Sterol-independent regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in tumor cells.

Elevated 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase expression supports synthesis of prenyl pyrophosphate intermediates required for tumor growth. In this study, the copy number of HMG-CoA reductase mRNA was determined in solid tumor and leukemic cell lines using competitive reverse transcriptase-polymerase chain reaction. Reductase mRNA was increased about eight-fold in Caco2 human colon adenocarcinoma cells compared with that in CCD18 normal colon cells. We also found a 50-fold enhancement of reductase mRNA in stimulated human lymphocytes compared with unstimulated cells. In CEM human leukemia cells, reductase mRNA was increased 8.6 times compared with that in stimulated lymphocytes. Greater low density lipoprotein receptor mRNA was also observed in tumor cells compared with normal counterparts. We hypothesized that elevated reductase mRNA was due to attenuation of sterol-mediated control of tumor reductase promoter activity. We first compared the methylation status of CpG dinucleotides in the promoters of reductase and p16 tumor suppressor genes from solid tumor, leukemic, and normal cells. As reported for other tumor cells the p16 promoter region was hypermethylated in Caco2 and CEM cells but was hypomethylated in corresponding normal cells. However, reductase promoter sequences in both normal and tumor cells were hypomethylated, demonstrating that methylation is not involved in sterol-independent reductase regulation. We addressed altered transcription factor binding to the tumor cell reductase promoter by transiently transfecting Caco2 and CCD18 with a plasmid vector containing a hamster HMG-CoA reductase promoter fused to the luciferase gene. We found that increased reductase mRNA was partially due to an approximately three-fold higher reductase promoter activity in Caco2 than in CCD18, measured by luciferase reporter assays. Thus, differential binding of transcription factor or factors on the tumor cell reductase promoter attenuates normal sterol-mediated regulation of reductase activity.

Isoprenoid-mediated inhibition of mevalonate synthesis: potential application to cancer.

Pure and mixed isoprenoid end products of plant mevalonate metabolism trigger actions that suppress 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase activity. These actions modulate HMG CoA reductase mRNA translation and the proteolytic degradation of HMG CoA reductase. Such post-transcriptional events, we propose, are activated directly by acyclic isoprenoids and indirectly by cyclic isoprenoids. Isoprenoids, acting secondarily to the dominant transcriptional effector of sterologenesis, modestly lower cholesterol levels, if and only if, sterologenesis is not repressed by a saturating imput of dietary cholesterol. An anomaly associated with tumor growth-a sterol feedback-resistant HMG CoA reductase activity-ensures a pool of sterologenic pathway intermediates. Such intermediates provide lipophilic anchors essential for membrane attachment and biological activity of growth hormone receptors, nuclear lamins A and B, and oncogenic ras. Tumor HMG CoA reductase retains high sensitivity to the isoprenoid-mediated secondary regulation. Repression of mevalonate synthesis by plant-derived isoprenoids reduces ras and lamin B processing, arrests cells in G1, and initiates cellular apoptosis. This unique tumor cell-specific sensitivity allows isoprenoids to be used for tumor therapy, an application emulating that of the statins, but one free of adverse effects. When evaluated at levels provided by a typical diet, isoprenoids individually have no impact on cholesterol synthesis and tumor growth. Nonetheless, isoprenoid-mediated activities are additive, and, sometimes synergistic. Therefore, the combined actions of the estimated 23,000 isoprenoid constituents of plant materials, acting in concert with other chemopreventive phytochemicals, may explain the lowered cancer risk associated with a diet rich in plant products. In contrast, that lowering of cancer risk does not correspond to supplemental intake of other dietary factors associated with fruits, vegetables, and cereal grains, namely fiber, beta-carotene, vitamin C, and vitamin E, and only weakly to supplemental folate.

Down-regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA levels and synthesis in syrian hamster C100 cells by the oxidosqualene cyclase inhibitor [4'-(6-allyl-ethyl-amino-hexyloxy)-2'-fluoro-phenyl]-(4-bromophenyl)-me thanone (Ro 48-8071): comparison to simvastatin.

In vivo inhibition of 2,3-oxidosqualene:lanosterol cyclase (OSC, E.C. 5.4.99.7)--the enzyme which catalyzes the cyclization of monooxidosqualene to lanosterol--does not result in elevated 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) activity. This trait is attributed to increased levels of oxysterols, produced upon partial inhibition of OSC, that suppress HMGR and other sterol-responsive genes. The OSC inhibitor [4'-(6-allyl-ethyl-amino-hexyloxy)-2'-fluoro-phenyl]-(4-bromopheny l)-methanone (Ro 48-8071) was shown earlier to lower low-density lipoprotein (LDL) cholesterol in hamsters with no increase in hepatic HMGR, in contrast to simvastatin. To delineate the regulatory mechanism(s) by which Ro 48-8071 reduces cholesterol synthesis without raising HMGR levels, Syrian hamster C100 cells were incubated with either Ro 48-8071 or simvastatin, and their effects on cholesterol synthesis and LDL uptake, as well as on HMGR mRNA levels and rates of synthesis, were determined. Using RNase protection and radioimmunoprecipitation assays, we found that, in the absence of LDL in the culture medium, both HMGR mRNA levels and synthesis were reduced with concentrations of Ro 48-8071 inhibiting cholesterol synthesis by 50-75%, whereas LDL uptake was either reduced or unchanged. In contrast, simvastatin, at concentrations inhibiting cholesterol synthesis by the same 50-75%, increased both HMGR mRNA levels and synthesis, as well as LDL uptake. In the presence of LDL, HMGR mRNA levels and synthesis along with LDL uptake were little affected after incubation with Ro 48-8071. Still, simvastatin markedly increased both HMGR mRNA levels and synthesis in cells incubated in the presence of LDL, leaving LDL uptake unaffected. These data suggest that inhibition of OSC by Ro 48-8071 results in an indirect down-regulation of HMGR mRNA levels and synthesis.

In vivo expression of an alternatively spliced human tumor message that encodes a truncated form of cathepsin B. Subcellular distribution of the truncated enzyme in COS cells.

Cathepsin B is a lysosomal cysteine protease whose increased expression is believed to be linked to the malignant progression of tumors. Alternative splicing and the use of alternative transcription initiation sites in humans produce cathepsin B mRNAs that differ in their 5'- and 3'-untranslated ends. Some human tumors also contain cathepsin B-related transcripts that lack exon 3 which encodes the N-terminal signal peptide and 34 of the 62-amino acid inhibitory propeptide. In this study we show that one such transcript, CB(-2,3), which is missing exons 2 and 3, is likely to be a functional message in tumors. Thus, CB(-2,3) was found to be otherwise complete, containing the remainder of the cathepsin B coding sequence and the part of the 3'-untranslated region that is common to all previously characterized cathepsin B mRNAs in humans. Its in vitro translation product can be folded to produce enzymatic activity against the cathepsin B-specific substrate, Nalpha-benzyloxycarbonyl-L-Arg-L-Arg-4-methylcoumaryl-7-amide. Endogenous CB(-2,3) from the metastatic human melanoma cell line, A375M, co-sediments with polysomes, indicating that it engages the eukaryotic translation machinery in these cells. Epitope-tagged forms of the truncated cathepsin B from CB(-2,3) are produced in amounts comparable to the normal protein after transient transfection into COS cells. Immunofluorescence microscopy and subcellular fractionation show this novel tumor form of cathepsin B to be associated with nuclei and other membranous organelles, where it is likely to be bound to the cytoplasmic face of the membranes. This subcellular distribution was different from the lysosomal pattern shown by the epitope-tagged, full-length cathepsin B in COS cells. These results indicate that the message missing exons 2 and 3 is likely to be translated into a catalytically active enzyme, and that alternative splicing (exon skipping) could contribute to the aberrant intracellular trafficking of cathepsin B that is observed in some human cancers.

Inhibition of squalene synthase but not squalene cyclase prevents mevalonate-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase synthesis at a posttranscriptional level.

Previously, we found that mevalonate-derived products together with an oxysterol regulated reductase synthesis at a posttranscriptional level. To determine which products were responsible for this regulation, either the squalene synthase inhibitor zaragozic acid A or the squalene cyclase inhibitor 4,4,10-beta-trimethyl-trans-decal-3beta-ol (TMD) was added to lovastatin-treated Syrian hamster cells in conjunction with mevalonate. Mevalonate alone decreased reductase synthesis 50% compared with lovastatin-treated cells. In contrast, when both zaragozic acid A and mevalonate were added to lovastatin-treated cells, there was no change in reductase synthesis. With either treatment, reductase mRNA levels did not change compared with lovastatin-treated cells. When both 25-hydroxycholesterol and mevalonate were added to lovastatin-treated cells, reductase synthesis and mRNA levels were decreased 95 and 50%, respectively. The 10-fold difference between changes in reductase synthesis and mRNA levels under these conditions reflects a specific effect of mevalonate-derived isoprenoids on reductase synthesis at the translational level. In contrast, coincubation of cells with mevalonate plus 25-hydroxycholesterol in the presence of zaragozic acid decreased reductase synthesis and mRNA levels 60 and 50%, respectively, compared with lovastatin-treated cells. Moreover, degradation of reductase was increased approximately 7-fold in cells treated with mevalonate alone but only 3-fold in cells treated with mevalonate and zaragozic acid A. These results indicate that isoprenoid products between mevalonate and squalene affect reductase at a posttranslational level by increasing degradation but do not regulate reductase synthesis at a posttranscriptional level. In contrast, when both TMD and mevalonate were added to lovastatin-treated cells, reductase synthesis was decreased approximately 50% with no corresponding decrease in reductase mRNA levels, similar to mevalonate only. Reductase degradation was increased approximately 7-fold under these conditions. Cellular incubation in TMD, mevalonate, and 25-hydroxycholesterol decreased reductase synthesis and mRNA levels 95 and 50%, respectively. From these results we concluded that mevalonate-derived nonsterols synthesized between squalene and lanosterol decrease reductase synthesis at a translational level-either alone or in combination with 25-hydroxycholesterol-and also increase reductase degradation.

The length of 5'-untranslated leader sequences influences distribution of 3-hydroxy-3-methylglutaryl-coenzyme A reductase mRNA in polysomes: effects of lovastatin, oxysterols, and mevalonate.

Transcripts for hamster 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase are heterogeneous in length. This heterogeneity is due to variations in the length of 5'-untranslated leader (UTL) sequences, which are generated by both alternate splicing within the first exon as well as alternate transcription start sites. Because mRNA 5'-UTL sequences have a role in regulating translational efficiency, the level and distribution of HMG-CoA reductase transcripts were measured in both total cellular RNA and polysomes from the Syrian hamster cell line C100. Cells were treated with either lovastatin alone, lovastatin and 25-hydroxycholesterol (25-OH C), or lovastatin, 25-OH C, and mevalonate, three treatment regimens used in an earlier study to demonstrate nonsterol-mediated translational control of HMG-CoA reductase synthesis [D. M. Peffley (1992) Somat. Cell Mol. Genet. 18, 19-32]. When reductase mRNA was measured by 5'-extension analysis under the same conditions, levels of transcripts with 5'-UTL regions ranging from 41 to 81 bases were reduced approximately four- to eightfold. In contrast, transcripts with 5'-UTL regions 93 to 100 bases in length were not reduced, and transcripts with 5'-UTL regions approximately 300-400 bases in length increased twofold. The addition of 25-OH C alone or both 25-OH C and mevalonate to lovastatin-treated cells lowered HMG-CoA reductase mRNA levels fivefold in total cellular RNA as determined by RNase protection assay. No comparable change was observed with control ribosomal protein S17 mRNA. Postmitochondrial supernatants representing both translationally inactive monosomes and translationally active polysomes were prepared by sucrose gradient fractionation from cells incubated with the standard three treatments. Because 5'-UTL sequences of many mRNAs have a role in regulating translational efficiency we isolated RNA from each fraction and measured levels of reductase transcripts by 5'-extension analysis. Under all three conditions, transcripts with 5'-UTL sequences 41-103 bases in length were primarily associated with dense sucrose fractions that contain polysomes. In contrast, reductase transcripts with leader sequences 300 to 400 bases were almost exclusively associated with the less dense sucrose fractions containing monosomes. These results indicate that both the level and polysome distribution of individual reductase transcripts are influenced by the length of 5'-UTL sequences.

Mevalonate regulates polysome distribution and blocks translation-dependent suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA: relationship to translational control.

We reported previously that 3-hydroxy-3-methylglutaryl coenzyme A reductase synthesis is regulated at the translational level by mevalonate. To determine at what stage mevalonate affects reductase synthesis, we examined the distribution of reductase mRNA in polysomes from cells treated with lovastatin alone; lovastatin and 25-hydroxycholesterol; or lovastatin, 25-hydroxycholesterol, and mevalonate. In lovastatin-treated cells, reductase mRNA was primarily associated with heavy polysome fractions. When 25-hydroxycholesterol was added to lovastatin-treated cells, reductase mRNA levels were reduced approximately fourfold in all polysome fractions, with no accompanying redistribution of reductase mRNA into lighter polysome fractions. However, addition of both 25-hydroxycholesterol and mevalonate to lovastatin-treated cells shifted reductase mRNA from heavier to lighter polysome fractions. No change in the distribution of control beta-actin or ribosomal protein S17 mRNA occurred with any of the treatments. These results suggest that mevalonate suppresses reductase synthesis at the level of initiation. When the translation inhibitor cycloheximide was added to all three regimens, reductase mRNA shifted into heavy polysome fractions. Treatment with either lovastatin alone or lovastatin plus 25-hydroxycholesterol resulted in a 50% greater loss of reductase mRNA from the heavy polysome fractions compared to the same fractions from noncycloheximide-treated cells. No loss of reductase mRNA occurred when cycloheximide was added to cells treated with both 25-hydroxycholesterol and mevalonate. beta-Actin mRNA levels and polysome distribution were not significantly changed by cycloheximide under any of these conditions. Translationally mediated suppression of reductase mRNA did not occur when protein synthesis was inhibited with puromycin. Our results indicate that regulation of reductase mRNA levels is translation-dependent and is linked to the rate of elongation.

3'-untranslated sequences mediate post-transcriptional regulation of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA by 25-hydroxycholesterol.

In an earlier study [Choi, Lundquist and Peffley (1993) Biochem. J. 296, 859-866], we determined that 25-hydroxycholesterol regulates 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase mRNA through a post-transcriptional mechanism that requires protein synthesis. To investigate whether 3'-untranslated sequences play a role in 25-hydroxycholesterol-mediated post-transcriptional control, we ligated approx. 1400 bp of the 3'-untranslated region of HMG-CoA reductase cDNA to the coding region of human beta-globin DNA. beta-Globin-3'-untranslated reductase fusion constructs were then transiently expressed in Chinese hamster ovary fibroblasts under conditions known to regulate reductase mRNA. There were no differences in beta-globin RNA levels in transfected cells incubated with or without lovastatin, a competitive inhibitor of reductase. However, in the presence of lovastatin and an oxysterol, 25-hydroxycholesterol, beta-globin RNA levels were decreased approx. 2-fold. Inhibition of protein synthesis with cycloheximide blocked the effects of 25-hydroxycholesterol on beta-globin RNA. Moreover, replacing the 3'-untranslated sequences with 1367 bp of the simian virus 40 enhancer region eliminated the regulatory effect of 25-hydroxycholesterol. Because the fusion construct has no sterol regulatory elements necessary for transcription, our results indicate that the change in beta-globin RNA occurred at a post-transcriptional level. In addition, we have shown that the 3'-untranslated region of HMG-CoA reductase cDNA imparted oxysterol-mediated post-transcriptional regulation to beta-globin RNA, an effect that required protein synthesis.

The appearance, distribution, and longevity of receptor-[125I]T3 complexes within the nuclei of isolated rat hepatocytes.

The nuclei of isolated rat hepatocytes were separable into three receptor compartments based upon their differential salt extractabilities: nucleoplasmic receptors (NP) extractable with 0.15 M KCl, high-salt extractable receptors (HSE) extractable with 0.4 M KCl, and salt-resistant receptors (SR) extractable with 0.4 M KCl/5 mM dithiothreitol. The receptor distribution among the three compartments was approximately NP, 45%; HSE, 30%; SR, 25%. The mean percent occupancy with endogenous T3 of the SR receptors (86%) was higher than the occupancies of the NP receptors (68%) and the HSE receptors (63%). When hepatocytes were pulsed with 3 nM [125I]T3 at 37 degrees C for brief intervals, receptor-[125I]T3 complexes were detectable in all three nuclear compartments within 15 sec. With increasing pulse intervals up to 120 sec, the receptor content of each nuclear compartment increased progressively and without evidence of preferential accumulation in any of the three compartments. To determine the life span and intercompartmental "migration" pattern of nuclear receptors, hepatocytes were pulsed with 3 nM [125I]T3 at 37 degrees C for 2.5 min or 5 min, followed by a chase with a 500-fold excess of nonlabeled T3. The population of receptor-[125I]T3 complexes generated during the pulse was serially recovered at increasing intervals after the chase. The complexes of each compartment dissociated with a half-life of approximately 3 min and manifested no predilection to accumulate in any of the compartments. Exposure of isolated hepatocytes to 3 nM T3 for 5 min or 10 min at 37 degrees C induced no change in the gross intercompartmental distribution of receptors compared to control hepatocytes incubated without T3.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of protein synthesis in baby-hamster kidney cells blocks oxysterol-mediated suppression of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA at a post-transcriptional level.

The effects of the protein-synthesis inhibitor cycloheximide on 25-hydroxycholesterol-mediated suppression of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase mRNA levels were evaluated in the baby-hamster kidney cell line C100. Cells cultured in medium supplemented with delipidized fetal bovine serum and 25 microM lovastatin for 12-24 h had a 5-fold higher level of HMG-CoA reductase mRNA than cells grown in medium supplemented with non-delipidized fetal bovine serum (FBS). The higher level was due to increased transcription, as determined by run-on assays with isolated nuclei. Addition of 25-hydroxycholesterol to lovastatin-treated cells lowered HMG-CoA reductase mRNA levels within 4 h of treatment to those of cells grown in FBS-supplemented medium. This decrease was due in part to a decrease in gene transcription. Cycloheximide added in conjunction with 25-hydroxycholesterol to lovastatin-treated cells blocked the suppression of mRNA levels, but did not block oxysterol-mediated suppression of transcription. In addition, cycloheximide added to cells grown in FBS-supplemented medium rapidly increased mRNA levels by 10-fold relative to untreated cells, with no comparable increase in transcription. No comparable increase in either the mRNA level or rate of transcription for beta-actin was observed under such conditions. These results indicate that cycloheximide specifically stabilizes HMG-CoA reductase mRNA in the presence of oxysterols and suggests that continuous synthesis of a short lived protein regulator is required for oxysterol-mediated suppression of HMG-CoA reductase mRNA at a post-transcriptional level.

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase synthesis in Syrian hamster C100 cells by mevinolin, 25-hydroxycholesterol, and mevalonate: the role of posttranscriptional control.

C100 is a baby hamster kidney cell line that expresses high levels of HMG-CoA reductase relative to its parental cell line SV28. In this study the effects of the oxysterol 25-hydroxycholesterol and mevalonate on the mRNA level and rate of synthesis for HMG-CoA reductase were evaluated in C100 cells treated with mevinolin, a competitive inhibitor of HMG-CoA reductase. The addition of 25-hydroxycholesterol to the cell culture medium resulted in a fourfold decrease in both the rate of synthesis and mRNA level for HMG-CoA reductase. Mevalonate at a concentration of 0.4 mM, when added to mevinolin-treated C100 cells, produced no apparent reduction in HMG-CoA reductase mRNA levels and only a small (25%) decline in HMG-CoA synthesis. Mevalonate (0.4 mM) added to 25-hydroxycholesterol-treated cells resulted in no further reduction in the HMG-CoA reductase mRNA level when compared to cells treated with 25-hydroxycholesterol alone, but produced an additional 30-fold decrease in the rate of HMG-CoA reductase synthesis. Degradation of HMG-CoA reductase was rapid in the presence (t1/2 = 1.34 h) or absence (t1/2 = 1.17 h) of mevinolin and was not changed significantly by adding either 25-hydroxycholesterol, alone (t1/2 = 1.30 h) or both 25-hydroxycholesterol and mevalonate (t1/2 = 1.30 h) to mevinolin-treated cells. This study demonstrates that mevalonate and 25-hydroxycholesterol act synergistically in the presence of mevinolin to achieve a greater degree of suppression in the rate of HMG-CoA reductase synthesis than can be accounted for by their individual effects on HMG-CoA reductase mRNA. In addition, the data suggest that mevalonate affects the synthesis of HMG-CoA reductase at a yet unidentified posttranscriptional control site.

A putative tRNATrp gene cloned from Dictyostelium discoideum: its nucleotide sequence and association with repetitive deoxyribonucleic acid.

Using a total tRNA population labeled with 32P, we have cloned a number of tRNA genes from Dictyostelium discoideum. A partial sequence of a cloned 1250-base-pair DNA insert, pDT-513, revealed the occurrence of a putative tRNATrp gene. In addition to the cloverleaf secondary structure, the tRNATrp gene contained all of the invariant and semiinvariant residues found in most tRNA sequences and has a 13-base-pair intron which is located one base removed from the 3' residue of the anticodon. The genomic distribution of the tRNA gene and its flanking sequences was examined via Southern annealing experiments. The structural gene is represented on at least six EcoRI fragments in the D. discoideum genome. Sequences flanking the 5' terminus of the cloned gene are repeated many times in the genome while the sequence flanking the 3' terminus of the pDT-513 DNA insert structural tRNA gene is present only once in the genome.