Primary gastric B-cell lymphoma: results of a prospective multicenter study. The German-Austrian Gastrointestinal Lymphoma Study Group.

BACKGROUND & AIMS: Appropriate management of primary gastric lymphoma is controversial. This prospective, multicenter study aimed to evaluate the accuracy of endoscopic biopsy diagnosis and clinical staging procedures and assess a treatment strategy based on Helicobacter pylori status and tumor stage and grade. METHODS: Of 266 patients with primary gastric B-cell lymphoma, 236 with stages EI (n = 151) or EII (n = 85) were included in an intention-to-treat analysis. Patients with H. pylori-positive stage EI low-grade lymphoma underwent eradication therapy. Nonresponders and patients with stage EII low-grade lymphoma underwent gastric surgery. Depending on the residual tumor status and predefined risk factors, patients received either radiotherapy or no further treatment. Patients with high-grade lymphoma underwent surgery and chemotherapy at stages EI/EII, complemented by radiation in case of incomplete resection. RESULTS: Endoscopic-bioptic typing and grading and clinical staging were accurate to 73% and 70%, respectively, based on the histopathology of resected specimens. The overall 2-year survival rates for low-grade lymphoma did not differ in the risk-adjusted treatment groups, ranging from 89% to 96%. In high-grade lymphoma, patients with complete resection or microscopic tumor residuals had significantly better survival rates (88% for EI and 83% for EII) than those with macroscopic tumor residues (53%; P < 0.001). CONCLUSIONS: There is a considerable need for improvement in clinical diagnostic and staging procedures, especially with a view toward nonsurgical treatment. With the exception of eradication therapy in H. pylori-positive low-grade lymphoma of stage EI and the subgroup of locally advanced high-grade lymphoma, resection remains the treatment of choice. However, because there is an increasing trend toward stomach-conserving therapy, a randomized trial comparing cure of disease and quality of life with surgical and conservative treatment is needed.

Effects of growth factors on cell cycle arrest in dolichyl phosphate-depleted cultures.

Previously we showed that CHO cell growth is arrested in the G1 or G0 phase within 24 h after the biosynthesis of mevalonic acid is blocked. The growth-limiting factor under these conditions appeared to be dolichyl phosphate or one of its glycosylated derivatives with consequent decrease in the synthesis of N-linked glycoproteins (Doyle, J.W., and A.A. Kandutsch, 1988, J. Cell Physiol. 137:133-140; Kabakoff, B., J.W. Doyle, and A.A. Kandutsch, 1990, Arch. Biochem. Biophys. 276:382-389). We show herein that cell surface glycoproteins are depleted in the inhibited cultures and that growth arrest is delayed when supraphysiological concentrations of insulin, insulin-like growth factor-1 (IGF-1) and bFGF are added to the culture medium. Apparently an elevated level of a growth factor increases the length of time during which a threshold level of occupied receptor is maintained as the number of glycosylated receptor molecules declines. The results support the idea that cellular levels of dolichyl phosphate and its derivatives may limit cell division by controlling the numbers of functional receptors for growth factors and of other glycoproteins on the cell surface.

Hepatic uptake and metabolism of ingested 24-hydroxycholesterol and 24(S),25-epoxycholesterol.

Although two hepatic sterol metabolites, 24(S)-hydroxycholesterol and 24(S),25-epoxycholesterol, are thought to be important regulators of cholesterol biosynthesis, nothing is known of their degradation and disposal in liver, nor of the mechanisms that regulate their levels. As an initial approach to these questions the two sterols were administered intragastrically, as a bolus, to mice and their hepatic accumulation and conversion to more polar compounds were examined as a function of time. These results were compared to those obtained for cholesterol and for the unnatural epimer of one of the oxysterols, 24(R)-hydroxycholesterol. Maximum concentrations of the three oxysterols in liver were reached by approx. 4 h and then declined to control levels by 8 h. More polar neutral and acidic metabolites were found in the liver extracts. Radiolabeled oxysterols and their metabolites were found in bile glands. In comparison, the amounts of hepatic free and esterified cholesterol and of acidic products formed from it increased gradually over the measured period of time. Rates of conversion of the two 24-hydroxycholesterol epimers into acidic compounds by a liver mitochondrial fraction in vitro exceeded those of 24(S),25-epoxycholesterol and cholesterol. 24(S)-Hydroxycholesterol did not lower the level of hepatic HMG-CoA reductase activity, consistent with the absence of any significant accumulation of the free sterol. Accumulation of appreciable amounts of free 24(S),25-epoxycholesterol was associated with lowered levels of reductase. The existence of hepatic systems for the rapid inactivation and degradation of the oxysterols is consistent with their postulated role in the regulation of cholesterol synthesis.

A proteolytic fragment of the oxysterol receptor which retains oxysterol binding activity.

The structural organization of the oxysterol receptor, postulated to be involved in the regulation of 3-hydroxy-3-methylglutaryl CoA reductase and cholesterol biosynthesis in mammalian cells, has been explored by limited proteolysis with trypsin, alpha-chymotrypsin, and endoproteinase GluC. Treatment with each of these proteases converts the receptor from a homodimer of approximately 95 kDa subunits to a 44-kDa form, based on hydrodynamic measurements by sucrose density gradient centrifugation and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of photoaffinity-labeled preparations indicates that the oxysterol binding site is on a 28-kDa fragment within the 44-kDa limit form of the receptor. The limit proteolytic form exhibits the high affinity and structural specificity for oxysterols of the native dimeric receptor with an increase in the rate constant of association for 25-hydroxycholesterol. The proteolytic form also shows an increased binding affinity for nonspecific DNA, but no sequence specificity for the oxysterol regulatory element from the reductase gene was detected.

Oxygenation of desmosterol and cholesterol in cell cultures.

In order to determine whether hydration of the delta 24 bond of desmosterol contributes to the formation of the regulatory oxysterol, 25-hydroxycholesterol, [3H]desmosterol was incubated with two cultured cell lines and the labeled products were analyzed. Small amounts of 25-hydroxycholesterol were formed with Chinese hamster lung (Dede) cell cultures, but not with mouse fibroblast (L) cell cultures. Apparently, desmosterol was converted into cholesterol, a process that does not occur in L cells, before 25-hydroxycholesterol takes place. No reliable evidence could be obtained for hydration of the delta 24 bond or for the reverse reaction upon incubation of [3H]25-hydroxycholesterol. Oxygenation of desmosterol occurred in both Dede and L cell cultures to give a mixture of 24(R)- and 24(S)-25-epoxy-cholesterol. This reaction, along with the production of 7-oxygenated sterols, may account for low levels of HMG-CoA reductase repressor activity previously found to be associated with delta 24 sterols.

Relationships among dolichyl phosphate, glycoprotein synthesis, and cell culture growth.

Following treatment of Chinese hamster ovary cells with inhibitors of mevalonate biosynthesis in the presence of exogenous cholesterol, the cellular concentration of phosphorylated dolichol and the incorporation of [3H]mannose into dolichol-linked saccharides and N-linked glycoproteins declined coincident with a decline in DNA synthesis. Addition of mevalonate to the culture medium increased rates of mannose incorporation into lipid-linked saccharides and restored mannose incorporation into N-linked glycoproteins to control levels within 4 h. After an additional 4 h, synchronized DNA synthesis began. Inhibition of the synthesis of lipid-linked oligosaccharides and N-linked glycoproteins by tunicamycin prevented the induction of DNA synthesis by mevalonate, indicating that glycoprotein synthesis was required for cell division. The results suggest that the rate of cell culture growth may be influenced by the level of dolichyl phosphate acting to limit the synthesis of N-linked glycoproteins.

Purification, subunit structure, and DNA binding properties of the mouse oxysterol receptor.

A cytosolic receptor protein for oxygenated sterols, postulated to be involved in the regulation of 3-hydroxy-3-methylglutaryl-CoA reductase and cholesterol biosynthesis, has been purified from mouse L cell cytosol greater than 3,600-fold in its undenatured form and to apparent homogeneity upon further electrophoresis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified 7.5 S receptor appears to be a dimer with similar or identical subunits of Mr 95,000. Proteolytic cleavage by an endogenous factor(s) gives rise to a 4.2 S form of the receptor which is resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into a heterogeneous mixture of ligand binding fragments of Mr 30,000-60,000. This 4.2 S form of the receptor retains high affinity for the oxysterol ligand and exhibits a more rapid oxysterol binding rate than the 7.5 S form. The 7.5 S form of the receptor binds to DNA-cellulose at low salt concentrations at neutral pH, and its affinity increases at low pH or in the presence of Zn2+. Receptor preparations from mouse liver were purified approximately 900-fold by the same purification procedure, but this was accompanied by conversion of the 7.5 S liver receptor to a approximately 4 S form, Mr approximately 55,000.

Prenylated proteins: demonstration of a thioether linkage to cysteine of proteins.

Prenylated amino acid fragments have been isolated from prenylated proteins of Chinese hamster ovary cells. Gel-exclusion chromatography indicates that these proteins are modified by two different prenyl groups. The prenyl-amino acid fragments are labeled by 35S from cysteine, and this bond is cleaved by Raney-Ni, proving that the prenyl residue is linked to protein via a thioether to cysteine. Hydrazinolysis has been used to demonstrate that the cysteine is carboxy terminal.

Metabolism of 25-hydroxycholesterol in mammalian cell cultures. Side-chain scission to pregnenolone in mouse L929 fibroblasts.

Three mammalian cell lines were examined for their ability to metabolize the regulatory oxysterol, 25-hydroxycholesterol, and derepress 3-hydroxy-3-methylglutaryl CoA reductase. In mouse L cell fibroblasts reductase activity was restored with the concomitant metabolism of 25-hydroxycholesterol via side-chain hydroxylation and scission of the C20-C22 bond. Chinese hamster lung cells did not appear to derepress the reductase and these cells and Chinese hamster ovary cells did not metabolize 25-hydroxycholesterol to a significant extent. Only 5-10% of the oxysterol became esterified with a fatty acid in any of the cell lines when grown in the described culture conditions.

Oxysterol regulators of 3-hydroxy-3-methylglutaryl-CoA reductase in liver. Effect of dietary cholesterol.

Hepatic regulatory oxysterols were analyzed to determine which oxysterols were present in livers of mice fed a cholesterol-free diet and whether repression of 3-hydroxy-3-methylglutaryl-CoA reductase following cholesterol feeding was accompanied by an increase in one or more oxysterols. Analysis of free and esterified sterols from mice fed a cholesterol-free diet resulted in the identification and quantitation of six regulatory oxysterols: 24-hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol, 7 alpha-hydroxycholesterol, 7 beta-hydroxycholesterol, and 7-ketocholesterol. Following the addition of cholesterol to the diet for 1 or 2 nights, hepatic 3-hydroxy-3-methylglutaryl-CoA reductase activity declined and the levels of oxysterols, especially those of the side-chain-hydroxylated sterols, increased. Total 3-hydroxy-3-methylglutaryl-CoA reductase repressor units attributable to identified free oxysterols increased 2.5- and 6-fold after 1 and 2 nights, respectively, of cholesterol feeding. The amounts of esterified 24-, 25-, and 26-hydroxycholesterol also increased, with the increase in esterified 24-hydroxycholesterol being the greatest. The 24-hydroxycholesterol was predominantly the 24S epimer and the 26-hydroxycholesterol was predominantly the 25R epimer, indicating enzymatic catalysis of their formation. The observed correlation between increased levels of regulatory oxysterols and repression of 3-hydroxy-3-methylglutaryl-CoA reductase in cholesterol-fed mice is consistent with a hypothesis that intracellular oxysterol metabolites regulate the level of the reductase.

Requirement for mevalonate in cycling cells: quantitative and temporal aspects.

In order to investigate a requirement for isoprenoid compounds in the cell cycle, DNA synthesis was examined in cultured Chinese hamster ovary cells in which mevalonate biosynthesis was blocked with mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Treatment of exponentially-growing cultures with mevinolin led to a decline in DNA synthesis and cell cycle arrest in G1. Synchronous DNA synthesis and cell division could be restored in the arrested cultures, in the absence of exogenous mevalonate, by removing the inhibitor from the culture thereby allowing expression of an induced level of HMG-CoA reductase. In order to quantitate the mevalonate requirement for entry into S phase, recovery of DNA synthesis was made dependent upon added mevalonate by preventing the induction of the enzyme using 25-hydroxycholesterol, a specific repressor of HMG-CoA reductase synthesis. When cultures were treated with both inhibitors, optimal recovery of DNA synthesis was obtained with 200 micrograms/ml mevalonate following an 8 h lag, whereas a progressively longer lag-time was found with lower concentrations of mevalonate. Exogenous dolichol, ubiquinone, or isopentenyladenine had no effect on the arrest or recovery of DNA synthesis. Cholesterol was required during the arrest incubation for cell viability, but was not sufficient for recovery in the absence of mevalonate. The recovery of DNA synthesis by 200 micrograms/ml mevalonate, which was maximal 14-16 h after the addition of mevalonate, only required that the mevalonate be present for the first 4 h, whereas more than an 8-h incubation was required for maximal recovery with 25 micrograms/ml mevalonate. Maximal recovery at either concentration of mevalonate was achieved after approximately 400 fmol mevalonate/micrograms protein was incorporated into non-saponifiable lipids. This quantity represents approximately 0.1% of the mevalonate required for the synthesis of total cellular isoprenoid compounds. The results indicate that production of a quantitatively minor product(s) of mevalonate metabolism is required during the first 4 h following release of the block before other cellular events necessary for entry into S phase can occur.

Inhibitors of sterol synthesis. Chemical syntheses, properties and effects of 4,4-dimethyl-15-oxygenated sterols on sterol synthesis and on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured mammalian cells.

The chemical syntheses of a number of 4,4-dimethyl substituted 15-oxygenated sterols have been pursued to permit evaluation of their activity in the inhibition of the biosynthesis of cholesterol and other biological effects. Described herein are the first chemical syntheses of 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-en-3 beta-ol-15-one, 3 beta,15 alpha-diacetoxy-4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene, 3 beta-acetoxy-4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-en-15 beta-ol, 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta,15 alpha-diol, 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta,15 beta-diol, 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-en-15 alpha-ol-3-one, 3 beta-benzoyloxy-4,4-dimethyl-5 alpha-cholest-8(14)-ene-7 alpha,15 alpha-diol, 7 alpha,15 alpha-diacetoxy-3 beta-benzoyloxy-4,4-dimethyl-5 alpha-cholest-8(14)-ene, 4,4-dimethyl-5 alpha-cholest-8(14)-en-3 beta-ol-15-one and 3 beta,7 alpha,15 alpha-tri-o-bromobenzoyloxy-5 alpha-cholest-8(14)-ene. Also prepared for use in the biological experiments were 4,4-dimethyl-5 alpha-cholest-7-ene-3 beta,15 alpha-diol, 4,4-dimethyl-5 alpha-cholest-8-ene-3 beta,15 alpha-diol and 4,4-dimethyl-5 alpha-cholest-8(14)-ene-3 beta,7 alpha,15 alpha-triol. The effects of twelve 4,4-dimethyl substituted 15-oxygenated sterols and of four 4,4-dimethyl substituted 32-oxygenated sterols on sterol synthesis and on the level of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity were evaluated in mouse L cells. With the exception of 4,4-dimethyl-5 alpha-cholest-8(14)-ene-3 beta,7 alpha,15 alpha-triol, all of the 4,4-dimethyl substituted 15-oxygenated sterols caused a 50% inhibition of sterol synthesis at less than 10(-6) M and six of the 4,4-dimethyl substituted 15-oxygenated sterols caused a 50% inhibition of sterol synthesis at less than 10(-7) M. 4,4-Dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta,15 alpha-diol caused a 50% decrease in sterol synthesis at 10(-8) M. The potencies of the 4,4-dimethyl substituted 15-oxygenated and C-32-oxygenated sterols with respect to inhibition of sterol synthesis and suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity have been compared with those of the corresponding sterols lacking the 4,4-dimethyl substitution.

Photoaffinity labeling of the oxysterol receptor.

A cytosolic receptor protein for oxygenated sterols, postulated to be involved in the regulation of 3-hydroxy-3-methylglutaryl-CoA reductase and cholesterol biosynthesis, can be labeled covalently by photoactivation of 7,7'-azo-[5,6-3H]cholestane-3 beta,25-diol. Other compounds tested for their potential as photoaffinity reagents were: 25-hydroxycholesta-4,6-dien-3-one, 3 beta,25-dihydroxycholest-5-en-7-one, and 3 beta-hydroxycholesta-8(14),9(11)-dien-15-one. These sterols did not bind to the receptor with adequate affinity, were not readily photolyzed, or did not react covalently with the receptor during photolysis. The successful photoaffinity label, 7,7'-azocholestane-3 beta,25-diol, binds to the receptor with high affinity (Kd = 9.1 nM). After activation of the partially purified oxysterol-receptor complex with UV light (greater than 300 nm), several covalently labeled proteins were found upon sodium dodecyl sulfate-gel electrophoresis. Labeling of one protein, Mr approximately 98,000, was much reduced when the binding reaction was carried out in the presence of an excess of unlabeled oxysterol. Under the reaction conditions investigated so far, approximately 1% of the specifically bound sterol was covalently linked after photolysis. These results are consistent with previous information suggesting that the Mr of the receptor subunit is approximately 97,000. The covalent labeling of the receptor reported herein should facilitate its further purification and characterization.

Inhibitors of sterol biosynthesis. Synthesis and activities of ring C oxygenated sterols.

The chemical syntheses of a number of C27 ring C oxygenated sterols have been pursued to permit evaluation of their activity in the inhibition of sterol biosynthesis in cultured mammalian cells. Thus, 5 alpha-cholest-7-ene-3 beta, 11 alpha-diol, 3 alpha-hydroxy-5 alpha-cholest-9(11)-en-12-one, and the previously unreported 11 alpha-hydroxy-5 alpha-cholest-7-en-3-one, 5 alpha-cholest-9(11)-ene-3,12-dione, and 3 beta-hydroxy-5 alpha-cholest-9 (11)-en-12-one have been synthesized. The effects of these compounds on the synthesis of digitonin-precipitable sterols from labeled acetate in mouse L cells and on the levels of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in the same cells have been investigated and compared with previously published data on other ring C oxygenated sterols. 5 alpha-Cholest-7-ene-3 beta, 11 alpha-diol was shown to be the most potent inhibitor of sterol synthesis.

Accumulation of regulatory oxysterols, 32-oxolanosterol and 32-hydroxylanosterol in mevalonate-treated cell cultures.

In a previous publication (Saucier, S.E., A.A., Taylor, F.R., Spencer, T.A., Phirwa, S., and Gayen, A.K., J. Biol. Chem. (1985) 260, 14571-14579), we demonstrated that cultured Chinese hamster lung (Dede) cells contain 24(S),25-epoxycholesterol and 25-hydroxycholesterol in cellular concentrations within the range required to repress 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. In this paper, we show that the addition to the culture medium of a concentration of mevalonate high enough to repress the reductase by 90% resulted in the appearance of two new regulatory oxysterols. The two new sterols are believed to be 32-oxolanosterol and 32-hydroxylanosterol on the basis of high performance liquid chromatography (HPLC) retention times and mass spectrometric and nuclear magnetic resonance spectroscopic data and by NaBH4 reduction of the putative aldehyde to material which had the HPLC retention time of the putative alcohol. The cellular concentrations of 24(S),25-epoxycholesterol and 25-hydroxycholesterol were not significantly changed by the presence of mevalonate. However, there was approximately a 30% increase in total HMG-CoA reductase repressor units which can be ascribed to the 32-oxolanosterol and 32-hydroxylanosterol, where 1 unit equals the repressor activity of 1 ng of 25-hydroxycholesterol. These results support the idea that the level of HMG-CoA reductase activity in growing cell cultures is determined by intracellular oxysterol metabolites and that relatively small changes in their numbers or concentrations can alter the level of HMG-CoA reductase activity.

15 beta-Methyl-5 alpha,14 beta-cholest-7-ene-3 beta,15 alpha-diol. Synthesis, structure, and inhibition of sterol synthesis in animal cells.

Treatment of 3 beta-benzoyloxy-14 alpha,15 alpha-epoxy-5 alpha-cholest-7-ene with methyl magnesium iodide gave, as the major product, 15 beta-methyl-5 alpha,14 beta-cholest-7-ene-3 beta,15 alpha-diol. The product was characterized as the free sterol and in the form of its 3 beta-acetoxy and 3 beta-p-bromobenzoate derivatives. Unambiguous assignment of structure was based upon X-ray analysis of the latter derivative. 15 beta-Methyl-5 alpha,14 beta-cholest-7-ene-3 beta,15 alpha-diol was found to be a potent inhibitor of sterol synthesis in cultured mammalian cells. The 15 beta-methyl-3 beta,15 alpha-dihydroxysterol caused a 50% reduction of the level of HMG-CoA reductase activity and a 50% reduction in the incorporation of labeled acetate into digitonin-precipitable sterols in L cells at a concentration of 3.0 x 10(-6) M.

Depression of hepatic dolichol levels by cholesterol feeding.

A study was conducted to determine whether repression of 3-hydroxy-3-methylglutaryl CoA reductase by a chronic high-cholesterol diet would deplete hepatic dolichol levels. Four-week-old male C57BL/6J mice were maintained on a control diet or a diet supplemented with 5% cholesterol. Animals from both groups were killed at various times and reductase activity and levels of free dolichol, dolichyl acyl ester, dolichyl phosphate, and ubiquinone were measured. The reductase activity was reduced by 90% within 1 week and remained depressed through 56 days. Initially, the levels of the free dolichol, acyl ester, phosphoryl ester, and ubiquinone were 7, 16, 5, and 80 micrograms/g liver, respectively. Early increases in the concentration of dolichyl phosphate and free dolichol were similar in both the cholesterol-fed and control groups. However, in the cholesterol-fed group the concentration of dolichyl acyl esters was only 50% of that in the control group by 7 days and it remained lower throughout the experiment. Total dolichol levels were lower by about 30%. Ubiquinone levels were transiently depressed at 7 days by 33% but returned to control levels by 4 weeks. After 56 days, the control values of dolichol and dolichyl phosphate remained constant whereas the dolichyl acyl ester levels continuously increased to a value of 133 micrograms/g of liver by 156 days. Subcellular fractionation of livers from 4-week-old mice indicated a lysosomal distribution of dolichol and dolichyl acyl ester and a lysosomal and microsomal distribution of dolichyl phosphate.

24,25-Epoxysterol metabolism in cultured mammalian cells and repression of 3-hydroxy-3-methylglutaryl-CoA reductase.

In view of the potential importance of 24,25-epoxysterols as intracellular regulators of 3-hydroxy-3-methylglutaryl-CoA reductase, the C-24 epimers of 24,25-oxidolanosterol and 24,25-epoxycholesterol were tested for their biological activity and metabolism in cell cultures. All four compounds produced repression of the reductase in cultured mouse fibroblasts (L cells), and both 24(S)- and 24(R),25-epoxycholesterol exhibited high affinity binding to the cytosolic oxysterol-binding protein. However, binding of the epimeric 24,25-oxidolanosterols was not detected. 24(S),25-Epoxycholesterol was not rapidly metabolized in either L cells or Chinese hamster lung (Dede) cells. 24(S),25-Oxidolanosterol was rapidly converted to 24(S),25-epoxycholesterol in both cell lines. 24(R),25-Oxidolanosterol was converted to 24(R)-hydroxycholesterol in Dede cells, but was converted instead to 24(R),25-epoxycholesterol in L cells, which lack sterol delta 24-reductase activity. Although 24(S),25-oxidolanosterol does not appear to accumulate in these cell cultures, it was found in human liver in about one-fifth the amount of 24(S),25-epoxycholesterol. 24(R),25-Epoxycholesterol was also converted to 24(R)-hydroxycholesterol in Dede cells, but not in L cells. Triparanol inhibited the reduction of the 24(R),25-epoxides in Dede cells, consistent with the idea that this reaction is catalyzed by the delta 24-reductase. 24(R)-Hydroxycholesterol and its 24(S) epimer exhibited affinity for the binding protein and repressed 3-hydroxy-3-methylglutaryl-CoA reductase.

24(S),25-Epoxycholesterol. Evidence consistent with a role in the regulation of hepatic cholesterogenesis.

Previously we showed that 24(S),25-epoxycholesterol is formed from acetate, via squalene 2,3(S),22(S),23-dioxide and 24(S),25-oxidolanosterol, during the normal course of cholesterol biosynthesis in S10 rat liver homogenate (Nelson, J. A., Steckbeck, S. R., and Spencer, T. A. (1981) J. Biol. Chem. 256, 1067-1068; Nelson, J. A., Steckbeck, S. R., and Spencer, T. A. (1981) J. Am. Chem. Soc. 103, 6974-6975). Herein we demonstrate that the nonsaponifiable extract from human liver tissue contains 24(S),25-epoxycholesterol in an amount approximately 10(-3) relative to cholesterol. We show that 24(S),25-epoxycholesterol, like many other oxygenated sterols, represses hydroxymethylglutaryl-CoA reductase activity in cultured cells and binds to the cytosolic oxysterol-binding protein. Furthermore, we show that this epoxide is not rapidly metabolized in cultured cells. These results suggest that 24(S),25-epoxycholesterol may participate in the regulation of hepatic cholesterol metabolism in vivo.

Identification of regulatory oxysterols, 24(S),25-epoxycholesterol and 25-hydroxycholesterol, in cultured fibroblasts.

Biosynthetically tritiated sterols from Chinese hamster lung (Dede) cells were fractionated by high performance liquid chromatography, and fractions were assayed for their ability to repress 3-hydroxy-3-methylglutaryl-CoA reductase in L cell cultures. Most of the activity found was associated with two oxysterols, 24(S),25-epoxycholesterol and 25-hydroxycholesterol. The identities of the two sterols were established by co-chromatography with authentic samples and by isotopic dilution and recrystallization. Only low levels of repressor activity were found in other fractions of the sterol extract. The endogenous concentrations of 24(S),25-epoxycholesterol (7.2 fg/cell) and 25-hydroxycholesterol (1.5 fg/cell) appear to be within the ranges required for the regulation of HMG-CoA reductase.