Cultured Rat Hepatoma Cells Research Articles

Induction of rat liver microsomal cytochrome P-450 isozymes and epoxide hydrolase by a series of 4′-substituted-2,3,4,5-tetrachlorobiphenyls

We have shown previously that 2,3,4,5-tetrachlorobiphenyl is ineffective as an inducer of rat liver microsomal cytochrome P-450. Addition of a single para-chloro substituent in the otherwise unsubstituted phenyl ring, to give 2,3,4,4′,5-pentachlorobiphenyl, produces a potent cytochrome P-450 inducer with both phenobarbital- and 3-methylcholanthrene-type characteristics. In the present study, 2,3,4,5-tetrachlorobiphenyl was substituted in the para(4′) position with 12 other functional groups. The 4′-X-C 12H 5Cl 4 derivatives were tested as inducers of cytochromes P-450 a– P-450 e and epoxide hydrolase, by immunochemical analysis of liver microsomes prepared 4 days after a single treatment (500 μmol/kg) of 1-month-old- male Long Evans rats. When the para′ substituents was a halogen (F, Cl, Br or I), the derivative induced both cytochromes P-450 b and P-450 e, and cytochromes P-450 c and P-450 d, which are the major phenobarbital- and 3-methylcholanthrene-inducible isozymes, respectively. A similar type of induction was observed with a second group of derivatives substituted with CN, NO 2 or CF 3. However, a derivative containing CH 3CO—(which is also a meta-directing, ring-activating substituent) failed to induce cytochromes P-450 a– P-450 e at the dosage and time tested. Members of a third group of derivatives, which contained an orto/para-directing, ring-activating substituent) were either ineffective inducers (OH, CH 3, CH 3O—), or were inducers of cytochromes P-450 c and P-450 d (isopropyl or t-butyl. Hence, 4′-substitution with a bulky lipophilic substituent conferred 3-methylcholanthrene- but not phenobarbital-type characteristics on 2,3,4,5-tetrachlorobiphenyl. Some of the derivatives tested, namely those substituted with Cl, Br, I and CF 3, were remarkably effective inducers of cytochrome P-450 a, causing a 10–11-fold induction of this isozyme. Data on the induction of cytochrome P-450 c were analyzed by multiparameter linear regression in an attempt to correlate the biological activity of the 4′-X-C 12H 5Cl 4 derivatives with the physiochemical properties of the various substituents. From these results, and those reported recently, we propose that binding of the 4′-X-C 12H 5Cl 4 derivatives to the rat cytosolic Ah receptor is favored by increasing the electronegativity, lipophilicity and hydrogen bonding characteristics of the 4′ substituent, whereas enzyme induction (both in vivo and in cultured rat hepatoma cells) is also governed by a fourth characteristic, the STERIMOL factor, which gives a measure of the width of the substituent. The importance of the size of the substituent in enzyme induction, but not receptor binding, may reflect a need for the Ah receptor to undergo a ligand-induced conformational change to initiate the chain of events leading to enzyme induction.

Phenotypic variability of cultured rat hepatoma cell population in respect to alpha‐fetoprotein synthesis

McA-RH 7777 hepatoma cells were cloned on 2 occasions a week apart (test cloning). Alpha-fetoprotein (AFP) phenotypes of primary and secondary clones from 55 primary clones were estimated. High interclonal heterogeneity of AFP expression was demonstrated. This variability exceeded by several orders of magnitude the mutation rate. We also found intraclonal differences in rates of variability. A "stabilizing" cloning was also carried out, aiming at the selection of AFP+ and AFP- stable cell lines. Stabilizing cloning included 7 cycles of cloning and selection of the most phenotypically stable clone. This stability was shown to be incomplete during the maintenance of clones in culture.

A role for divalent cations in specifying the start site for transcription from chromatin templates in vitro.

An assay that employs nucleoside 5'-O-(2-thiotriphosphates) was used to detect initiation of mouse mammary tumor virus (MMTV) RNA chains in preparations of isolated nuclei from cultured rat hepatoma cells containing stably integrated proviruses. RNA chains initiated with adenosine 5'-O-(2-thiotriphosphate), guanosine 5'-O-(2-thiotriphosphate), or uridine 5'-O-(2-thiotriphosphate) were separated from the remaining RNA by mercury-Sepharose column chromatography and analyzed for correctly initiated RNA chains with a T1 nuclease protection assay. Combined use of the thionucleotide transcription reaction with the T1 nuclease assay allowed precise localization of the transcription start sites. The majority of MMTV RNA chains were initiated with guanosine 5'-O-(2-thiotriphosphate) at a template site 133 nucleotides upstream from a PvuII site that coincides with the right end of the long terminal repeat. However, some RNA chains were also initiated with adenosine 5'-O-(2-thiotriphosphate) and uridine 5'-O-(2-thiotriphosphate) at template sites within three nucleotides of the primary guanosine start site. When Mn2+ was substituted for Mg2+ in the transcription reaction, MMTV RNA chains were initiated with approximately the same efficiency, but the start site was shifted to a position approximately 40 nucleotides downstream from the physiological start site; in the presence of Mn2+, MMTV RNA chains were initiated only with guanosine 5'-O-(2-thiotriphosphate). When the nuclei were exposed to both Mn2+ and Mg2+, transcription initiated at the manganese-dependent site. Mn2+ also caused the transcription start site for 45 S pre-rRNA to shift about 10 nucleotides upstream from the physiologically correct start site.

Metformin enhances certain insulin actions in cultured rat hepatoma cells.

The effect of the oral antidiabetic agent metformin on insulin regulation of glycogen metabolism, tyrosine-aminotransferase activity, and [1-14C]aminoisobutyric acid uptake was studied in H4IIE cultured rat hepatoma cells. Metformin enhanced both basal (from 0.213 +/- 0.016 to 0.262 +/- 0.024 nmol/mg protein, p less than 0.01) and insulin stimulated [3H] glucose incorporation into glycogen in a time-dependent and dose-dependent manner. A small effect of metformin was seen at 1 mumol/l, and its greatest effects were obtained at 10 mumol/l. At the same concentrations, metformin did not influence basal tyrosine-aminotransferase activity but it potentiated insulin stimulated tyrosine-aminotransferase activity (+29.2 +/- 1.4%, p less than 0.01) and prevented the loss of tyrosine-aminotransferase responsiveness to insulin in H4IIE cells desensitised by a previous exposure to insulin. In contrast, metformin had no effect on basal or insulin-stimulated [1-14C]aminoisobutyric acid uptake. Over the concentrations of metformin that enhanced insulin action in H4IIE cells, the drug had no significant effect on insulin binding to its receptor. These studies suggest, therefore, that metformin may influence cellular metabolism by potentiating certain insulin actions through mechanisms that may be beyond insulin receptor binding.

Evidence that 2,3,7,8-tetrachlorodibenzo-p-dioxin induces NADPH cytochrome c (P-450) reductase in rat hepatoma cells in culture

The lack of aryl hydrocarbon (benzo [a] pyrene) hydroxylase (AHH) (EC 1. 14. 14. 1) induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in a clone of rat heptoma (HTC cl-1) cells is not caused by the lack of nuclear Ah receptor or by a deficiency in the activity of NADPH-cytochrome c (P-450) reductase. Treatment of HTC cl-1 cell line with TCDD for 18 h in culture resulted in a reproducible 500–600% increase in reductase activity without concomitant expression in AHH activity. These data suggests that TCDD induces cytochrome c reductase activity and that the lack of inducible AHH activity in rat hepatoma cells could reflect a defect in the structural gene (s) encoding for cytochrome P 1-450, or an Ah receptor with a faulty DNA binding domain.

Role of the 4-5S binding protein in the induction of aryl hydrocarbon hydroxylase in the rat.

Analysis of male Sprague--Dawley rat hepatic cytosol from two commercial animal laboratories for the polycyclic aromatic hydrocarbon (PAH) 4-5S binding protein showed that in one group of animals no 4-5S protein was detectable (-4S) whereas the levels of this protein were 208 +/- 57 fmol/mg cytosolic protein in the +4S rats. The role of the 4-5S binding protein in the transregulation of the cytochrome P-450-dependent monooxygenase, aryl hydrocarbon hydroxylase (AHH), was therefore investigated in the -4S and +4S Sprague-Dawley rats. The dose-response curves for the induction of hepatic microsomal AHH by 3-methylcholanthrene (MC) were indistinguishable in both +4S and -4S rats and comparable results were observed for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as an inducer. Both MC and TCDD exhibit high binding affinities for the aryl hydrocarbon (Ah) 8-9S receptor protein, whereas MC but not TCDD bound with high affinity to the 4-5S binding protein. Benzo[a]pyrene (B[a]P) binds with moderate affinity to both the Ah receptor and 4-5S binding protein and induces AHH in both -4S and +4S rats. Perylene binds with moderate affinity to the 4-5S binding protein but does not interact with the Ah receptor. This PAH was inactive as an inducer of AHH in +4S and -4S Sprague-Dawley rats. These results show that there was a correlation between the Ah receptor binding affinities of MC, B[a]P and perylene and their potencies as AHH inducers in Sprague-Dawley rats, and this corresponds to previous correlations for the induction of AHH in rat hepatoma H-4-II E cells in culture. In contrast no such correlations existed between the AHH induction potencies of these polynuclear aromatic hydrocarbons and their affinities for the 4-5S binding protein. These data, coupled with the fact that the absence of the 4-5S binding protein in the -4S Sprague-Dawley rats did not affect AHH inducibility by MC, B[a]P or perylene, suggests that the 4-5S binding protein does not play a role in the transregulation of cytochrome P-4501A1 in the rat or rat hepatoma cells in culture.

Iron regulates ferritin mRNA translation through a segment of its 5' untranslated region.

In previous studies, we showed that acute administration of iron to intact rats or to rat hepatoma cells in culture induces synthesis of the iron-storage protein ferritin by activating translation of inactive cytoplasmic ferritin mRNAs for both the heavy (H) and the light (L) subunits. In the course of activation, these ferritin mRNAs are recruited onto polysomes. To elucidate the structural features of these mRNAs involved in the translational response to iron, a chimera was constructed from the 5' and 3' untranslated regions (UTRs) of ferritin L subunit mRNA fused to the reading frame of the mRNA of bacterial chloramphenicol acetyltransferase (CAT). This chimera and deletion constructs derived from it were introduced into a rat hepatoma cell line by retrovirus-mediated gene transfer. The complete chimera showed increased CAT activity in response to iron enrichment of the medium, whereas deletion of the first 67 nucleotides of the 5' UTR, which contain a highly conserved sequence, caused loss of regulation by iron. Whereas cis-acting sequences located in the 5' flanking regions of many genes have been repeatedly implicated in modulating their transcriptional expression, we report here a specific regulatory translational sequence found within the 5' UTR of a eukaryotic mRNA.

Regulation of genes for glycolytic enzymes in cultured rat hepatoma cell lines.

We examined the control by hormones and culture conditions of the expression of pyruvate kinase L, aldolase B, and a liver-specific 5.4-kb mRNA species [Pichard, A. L. et al. (1985) Biochem. J. 226, 637-644] in three rat hepatoma cell lines, MH1C1, Fao and Faza. The expression level of these markers ranges from 2% (for pyruvate kinase L mRNA) to 10-12% (for 5.4-kb mRNA species) of the glucose-induced mRNA values found in rat liver. The mRNAs of the three liver-specific genes strongly decrease after treatment of the hepatoma cells with cyclic 8-bromo-AMP, cyclic dibutyryl-AMP or forkolin, pyruvate kinase L mRNA being the most sensitive to this inhibiting effect. In contrast, the concentration of pyruvate kinase L mRNA nuclear precursors is not modified by the cyclic AMP analogues, indicating that these agents do not act at the transcriptional level but, instead, probably destabilize the transcripts. Glucose or fructose does not modify the expression of these three marker genes in any of the studied cell lines. Insulin is inefficient in modifying concentrations of the mRNAs for pyruvate kinase L and aldolase B, alone or in the presence of carbohydrates. In contrast, it stimulates about fivefold the expression of the 5.4-kb mRNA species in the MH1C1 cell line; this stimulation is carbohydrate-independent. The hepatoma cell lines mimic, therefore, the effect of cyclic AMP on the inhibition in vivo of the expression of genes encoding glycolytic or lipogenic enzymes [Vaulont, S. et al. (1984) Biochem. Biophys. Res. Commun. 125, 135-147]. In contrast, the effect of carbohydrates [Munnich, A. et al. (1984) J. Biol. Chem. 259, 10228-10231] is undetectable. The insulin sensitivity of the liver-specific genes is conserved for the 5.4-kb mRNA species only, especially in the MH1C1 cell line, but not for the other investigated mRNAs, which seems to reflect a fundamental difference in the in vivo effect of insulin on these genes. Finally, S1 nuclease mapping of the start-site of pyruvate kinase L gene transcription shows that the normal site used in vivo is also used in the Fao and Faza lines while, in the MH1C1 line, it coexists with multiple aberrant upstream initiation sites.

Translation of ferritin light and heavy subunit mRNAs is regulated by intracellular chelatable iron levels in rat hepatoma cells.

Acute administration of iron to rats has been previously shown to induce liver ferritin synthesis by increasing the translation of inactive cytoplasmic ferritin mRNAs for both heavy (H) and light (L) subunits by mobilizing them onto polyribosomes. In this report rat hepatoma cells in culture are used to explore the relationship of this response to intracellular iron levels. After adding iron as ferric ammonium citrate to the medium, latent ferritin H- and L-mRNAs were extensively transferred to polyribosomes, accompanied by increased uptake of [35S]methionine into ferritin protein. Because total cellular levels of L- and H-mRNA were not significantly changed by exposure to iron, the increased ferritin mRNAs on polyribosomes most probably come from an inactive cytoplasmic pool, consistent with the inability of actinomycin-D and of cordycepin to inhibit iron-induced ferritin synthesis. When deferoxamine mesylate, an intracellular iron chelator, was added after the addition of iron to the medium, ferritin mRNA on the polyribosomes was reduced, while the free messenger pool increased, and ferritin synthesis diminished. In contrast, the extracellular iron chelator diethylenetriaminepentaacetic acid failed to inhibit the induction of ferritin protein synthesis. Addition of iron in the form of hemin also caused translocation of mRNA to polyribosomes, a response that could be similarly quenched by deferoxamine. Because hemin does not release chelatable iron extracellularly, we conclude that the level of chelatable iron within the cell has a regulatory role in ferritin synthesis through redistribution of the messenger RNAs between the free mRNA pool and the polyribosomes.

Concanavalin A changes not only the number of insulin binding sites but also the binding affinity in rat hepatoma cells in culture.

When rat hepatoma cells (R-Y121B) were first incubated with labeled insulin, followed by concanavalin A, at 500 micrograms/ml at 23 degrees C, the total cell-associated radioactivity increased. At 4 degrees C, however, this increase was not observed. Scatchard analysis revealed that concanavalin A increased insulin binding affinity. The bound insulin was internalized together with concanavalin A. When the cells were incubated with concanavalin A prior to insulin addition, however, the total cell-associated radioactivity decreased at both temperatures. This was caused by the masking of insulin binding sites by the lectin.

Increase in Insulin Binding Affinity by Chloroquine in Cultured Rat Hepatoma Cells

Chloroquine increased the binding of 125I-labeled insulin to rat hepatoma cells (R-Y121B) at 4 degrees C. The effect of chloroquine on insulin binding was amplified at 23 degrees C, and a large increase in cell-associated radioactivity was observed. Scatchard analysis showed that chloroquine increased the affinity of insulin for cells at both 4 degrees C and 23 degrees C, and that this increase eventually caused the accumulation of insulin internalized by cells at 23 degrees C.

Cytotoxic activity of polyindoline alkaloids of Psychotria forsteriana (Rubiaceae) (1).

We have shown that the planar formula of the polyindoline alkaloids (C, B, E and D) isolated from the leaves of Psychotria forsteriana agree respectively with Quadrigemine A, Quadrigemine B, Psychotridine, and Isopsychotridine C. Their cytotoxic activity on cultured rat hepatoma cells (HTC line) is reported in this paper. These alkaloids showed a higher toxicity on HTC cells than vincristine, a bisindole alkaloid currently used in antitumor chemotherapy.

Formation of functional asialoglycoprotein receptor after transfection with cDNAs encoding the receptor proteins.

The rat asialoglycoprotein receptor (ASGP-R) has been expressed in cultured rat hepatoma cells (HTC cells) after transfection with cloned cDNAs. Fluorescence-activated cell sorting of transfected cells was used to identify the functional cDNA clones and to isolate cells expressing the ASGP-R. Simultaneous or sequential transfections with two cloned cDNAs that encode related but distinctive polypeptide chains were needed to obtain ASGP-R activity; transfection with either cDNA alone failed to produce detectable ASGP-R. The affinity of transduced ASGP-R for asialo orosomucoid is less than that of the native rat ASGP-R, and the number of surface receptors in clones expressing ASGP-R is about one-fifth that found on rat hepatocytes.

Lipid peroxidation and activities of tyrosine aminotransferase and glutamine synthetase in hepatoma and glioma cells grown in bovine colostrum-supplemented medium.

The growth stimulating properties of bovine serum and colostrum were compared in rat hepatoma (HTC) and glioma (C6) cell cultures. A colostrum concentration of 2% was optimal for HTC cells, which then reached a terminal density 40% of that in serum-supplemented medium. The corresponding figures for C6 cells were 10 and 81%, respectively. After 4 d in culture, levels of lipid hydroperoxides were measured and compared. Highest levels of lipid hydroperoxides were found in HTC and C6 cells grown in unsupplemented medium. HTC and C6 cells grown in serum supplemented medium contained levels of 52 and 64%, respectively, of that in unsupplemented medium. The corresponding levels for cells grown in presence of colostrum were 40% for HTC and 44% for C6 cells. To obtain information on any functional alterations in the cells due to the presence of colostrum the induction of tyrosine aminotransferase (EC 2.6.1.5) and glutamine synthetase (EC 6.3.1.2) by dexamethasone was studied. Although colostrum seemed to increase the basal activities of the enzymes, no significant effects on the degree of induction could be detected.

An ultrastructural study of rat hepatoma cells in culture, their variants and revertants

We compared the ultrastructure of a well-differentiated rat hepatoma line (H4II) and its clonal progeny, including dedifferentiated variant cells, and revertants of the variants in which the spectrum of hepatocyte-specific functions is again expressed. The cells of the original differentiated lines and the revertants were very similar to one another. In addition, they exhibited some of the characteristics of fetal and neonatal hepatocytes. Variant cells which fail to express hepatocyte functions showed a wide range of morphological alterations accompanied by generalized disorganization. It is concluded that the loss of hepatocyte differentiation in the variants is not associated with a uniform morphological type, and that a wide range of ultra-structural phenotypes can be generated in the progeny of a single neoplastic but well-differentiated hepatocyte. Also, the expression of hepatocyte functions only occurs within a limited and organized morphological framework that includes features of young hepatocytes.

Plasma-protein production by rat hepatoma cells in culture, their variants and revertants

A series of subclones of the H4II line of the Reuber H35 rat hepatoma produce substantial amounts of three plasma proteins, transferrin, α 1-antitrypsin and fibrinogen. Immunocytochemical staining demonstrated that each of these proteins is synthesized by essentially every cell of these cell populations. Cells of dedifferentiated variant clones either cease to produce the proteins, or exhibit a substantial reduction that is accompanied by variability in the synthetic activity of individual cells of the population, As previously observed with regard to angiotensinogen production, the variant clones clearly divide into two categories: those that show only a reduction in synthesis are able to give rise to revertants, whereas the negative clones fail to do so. Revertant cells exhibit a dramatic restoration of the synthesis of plasma proteins, which in some cases, exceeds by severalfold the rates seen in the differentiated clones of origin. In addition, the revertant cells synthesize α-fetoprotein, a function that is not expressed by H4II cells or its daughter subclones. Immunocytochemical staining revealed that, with regard to several plasma proteins including albumin, fibrinogen and α-fetoprotein, the cell populations of revertant clones are very heterogeneous, for only a fraction of the cells synthesizes each protein. Hybrid cells resulting from several types of crosses, exhibited extinction of the plasma proteins, the exception being transferrin, whose production was maintained, but at a reduced level and in only a fraction of the cells. Taken together, our results show that the expression of albumin and transferrin can be dissociated from one to another, and from that of fibrinogen, α 1-antitrypsin and angiotensinogen.

Trace element uptake in liver cells. 2. Effect of different proteins in the medium on the uptake of copper and zinc by hepatoma cells

Cultured rat hepatoma cells (HTC-cells) were used to study the uptake of copper and zinc from a minimal salt-glucose medium, supplemented with albumin from different species or with ovalbumin. Competitive equilibrium dialysis showed that at low molar ratios of metal/protein ( < 1) the affinity for copper of human and bovine albumin was about equal, but that of dog albumin or ovalbumin was much lower. Only a small difference in affinity for zinc could be detected between human albumin and ovalbumin. Supplementing the medium with the different proteins the rate of copper uptake in the cell at a given molar Cu/protein ratio increased as follows: human albumin ⋍ bovine albumin < dog albumin < ovalbumin. When the molar Cu/protein ratio was increased, a discontinuity was seen with all three albumin species at a ratio of about 1. In contrast, the zinc uptake mimics that of Cu/ovalbumin, and no discontinuity was observed using different molar Zn/protein ratios. These results indicate that the rate of copper and zinc uptake depends strongly on its affinity for the protein: a low affinity leads to a high uptake. The results suggest further that at physiologic concentrations zinc is taken up by a mechanism different from that for copper.

Mechanism of the hepatic lipase induced accumulation of high-density lipoprotein cholesterol by cells in culture.

Hepatic lipase can enhance the delivery of high-density lipoprotein (HDL) cholesterol to cells by a process which does not involve apoprotein catabolism. The incorporation of HDL-free (unesterified) cholesterol, phospholipid, and cholesteryl ester by cells has been compared to establish the mechanism of this delivery process. Human HDL was reconstituted with 3H-free cholesterol and [14C]sphingomyelin, treated with hepatic lipase in the presence of albumin to remove the products of lipolysis, reisolated, and then incubated with cultured rat hepatoma cells. Relative to control HDL, modification of HDL with hepatic lipase stimulated both the amount of HDL-free cholesterol taken up by the cell and the esterification of HDL-free cholesterol but did not affect the delivery of sphingomyelin. Experiments utilizing HDL reconstituted with 14C-free cholesterol and [3H]cholesteryl oleoyl ether suggest that hepatic lipase enhances the incorporation of HDL-esterified cholesterol. However, the amount of free cholesterol delivered as a result of treatment with hepatic lipase was 4-fold that of esterified cholesterol. On the basis of HDL composition, the cellular incorporation of free cholesterol was about 10 times that which would occur by the uptake and degradation of intact particles. The preferential incorporation of HDL-free cholesterol did not require the presence of lysophosphatidylcholine. To correlate the events observed at the cellular level with alterations in lipoprotein structure, high-resolution, proton-decoupled 13C nuclear magnetic resonance spectroscopy (90.55 MHz) was performed on HDL3 in which the cholesterol molecules were replaced with [4-13C]cholesterol by particle reconstitution.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure-function relationships of retinoids in their effects on retinol-binding protein metabolism in cultured H 4II EC 3 liver cells

Studies were conducted to explore the structural features of retinoids that may be required to stimulate the secretion or production of retinol-binding protein (RBP) by H 4II EC 3 rat hepatoma cells in culture. Sixteen retinoids, that differed from all- trans-retinol in the cyclohexene ring, the polyene side chain, and/or the functional end group, were each incubated with H 4II EC 3 cells, and RBP secretion and accumulation were determined by radioimmunoassay. A number of retinoids, in addition to retinol, effectively stimulated RBP secretion. The results suggest that an intact cyclohexene ring may be necessary for the stimulation of RBP secretion. In contrast the system did not exhibit much specificity with regard to either the structure of the side chain or the nature of the end group. No relationship was found between the ability of a retinoid to stimulate RBP secretion and production and its biological activity. The biologically active retinoid, 13- cis-retinoic acid, was inactive in the present system, whereas the biologically inactive perhydromonoeneretinol was moderately effective in stimulating both RBP secretion and accumulation. In contrast, there appeared to be some relationship between the ability of different retinoids to stimulate RBP secretion and their ability to bind to RBP. In general, retinoids that had previously been shown to bind to RBP produced a greater stimulation of RBP secretion than those that did not bind to RBP. The secretion of RBP obtained with a given retinoid was not well correlated with the net accumulation of RBP. For example, retinoyl amide did not stimulate RBP secretion but was moderately effective in stimulating RBP accumulation. Thus, the secretion of RBP does not appear to be necessary for the stimulation of the net accumulation of RBP.

Association of the glucocorticoid hormone receptor with ribonucleic acid

The hypothesis that the glucocorticoid hormone receptor interacts with RNA has been tested in cultured rat hepatoma cells. The receptor was covalently labeled with radioactive dexamethasone mesylate, and putative RNA-receptor complexes were stabilized by either cell-free crosslinking using formaldehyde or irradiation of intact cells. After chemical cross-linking in vitro, the receptor displayed the buoyant density of a ribonucleoprotein in CsCl gradients. After photochemical crosslinking in cells labeled with radioactive uridine, the receptor analysed by polyacrylamide gel electrophoresis was carrying labeled ribonucleotides.