PubMed Health⌕ Search

Biomedical subjects

C J Steer

Publications and source records attributed to C J Steer.

At least 19 recordsLinked to original sources
PubMed1997

A novel link between REC2, a DNA recombinase, the retinoblastoma protein, and apoptosis.

The REC2 recombinase is essential for recombinational repair following DNA damage as well as for successful meiosis and gene targeting in the corn smut Ustilago maydis. Here we report that overexpression of REC2 induced apoptotic cell death in human HuH-7, Hep G2, and Hep 3B hepatoma cells. Apoptosis was related to recombinase activity and was significantly increased by inhibition of retinoblastoma (Rb) expression with transforming growth factor-beta1. REC2-induced apoptosis was associated with a significantly reduced percentage of cells in the G1 phase of the cell cycle and a significant reduction in G2/M only in the Rb(-/-) Hep 3B cells. Overexpression of REC2 resulted in increased abundance of the hyperphosphorylated form of Rb. However, by immunoprecipitation REC2 was associated primarily with hypophosphorylated Rb, suggesting that REC2 may be involved in modulating the phosphorylation state of Rb. The A and B pocket domains with the spacer amino acid sequence and the carboxyl-terminal region of Rb were required for maximal binding to REC2. Overexpression of Rb significantly inhibited REC2-induced apoptosis even in the presence of transforming growth factor-beta1. Taken together, these data suggest a novel interaction of Rb with the recombinase REC2 and a role for this complex in bridging DNA recombination and apoptosis.

Apoptosis↗
PubMed1997

Developmental regulation of CRD-BP, an RNA-binding protein that stabilizes c-myc mRNA in vitro.

We previously isolated and characterized a coding region determinant-binding protein (CRD-BP) that might regulate c-myc mRNA post-transcriptionally. CRD-BP binds specifically to the coding region of c-myc mRNA and might stabilize c-myc mRNA in vitro by protecting it from endonucleolytic cleavage. Since c-myc abundance is regulated during embryonic development and cell replication, we investigated whether CRD-BP is also regulated in animal tissues. We focused on CRD-BP expression during rat liver development and liver regeneration, because c-myc mRNA is regulated post-transcriptionally in both cases. CRD-BP expression parallels c-myc expression during liver development; the protein is present in fetal and neonatal liver but is absent or in low abundance in adult liver. In contrast, the up-regulation of c-myc mRNA following partial hepatectomy is not accompanied by up-regulation of CRD-BP. To our knowledge, CRD-BP is the first example of a putative mammalian mRNA-binding protein that is abundant in a fetal tissue but either absent from or scarce in adult tissues. Its expression in fetal liver and in transformed cell lines suggests CRD-BP is an oncofetal protein.

Animals↗
PubMed1997

The effect of changes in hepatocyte membrane potential on immediate-early proto-oncogene expression following partial hepatectomy in rats.

The stimulus responsible for inducing hepatocytes to enter the cell cycle following partial hepatectomy (PHx) remains to be identified. One suggested candidate is the change in hepatocyte membrane potential (PD) that occurs immediately following PHx. To test this possibility, we monitored changes in hepatocyte PD and immediate-early proto-oncogene expression in rats pretreated with saline or gamma aminobutyric acid (GABA), an amino-acid neurotransmitter that hyperpolarizes isolated hepatocytes. Intraperitoneal injections of saline or GABA (500 microg/g body weight) were administered to adult, male Sprague-Dawley rats 1 hour prior to 70% PHx. Rats were sacrificed and the livers excised at various times until 180 minutes post-PHx for messenger RNA (mRNA) and protein analyses. In additional groups of saline- and GABA-treated rats, PD changes were recorded continuously from -260 to 180 minutes post-PHx. Serum GABA concentrations were determined by ion-exchange chromatography with orthopthaldehyde fluorescence detection. Hepatocyte PD's were recorded in situ by intracellular microelectrodes with an Axoprobe-1A amplifier. Steady-state levels of c-fos, c-jun, jun-B, and c-myc transcripts and proteins were documented by Northern blots of poly(A)-enriched RNA derived from resected livers and Western blots of total nuclear protein, respectively. Serum GABA concentrations remained unchanged in saline-treated controls but increased 10- to 20-fold above baseline values in GABA-treated rats. In saline-treated controls, hepatocyte depolarization occurred immediately and was maintained throughout the 180 minutes post-PHx period (PD pre-PHx, -36.8 +/- 5.1; 15 minutes post-PHx, -27.5 +/- 5.7; and 180 minutes post-PHx, -28.3 +/- 4.4 mV, mean +/- SD); whereas in GABA-treated rats, hepatocyte PD remained unchanged (-37.0 +/- 1.1; -36.4 +/- 3.1 and -39.2 +/- 2.7 mV, respectively). Despite abrogation of hepatocyte PD changes, proto-oncogene mRNA and protein levels in saline- and GABA-treated rats were either similar or, in the case of c-fos and c-jun, increased five- to sevenfold in GABA-treated rats. The results of this study indicate the following: 1) hepatocytes depolarize immediately post-PHx and remain depolarized throughout the priming phase of the cell cycle; 2) elevated serum GABA concentrations prevent PHx-induced hepatocyte depolarization; and 3) depolarization is not the stimulus responsible for priming hepatocytes into replicative competence.

Animals↗
PubMed1997

Targeted nucleotide exchange in the alkaline phosphatase gene of HuH-7 cells mediated by a chimeric RNA/DNA oligonucleotide.

Although a variety of methods has been devised for modification of hepatic genes, none has been effective for long-term correction of genetic disorders. In this study, we employed a recently described novel experimental strategy for site-directed nucleotide exchange in genomic DNA of HuH-7 human hepatoma cells. A chimeric 2'-O-methylated-RNA/DNA oligonucleotide containing sequences complementary to 25 bases of the alkaline phosphatase gene was constructed as a duplex containing a G to A substitution at nucleotide 935. Cells were transfected with oligonucleotides for 48 hours, then harvested for DNA isolation and polymerase chain reaction (PCR) amplification of exon 6 of the alkaline phosphatase gene. Colony lifts were hybridized to 17 mer 32P-labeled oligonucleotide probes specific to the 935-G and 935-A sequences. Hybridizing colonies were grown, plasmid DNA isolated, and sequenced. Transfection efficiency was determined at 24 hours by nuclear uptake of fluorescein-12-dUTP-labeled chimeric oligonucleotides. Colonies hybridizing with the 935-A probe were identified only from cells transfected with the specific chimeric oligonucleotide; and there was no evidence of cross-hybridization. Conversion of G to A at nucleotide 935 occurred at an overall frequency of up to 11.9% and when corrected for transfection efficiency approached 43%. No other alterations were detected in the sequence of exon 6 with the targeted nucleotide exchange. These results show that a single base pair alteration in the alkaline phosphatase gene of HuH-7 cells can be introduced at a relatively high frequency following transfection with chimeric RNA/DNA oligonucleotides. This technique offers a novel and potentially powerful strategy for site-directed hepatic gene alteration without the use of viral-based vectors.

Alkaline Phosphatase↗
PubMed1996

The retinoblastoma gene product inhibits TGF-beta1 induced apoptosis in primary rat hepatocytes and human HuH-7 hepatoma cells.

Transforming growth factor-beta1 (TGF-beta1) can induce rapid growth arrest and apoptosis in hepatic cells. Its growth suppressive effects appear to be linked to decreased phosphorylation of the protein product of the retinoblastoma gene, pRb. To characterize the role of pRb in apoptosis, we examined endogenous retinoblastoma gene (Rb) expression following treatment with TGF-beta1, okadaic acid, or antisense Rb S-oligonucleotides in cultured primary rat hepatocytes and human hepatoma HuH-7 cells. We also investigated the effects on apoptosis of Rb overexpression following transfection with vectors containing wild-type Rb in HuH-7 cells. Our results indicated that transfection with Rb antisense S-oligonucleotides blocked the expression of pRb in cultured primary hepatocytes and induced apoptosis. Treatment of HuH-7 cells with TGF-beta1 inhibited expression and phosphorylation of pRb, and also induced apoptosis. Furthermore, 93% of viable preapoptotic cells were arrested in the G1 phase of the cell cycle. Incubation with the phosphatase inhibitor okadaic acid maintained pRb in its phosphorylated state, and resulted in significant apoptosis. Overexpression of wild-type Rb inhibited TGF-beta1 induced apoptosis in HuH-7 cells. In contrast, overexpression of transcription factor E2F-1, a known target for the activity of pRb, caused significant apoptosis. However, coexpression of Rb suppressed E2F-1 induced apoptosis in HuH-7 cells. Our results suggest that inhibition of pRb expression is associated with hepatocyte apoptosis. Furthermore, E2F-1 appears to be a target in the pathway through which pRb modulates the apoptotic threshold in hepatic cells. Finally, the data suggest that these cells exit the cell cycle during the G1 phase before progressing into apoptosis and pRb may be a negative regulator of this process.

Animals↗
PubMed1996

Posttranscriptional regulation of gene expression in liver regeneration: role of mRNA stability.

The rentry of hepatocytes and nonparenchymal cells from the normal quiescent G0 phase into the cell cycle during liver regeneration after 70% partial hepatectomy results in the discrete modulation of mRNA transcripts for many different genes. The modulation of steady-state levels of transcripts for genes involved in hepatocyte growth and replication during liver regeneration indicates that gene expression is regulated not only transcriptionally but also posttranscriptionally. In fact, posttranscriptional control appears to be the primary mechanism of regulating gene expression after the first 3 h after partial hepatectomy. Alteration in transcript stability is a key posttranscriptional regulatory mechanism used by the regenerating liver to modulate the steady-state transcript levels of multiple genes. Even genes that are transcriptionally activated during liver regeneration exhibit posttranscriptional control at the level of transcript stability. Moreover, the abundance of mRNA binding proteins, as well as translational activity and rate of poly(A) tail removal, are modulated and appear to influence transcript stability during liver regeneration. However, alteration of transcript stability is not the sole posttranscriptional mechanism regulating steady-state levels. Posttranscriptional control also occurs at the level of alternative splicing, stabilization of heterogeneous nuclear (hn) RNA, and hnRNA processing. Moreover, the role of nucleocytoplasmic transport of mature mRNA during liver regeneration is still undefined. Thus, during liver regeneration gene expression is regulated at multiple levels after the initial synthesis of hnRNA. By understanding the role of posttranscriptional mechanisms in regulating steady-state transcript levels in an in vivo model of normal growth, we will begin to appreciate its role in the genesis of abnormal growth.

Animals↗
PubMed1996

Alterations in mRNA stability during rat liver regeneration.

We examined the in vivo transcriptional and posttranscriptional regulation of various genes involved in hepatocyte growth and replication that exhibited changes in steady-state mRNA levels after 70% partial hepatectomy (PH). Of the 19 genes examined by nuclear run-on assay, 17 demonstrated no change in transcriptional activity through the first 96 h of regeneration. However, results from in vivo half-life determinations indicated that changes in mRNA stability played a critical role in regulating transcript levels during liver regeneration. For many of the genes, alterations in transcript abundance correlated with similar changes in mRNA half-lives. Inhibition of protein synthesis by cycloheximide was generally associated with increased levels of mRNA expression, but no detectable changes in transcriptional rates in both control and regenerating rat liver. Finally, genomic methylation status was investigated by Southern analysis for several genes that displayed changes in mRNA stability. Interestingly, increases in mRNA half-lives for the genes p53, c-myc, H-ras, and ornithine decarboxylase were associated with decreased genomic methylation. In conclusion, regulation of gene expression beyond the immediate early phase of te cell cycle during rat liver regeneration after PH occurs predominantly at the posttranscriptional level. mRNA stability appears to be a significant factor in this control, and may itself be modulated by the methylation status of the corresponding genomic DNA.

Animals↗
PubMed1996

Formation of delta 22-bile acids in rats is not gender specific and occurs in the peroxisome.

We recently demonstrated that the formation of delta 22-bile acids is a quantitatively major pathway for normal bile acid synthesis in the adult male Sprague-Dawley rat. This pathway is specific for 7 beta-hydroxy bile acids and, when ursodeoxycholic acid is administered, delta 22-ursodeoxycholic acid appears as a major metabolite in the liver tissue, bile, intestinal contents, and plasma. The aims of this study were, therefore, to determine whether this metabolic pathway was gender specific, and to establish that the peroxisome is a site of formation of delta 22-bile acids. Bile acids were determined by gas chromatography-mass spectrometry in liver tissue, jejunum, and plasma of adult female rats and in animals fed a diet containing 0.4% and 1% ursodeoxycholic acid. Bile acid metabolism in female rats was found to be similar to that of male rats, and delta 22-beta-muricholic acid, rather than beta-muricholate, was likewise confirmed as the major muricholic acid synthesized. Ursodeoxycholic acid administration resulted in the appearance of delta 22-ursodeoxycholic acid as a major metabolite. When adult male Sprague-Dawley rats were treated with clofibrate, a drug that induces peroxisomal proliferation, liver weight increased 40-60% and total bile acid synthesis decreased markedly, but the relative composition of individual bile acids was unchanged. When ursodeoxycholic acid was added to the diet, the proportion of delta 22-bile acids relative to the corresponding saturated analogues increased significantly compared with untreated rats, indicating that clofibrate had "amplified" the pathway for formation of delta 22-bile acids. When UDCA was incubated in vitro with a peroxisomal-enriched fraction from normal adult male rat liver, delta 22-ursodeoxycholic acid was formed in proportions comparable to that observed in vivo when this bile acid was given orally. These studies establish that the pathway for the formation of delta 22-bile acids is not gender specific and mainly occurs in hepatic peroxisomes.

Animals↗
PubMed1996

Differential regulation of cyclin B1 RNA and protein expression during hepatocyte growth in vivo.

Cyclin genes and their products are important regulatory participants in the eukaryotic cell cycle. It is well established that cyclin B1 protein forms a complex with cyclin-dependent kinase 1 (CDK1), which, when activated, initiates mitosis. We have previously established that cyclin B1 gene expression is posttranscriptionally regulated in regenerating rat liver after 70% partial hepatectomy (PH). We now report further characterization of cyclin B1 gene expression, as well as that of CDK1 and cdc25B, in this unique in vivo model of cell proliferation. Cyclin B1 transcripts were detected by RNase protection through 96 h of liver regeneration and exhibited dramatic changes in steady-state levels. Peak expressions occurred at 24-30 h, more significantly at 42-48, and at 72 h. By Northern blot analysis, single transcripts for CDK1 and cdc25B were detected, and the temporal expression of both transcripts during liver regeneration mirrored that of cyclin B1. By Western blot and immunohistochemical analyses, cyclin B1 protein levels did not change significantly in either nuclear or cytoplasmic fractions, whereas CDK1 protein levels paralleled their associated RNA expression. Cdc25B protein levels steadily decreased from 0 to 96 h after PH. In addition, cytoplasmic protein levels of cyclin B1 exhibited a constant distribution in subfractions of microsome- and polysome-associated and free proteins. Cyclin B1 RNA also localized to these three cytoplasmic subfractions. Finally, the apparent translational activity of cyclin B1 transcripts was very similar at both 24 and 48 h after PH, in contrast to their respective mRNA half-lives. In a peroxisome proliferation model of hepatocyte growth and apoptosis, cyclin B1 and CDK1 proteins were induced in the absence of transcript up-regulation. Our results demonstrate that cyclin B1 mRNA steady-state levels are regulated posttranscriptionally in regenerating rat liver. Furthermore, the pattern of cyclin B1 transcript expression is paralleled by that of the CDK1 gene, whereas their respective protein steady-state levels provide a striking contrast. Finally, cyclin B1 is differentially regulated by an uncoupling of transcript abundance and translational processing in two in vivo models of hepatocyte growth. The abundance of cyclin B1 protein in nonreplicating cells suggests that cyclin B1 may be available for other cellular pathways in the hepatocyte.

Animals↗
PubMed1996

Modulation of apoptosis-associated genes bcl-2, bcl-x, and bax during rat liver regeneration.

Liver regeneration (LR) after 70% partial hepatectomy (PH) represents a unique in vivo model of cell cycle and gene regulation. This study was conducted to characterize apoptosis-associated gene expression during LR. The results indicated that transcripts for both bcl-x and bcl-2 exhibited similar patterns of expression during LR with peaks at 6 h post-PH. In contrast, the major 1.1-kb bax transcript exhibited peaks at 18 (P < 0.05) and 72 h (P < 0.001) post-PH. Nuclear run-on analyses for all three genes indicated no detectable transcription rate changes during LR. At 6 h post-PH, when bcl-x mRNA levels were increased by 25-fold (P < 0.001), bcl-x mRNA half-life was elevated 4-fold (P < 0.001). Similarly, bax transcript half-life increased from 2.8 h at 0 h to 4.3 h at 24 h (P < 0.001) and > 8 h at 40 h (P < 0.001) post-PH, coincident with increases in steady-state levels of mRNA. Western blot analyses of Bcl-2 and Bcl-x proteins showed no significant change through 96 h of LR, whereas Bax protein levels cycled in parallel with its mRNA. Interestingly, novel Bax- and Bcl-2-cross-reactive proteins of 31 and 32 kDa, respectively, were detected in nuclei isolated from quiescent liver. When liver growth was induced by the peroxisome proliferator clofibrate, transcript and protein levels were coupled for bcl-x but not for bax. In conclusion, the apoptosis-associated genes bcl-2, bcl-x and bax are modulated at the transcript and protein levels during LR, suggesting a role for these gene products in normal liver growth. The alterations in transcript levels occur posttranscriptionally and involve changes in mRNA stability. Furthermore, unlike bax, steady-state protein and transcript levels are uncoupled for both bcl-2 and bcl-x, suggesting a role for translational regulation during LR after PH.

Animals↗
PubMed1995

delta 22-Ursodeoxycholic acid, a unique metabolite of administered ursodeoxycholic acid in rats, indicating partial beta-oxidation as a major pathway for bile acid metabolism.

We describe for the first time the identification of 3 alpha, 7 beta-dihydroxy-5 beta-chol-22-en-24-oic acid (delta 22-UDCA) in the plasma, bile, intestinal contents, and liver tissue of Sprague-Dawley rats after intravenous and oral administration of ursodeoxycholic acid (UDCA). Infusion of [2,2,4,4-2H4]UDCA confirmed delta 22-UDCA to be a specific metabolite of UDCA. Definitive confirmation of this unique and major metabolite was established by liquid secondary ionization mass spectrometry and gas chromatography-mass spectrometry by comparison of the retention index and mass spectrum with an authentic standard of delta 22-UDCA. When rats were fed a diet containing 1.0% UDCA, high concentrations of delta 22-UDCA were found in the plasma (40.3 +/- 11.8 mumol/L) and liver tissue (300.9 +/- 64.2 nmol/g of tissue), and these represented 36% and 57%, respectively, of the UDCA concentration. In animals fed 0.4% and 1.0% UDCA, the mass of delta 22-UDCA in the jejunum was high (7.5 +/- 0.9 and 6.6 +/- 0.6 mg, respectively), accounting for 50-60% of the total UDCA, but diminished markedly along the intestine, accounting for < 3% of the total UDCA in the colon. Although delta 22-UDCA was not found in biological samples from control rats, delta 22-beta-muricholic and delta 22-omega-muricholic acids were normal constituents of plasma and intestinal contents and were major muricholate isomers in liver tissue and bile. Synthesis of delta 22-bile acids appears to be highly specific toward bile acids possessing a functional 7 beta-hydroxyl group. We presume that, in common with pathways for endogenous bile acid synthesis, partial side-chain oxidation of UDCA occurs in the peroxisome with formation of alpha/beta unsaturation; since UDCA has only a 5-carbon side chain, release of propionic or acetic acid is not possible, beta-oxidation proceeds no further, and delta 22-UDCA is formed. While the mechanism of formation and physiological significance of delta 22-bile acids remain to be established, our data indicate that partial beta-oxidation is a quantitatively important pathway for endogenous bile acid synthesis and for UDCA metabolism in this species.

Animals↗
PubMed1995

Tauroursodeoxycholate increases rat liver ursodeoxycholate levels and limits lithocholate formation better than ursodeoxycholate.

BACKGROUND & AIMS: To explain the greater hepatoprotective effect of tauroursodeoxycholic acid vs. ursodeoxycholic acid, the absorption, hepatic enrichment, and biotransformation of these bile acids (250 mg/day) were compared in rats. METHODS: Bile acids were determined in intestinal contents, feces, urine, plasma, and liver by gas chromatography-mass spectrometry. RESULTS: The concentration of ursodeoxycholate in the liver of animals administered tauroursodeoxycholic acid (175 +/- 29 nmol/g) was greater (P < 0.05) than in animals administered ursodeoxycholic acid (79 +/- 19 nmol/g). Hepatic lithocholate was substantially higher after ursodeoxycholic acid administration (21 +/- 10 nmol/g) than after tauroursodeoxycholic acid administration (12 +/- 1 nmol/g). A concomitant reduction in the proportion of hydrophobic bile acids occurred that was greatest during tauroursodeoxycholic acid administration. In the intestinal tract, the mass of ursodeoxycholate and its specific metabolites was greater in rats administered tauroursodeoxycholic acid (27.2 mg) than those administered ursodeoxycholic acid (13.2 mg). In feces, the proportion of lithocholate was 21.9% +/- 4.9% and 5.4% +/- 4.0% after ursodeoxycholic acid and tauroursodeoxycholic acid administration, respectively. CONCLUSIONS: Compared with ursodeoxycholic acid, tauroursodeoxycholic acid induces a greater decrease in the percent composition of more hydrophobic bile acids within the pool, limits lithocholate formation, and increases hepatic ursodeoxycholate concentration. These differences are explained by increased hepatic extraction and reduced intestinal biotransformation and not by enhanced absorption of the amidated species.

Absorption↗
PubMed1995

The site-specific delivery of ursodeoxycholic acid to the rat colon by sulfate conjugation.

BACKGROUND & AIMS: Because ursodeoxycholate has been shown to act as a tumor-suppressive agent in the colon, the absorption and metabolism of its sulfate conjugates were examined in rats to show that sulfation would facilitate the site-specific delivery of ursodeoxycholate to the colon. METHODS: Bile acids were measured in intestinal contents, feces, urine, plasma, and liver tissue after oral administration of ursodeoxycholate and its C-3, C-7, and C-3,7 sulfate derivatives. RESULTS: Ursodeoxycholate was found in the jejunum after administration of all bile acids, but the mass was greatest for ursodeoxycholic acid administration. In the colon, lithocholic acid, normally found in negligible amounts, became the major bile acid after ursodeoxycholate administration. In contrast, reductions in mass and proportions of lithocholate and deoxycholate occurred after administering the C-7 sulfates. The fecal lithocholate/deoxycholate ratio, a risk marker for colon cancer, increased markedly after administration of ursodeoxycholate and its C-3 sulfate, but did not change after administering the C-7 sulfates. Unlike ursodeoxycholate or its C-3 sulfate, which increased liver concentrations of lithocholate and ursodeoxycholate, the C-7 sulfates had the opposite effect, which was consistent with poor absorption. CONCLUSIONS: Sulfation of ursodeoxycholate, specifically at the C-7 position, protects the molecule from bacterial degradation and inhibits its intestinal absorption, thereby facilitating delivery to the colon.

Animals↗
PubMed1995

Regulation of bile acid synthesis by deoxycholic acid in the rat: different effects on cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase.

We examined the effects of feeding deoxycholic acid (1% and 0.4% of diet), alone and in combination with ursodeoxycholic acid, on serum and biliary bile acid concentrations, hepatic morphology, and the activities and steady-state messenger RNA (mRNA) levels of HMG-CoA reductase and cholesterol 7 alpha-hydroxylase in the rat. Feeding 1% deoxycholic acid increased serum bile acid concentrations (cholestasis), produced portal triad inflammation, bile duct proliferation, and severe hepatocyte necrosis with nuclear pleomorphism. Hepatic damage was prevented when ursodeoxycholic acid (1%) was combined with the deoxycholic acid (1%), or when deoxycholic acid intake was reduced to 0.4%. HMG-CoA reductase and cholesterol 7 alpha-hydroxylase activities were markedly inhibited (-56% and -55%, respectively) with either 1% or 0.4% deoxycholic acid. Ursodeoxycholic acid alone produced an insignificant decline in HMG-CoA reductase and cholesterol 7 alpha-hydroxylase activities, and when combined with 1% deoxycholic acid did not lessen the inhibitory effect of the latter. Steady-state mRNA levels increased 20-fold for HMG-CoA reductase and 53-fold for cholesterol 7 alpha-hydroxylase in rats fed 1% deoxycholic acid. In contrast, 0.4% deoxycholic acid decreased HMG-CoA reductase mRNA levels 76%, and cholesterol 7 alpha-hydroxylase mRNA levels 82%. Ursodeoxycholic acid alone did not affect HMG-CoA reductase or cholesterol 7 alpha-hydroxylase steady-state mRNA levels. Steady-state mRNA levels and activities of sterol 27-hydroxylase, a key enzyme in the alternative acidic pathway of bile acid synthesis, did not change with either high or low doses of deoxycholic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Posttranscriptional regulation of mRNA levels in rat liver associated with deoxycholic acid feeding.

We investigated the effects of bile acid feeding on the mRNA levels and transcriptional activity of genes involved in various facets of hepatic cell function. Rats were maintained for 10 days on standard diet supplemented with combinations of 1 and 0.4% deoxycholic acid and ursodeoxycholic acid. Significant reductions in mRNA levels for liver fatty acid binding protein, albumin, the asialoglycoprotein receptor, connexins 32 and 26, and cytochromes P-450IIB1 and P-450IIE1 were associated with 1% deoxycholic acid feeding. Conversely, the 1% deoxycholic acid-fed animals exhibited increased mRNA levels for cholesterol 7 alpha-hydroxylase, 3-hydroxy-3-methylglutaryl-CoA reductase, multidrug resistance, procollagens, extracellular matrix, protooncogenes, tumor suppressors, and cyclins. The 0.4% deoxycholic acid-fed animals exhibited increased mRNA levels for c-jun, H-ras, p53, cyclins D1 and D3, fibronectin, and procollagens alpha 1(I) and alpha 1(III). Transcriptional rate changes could not account for the observed changes in steady-state mRNA levels. Ursodeoxycholic acid feeding had no significant effect on gene expression and almost completely inhibited the changes associated with 1% deoxycholic acid when coadministered. The results indicate that dietary ingestion of deoxycholic acid profoundly affects hepatic gene expression in the rat, and regulation occurs primarily at the posttranscriptional level.

Animals↗
PubMed1995

Modulation of retinoblastoma and retinoblastoma-related proteins in regenerating rat liver and primary hepatocytes.

Protein expression of the retinoblastoma (Rb) tumor suppressor gene product was examined by immunoblot analysis of nuclei isolated from regenerating rat liver after 70% partial hepatectomy (PH). Levels were almost undetectable in quiescent 0-h livers but increased 15- to 60-fold 3 to 24 h post-PH, 105-fold at 30 h, and 20- to 50-fold at 60 to 72 h post-PH. Expression returned to near baseline levels at 18, 42, and 48 h post-PH. A similar pattern of Rb protein expression in the regenerating liver was observed by indirect immunofluorescence microscopy, with peak nuclear expression at 30 h post-PH. Rb-related proteins with apparent molecular masses of 300, 156, and 74 kDa were detected in regenerating liver using mAbs to the Rb protein. Their expression increased 6- to 8-fold during regeneration, and only p156 returned to baseline levels at 60 h post-PH. Rb and its related proteins were detected in cultured primary hepatocytes, and although total protein levels did not change appreciably, there was a dramatic shift from cytosol into nuclei through 96 h. The half-life of the Rb protein was determined to be 1.9 h in regenerating liver and 2.2 h in cultured primary hepatocytes. Rb protein abundance in synchronized HuH-7 human hepatoma cells was cell cycle dependent and exhibited peak nuclear expression during S phase. Rb protein was detected primarily in its hyperphosphorylated state during liver regeneration and through the cell cycle of the HuH-7 cells. In vivo administration of transforming growth factor beta 1, an inhibitor of DNA synthesis in regenerating liver, resulted in reduced expression of Rb as well as its protein partners, cell cycle-dependent kinase 4 and cyclin E. The results suggest that in the regenerating rat liver and in synchronized HuH-7 cells, expression of Rb protein is modulated in a cell cycle-dependent fashion, remains primarily in a hyperphosphorylated state, and exhibits a relatively short half-life. The inhibition of Rb protein expression by transforming growth factor beta 1 may be linked to its simultaneous suppression of cell cycle-dependent kinase 4 and cyclin E protein levels.

Animals↗