liver regeneration induced by partial hepatectomy or CCl4 injury. In either model of liver regeneration, the proportion of AFP mRNA in polysomal RNA is highest approximately 24 h after the peak of DNA synthesis. The increase in the proportion of AFP mRNA in polysomal RNA is relatively small during liver regeneration (2-4-fold) but is larger (30-50-fold) in preneoplastic livers of rats fed a choline-deficient diet containing 0.1% ethionine. In contrast to those changes in AFP mRNA, albumin mRNA levels remain unchanged during liver regeneration and double in preneoplastic livers. Our results indicate that the concept of "retrodifferentiation" as it applies to liver regeneration and certain types of hepatic neoplasia needs reevaluation.     

Regulation of liver metallothionein and plasma zinc by the glucocorticoid dexamethasone.

Administration of the glucocorticoid dexamethasone to adrenalectomized rats significantly decreased the serum zinc concentration within 14 hr. Dexamethasone did not detectably alter the liver zinc content, but markedly increased the proportion of zinc associated with liver metallothionein. The rate of incorporation of ³⁵S-cystine into this protein was stimulated to a maximal extent 7 hr after administration of the glucocorticoid. Poly(A)⁺ mRNA from liver polysomes was isolated and translated in a cell-free protein synthesizing system. Nearly twice as much polysomal metallothionein mRNA was found 7 hr following treatment with dexamethasone. These results suggest that glucocorticoids can regulate the plasma zinc concentration by a process that is related to the biosynthesis of the hepatic zinc-binding protein, metallothionein.     

Ornithine decarboxylase activity in lymphoid tissues of rats: Effects of glucocorticoids

The activity of ornithine decarboxylase was measured in several tissues of young female rats after treatment with cortisone acetate or dexamethasone. The expected increase in activity observed in liver and kidney was in marked contrast to the profound decrease found in thymus and spleen. Initially high activity in thymus was decreased to very low levels, sometimes below the limit of the assay procedure, 5 hours after treatment with dexamethasone or cortisone. There was also a large decrease in activity of the enzyme in spleen of hormone-treated rats. In both tissues, the marked effect was still evident 12 hours after treatment.     

Poly(A) polymerase activity in ethionine-intoxicated rats: the relative effectiveness of disaggregated and intact polyribosomes as primers for poly(A) polymerase

Ethionine intoxication causes a change in the metabolism of poly(A) sequences on the 3' OH terminus of mRNA in rat liver in vivo. In an attempt to determine the factors responsible for these changes, nuclear and cytoplasmic poly(A) polymerase activities and the state of the primer were examined in vitro. Requirements for optimal enzyme activities were determined. The nuclear and cytoplasmic enzymes had different K+, Mn2+, and poly(A) primer optima. The levels of nuclear and cytoplasmic poly(A) polymerase activity were shown to decrease following ethionine intoxication. Poly(A)+ RNA isolated from the livers of saline- and ethionine-treated rats served equally well as primers for the cytoplasmic poly(A) polymerase.Disaggregated polysomes were seven times more effective as primers than were intact polysomes. The results suggest that the mRNP particle which is released from polysomes as a result of ethionine intoxication functions better as a poly(A) polymerase primer than does the intact polysome.     

The induction and characterization of rat liver stearyl-CoA desaturase mRNA

Poly(A+) RNA isolated from livers of rats induced for stearyl-CoA desaturase contains elevated levels of mRNA for this enzyme which is translated in a rabbit reticulocyte system. The protein is immunologically and by peptide fingerprinting following Staphylococcus aureus V8 protease digestion identical to the isolated enzyme and, therefore, not synthesized in a detectable larger precursor form. The desaturase mRNA is selectively translated on free cytoplasmic polysomes from rat liver and represents at least a 40-fold increase in translatable mRNA in livers of induced animals. Northern blot analysis, using a cDNA probe complementary to rat liver desaturase mRNA, demonstrated that the desaturase is encoded by a 4900-base mRNA which is elevated approximately 50-fold in induced liver.