Effect of β-carotene on the expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase in rat liver

Cancer Letters 96 (1995)

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201-208

CAKE& LETTERS

Effect of p-carotene on the expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase in rat liver Fernando S. Morenoav*, Maria Rosaria Rossiellob, Sharmila Manjeshwarb, Ravindra Nathb, Prema M. Raob, Srinivasan Rajalakshmib, Dittakavi S.R. Sarmab aDepartamento de Alimentos e Nutricao, Experimental, Faculdade de Citncias, Farmac&ticas, Universidade de S:o Paula, Av. Lineu Prestes 480, SCTOPaul0 06389-970, Brazil bDepartment of Pathology, Medical Sciences Building. University of Toronto, Toronto, Ontario, M5S IA8. Canada

Received10 May 1995;revision received25 July 1995;accepted26 July 1995

Abstract

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), is a rate-limiting enzyme in the biosynthesis of not only cholesterol but also a variety of non-sterol isoprenoids. It is subjected to multivalent feedback suppression by transcriptional and post-transcriptional control mechanisms mediated by sterols and non-sterol substances. In the present study, the effect of a plant isoprenoid, &carotene, on the expression of HMG-CoA reductase in rat liver was investigated. In control rats the hepatic levels of mRNA transcripts of HMG-CoA reductase increased following U3 partial hepatectomy with two peaks, one at 8 h and the other at 24 h. Administration of the carotenoid (70 mg/kg, given every alternate day for 3 consecutive weeks) partially inhibited the increase in the transcript level with a 50% reduction at 8 h and 30% reduction at 24 h post partial hepatectomy. Nuclear run-off assays with nuclei isolated from the resting liver and from livers of control rats and rats exposed to B-carotene for 3 consecutive weeks and killed 8 h after partial hepatectomy indicated that p-carotene did not inhibit the rate of transcription of HMG-CoA reductase gene. These observations suggest that B-carotene regulates the expression of HMG-CoA reductase by some post-transcriptional mechanisms. Ikyvordr:

Rat

liver; Partial hepatectomy; &Carotene; HMG-COA reductase; Gene expression

1. Introduction 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase; mevalonate:NADPt oxidoreductase (CoA-acylating), EC 1.1.1.34) catalyzes the conversion of HMG-CoA to mevalonate, a precursor not only for sterols like cholesterol, but also for non-sterol isoprenoids such as ubiquinone, doli-. * Corresponding

author.

0304-3835/95/$09.50 0 1995 Elsevier SSDI 0304-3835(95)03933-N

Science

Ireland

chol, isopentenyladenine, and prenyl moieties of proteins. It is a classic example of a rate-limiting enzyme in a biosynthetic pathway that is controlled by end-product repression [ 11. HMG-CoA reductase activity in animal cells is subject to multivalent control and is sensitive to negative regulation by both sterol and non-sterol products of mevalonate metabolism [2]. The available evidence suggests roles for both transcriptional and post-transcriptional mechanisms for the multivalent regulation of HMG-

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CoA reductase [3-53. Sterol and non-sterol metabolites act at different levels. Sterols partially repress transcription through an effect on the sterol regulatory element-l (SRE-1) and non-sterol isoprenoids further reduce the enzyme by inhibiting translation of the mRNA. In addition, degradation of the reductase protein is accelerated by sterol and non-sterol products acting together [2,3]. A direct association between mevalonate synthesis, cholesterol homeostasis and cell proliferation has also been demonstrated by several investigators [6,7]. Cholesterol is required for cell growth during the early Gl phase of the cell cycle, whereas a non-sterol metabolite of mevalonate allows entry into the DNA synthetic phase [8]. However, unlike normal cells, both preneoplastic and neoplastic lesions of the liver exhibit a loss of down-regulation of HMG-CoA reductase and of cholesterogenesis [6]. B-Carotene (BC) is a polyisoprenoid compound synthesized in plants from mevalonate by the HMGCoA reductase pathway. We have recently shown that BC, the carotenoid with most provitamin A activity, has inhibitory effects on the induction of preneoplastic lesions in rats by the resistant hepatocyte model of hepatocarcinogenesis [9]. Epidemiological evidence as well as experimental results from animal studies and cell culture indicates that BC has cancer chemopreventive properties [l&-12]. Since HMGCoA reductase is regulated by end-product repression both in animal [I] and plant [ 131 systems, it is speculated that one of the mechanisms by which BC exerts its cancer chemopreventive effects may be by inhibiting HMG-CoA reductase. Therefore, the present study was designed to examine the effects of BC on rat liver HMG-CoA reductase induced by 2/3 partial hepatectomy (PH). 2. Materials

and methods

2.1. Animals and treatments Male Fischer 344 rats (Charles River Breeding Laboratoties, St. Constant, Quebec) weighing 140 g were housed in groups of two animals per cage and were maintained on a 12 h light/dark cycle with food (Purina Chow) and water available ad libitum. They were acclimatized to this environment for 1 week before use. The rats were divided into two groups. To one group (group /3>, BC (70 mg/kg body wt; trans-

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B-carotene type I; Sigma Chemical Co., St. Louis, MO) dissolved in corn oil was administered by gavage every alternate day for 3 consecutive weeks, while rats in group C received only corn oil and served as controls. Our earlier experiments indicated that this dose and protocol of administration of pcarotene exerted inhibitory effects on the induction of preneoplastic lesions in rats by the resistant hepatocyte model of liver carcinogenesis [9]. Twenty-four hours after the last gavage, all animals from both groups @ and C) were submitted to 2/3 PH to provide a proliferative stimulus. Liver removed at the time of PH served as the resting liver (RL). The rats were killed under ether anesthesia at 8, 18, 22, 24, 26 or 30 h after PH. At least three animals were included for each time point. Immediately after removal, the livers were either snap frozen in liquid nitrogen and kept at -70°C until use or immediately used for isolating nuclei. FOF Northern analysis and for nuclear run-off assays livers from three rats were pooled. 2.2. Isolation oj’KNA and Northern blot unaiysis Total RNA from 1 g pooled liver was isolated by guanidine thiocyanate method [ 141. Polyadenylated RNA was isolated by two cycles of oligo (dT)cellulose (Boehringer Mannheim) chromatography [ 151. Ten microgram aliquots of polyA+RNA were denaturated and electrophoresed on 1% agaroseformaldehyde (2.2 M) gels in morpholinopropanesulfonic acid (MOPS) buffer (0.02 M MOPS (pH 7.0) 50 mM sodium acetate, 10 mM EDTA (pH 8.0)). The separated polyA+RNAs were transferred to DuraloseUV (Strategene) membranes by capillary blotting, and fixed by baking to 80°C under vacuum for 2 h. Prehybridization and hybridization were carried out as described by Sambrook et al. [ 161. Radiolabelling of probes was done according to Feinberg and Vogelstein [ 171, with the Multiprime DNA Labelling System Kit and [u-32P]dCTP (6000 Ci/mmol) (Amersham) and the labeled probes purified using NickTM columns (Pharmacia). Routinely, probes with specific activities of (l-3) X IO9 cpm&g DNA were obtained. The probe for HMG-CoA reductase was the 4.5 kb BamHI fragment from the plasmid pRed 227 (ATCC). It represents the cDNA for hamster HMGCoA reductase and contains the entire coding region as well as 163 bp of the S-untranslated region and 1.65 kbp of the 3’-untranslated sequence 1181. The

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filters were washed twice in 2~ SSC plus 0.5% SDS for 10 min at room temperature and once in 0.1 x SSC plus 0.5% SDS for 15 min at 52°C. The membranes were then air-dried briefly and autoradiography was carried out at -70°C using Kodak XARJ films in an X-ray cassette fitted with intensifying screens. The filters were stripped of the HMG-CoA reductase probe by boiling in 0.1 X SSC plus 0.1% SDS for 10 min and were reprobed with a v-Ha-ras (rat) probe (Oncor, Gaithersburg, MD). Quantitative densitometry of the autoradiograms was performed using an LKB Ultrascan XL Enhanced Laser Densitometer. 2.3. Nuclear run-off assay

Rat liver nuclei were isolated from 6 g of freshly excised pooled liver essentially as described by Schibler et al. [19]. The purified nuclei were either used immediately or stored at -70°C after suspending in glycerol storage buffer (50 mM Tris, pH 8.0, 5 mM MgC12, 0.1 mM EDTA, 40% glycerol). In vitro elongation of nascent transcripts was performed using the method of McKnight and Palmiter [20] with minor modifications. Nuclear suspensions (routinely 2 x lo7 nuclei per reaction) were used per assay in 150~1 of reaction mixture (10 mM Tris (pH 8.0), 5 mM MgCl*, 300 mM KCl, 5 mM DTT, 0.5 mM each of ATP, CTP and GTP, 100,&i [~z-~~P]UTP (650 Ci/mmol, ICN), 130 U/ml of RNase inhibitor) and incubated at 30°C for 30 min. The reactions were terminated by the addition of 20 units of

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RNase-free DNase I (Pharmacia) per sample and further incubated at 37°C for 15 min. Following lysis of the nuclei, the RNA was purified by treatment with Proteinase K and phenol-isoamyl alcoholchloroform extraction and by precipitation with absolute ethanol in the presence of 0.3 M potassium acetate. After a second round of DNase I treatment, the final RNA pellet was resuspended in TE buffer (10 mM Tris (pH 8.0), 1 mM EDTA) and further purified by one passage through a Sephadex G-50 column. Plasmids containing cDNAs for HMG-CoA reductase (ATCC 37365) and glyceraldehyde-3-phosphate dehydrogenase (GAPD; ATCC 57090) were linearized with BamHI or PstI, respectively and then denaturated in the presence of 0.2 N NaOH [21]. The denaturated plasmids (1 or 3 pg) were then slot-blotted onto nitrocellulose membranes using a Schleicher and Schuell minifold and baked at 80°C for 2 h under vacuum. Hybridization was carried out at 42°C for 72 h using 7 X 106 cpm of the labeled nascent RNA transcripts prepared above. Washing of filters, autoradiography and densitometric analysis were carried out as described above. 3. Results and discussion 3.1. Effect of BC on HMG-CoA reductaseexpression

In order to investigate the effect of BC on HMGCoA reductase expression, F344 rats were treated for 3 consecutive weeks with the carotenoid and sacri-

HMGCoA Reductase 8h

18h

22h

24h

26h

30h

Fig. 1. Effect of BC on the level of hepatic HMG-CoA teductase mRNA transcripts induced by Y3 PH. Polyadenylated RNA was prepared from livers of rats tmated with BC @) or corn oil (C) and subjected to 2/3 PH and from the livers of control rats taken at the time of PH (RL). Aliquots of each RNA (1Opg) were denaturated and electrophoresed in 1% agarose-formaldehyde gels. The RNA was transferred onto a Duralose-UV membrane, and the filter was probed with a 32P-lahelled cDNA for HMG-CoA reductase (1.3 X lo9 cpm+g) for 48 h at 42°C. The autoradiogram showed a single band at 4.3-4.5 kb. Details concerning the treatment of rats with BC and pattial hepatectomy are given in the text.

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Fig. 2. Effect of BC on the level of hepatic HMG-CoA reductase mRNA transcripts induced by U3 PH. The figure represenLs quandtative analysis obtained by laser densitometry of the autoradiograph shown in Fig. 1. The area of the band is expressed in arbitrary units. Solid and hatched bars reptesent, respectively, reductase mBNA levels from corn oil and BC treated animals sacrificed at 8 and 24 h after PH. Each bar represents the results of one experiment. The entire experiment was repeated twice with a similar pattern of results.

ficed at 8, 18, 22, 24, 26 or 30 h after 2i3 PH. The kinetics of expression of the 4.3-4.5 kb transcripts for HMG-CoA reductasefollowing 2/3 PH measured as polyA+mRNA are shown in Fig. 1. The results show that compared to the expressionof the gene in

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resting livers (RL), the expression of HMG-CoA reductasein the control rat liver increasesafter 2/3 PH with two peaks of activity, a major one at 8 h and a smallerone at 24 h after PH. These resultsare consistent with the reported findings that the activity of HMG-CoA reductasein rat liver increasedas early as 8 h following PH [22], and that in synchronized reductase BHK-21 cells a marked rise in HMG-COA activity consistently occurred at or just preceding the S phase of the cell cycle [23]. In contrast, pretreatment with BC for 3 consecutive weeks (Group fi) partially repressed the increase in the levels of mRNA transcripts of HMG-CoA reductasein the rat liver both at 8 and 24 h after PH (Fig. 1). Densitometric analysisindicated a decreaseof 50 and 30% in BC treated animals (Group /?) at the 8 and 24 h time points, respectively, compared to the corresponding controls (Fig. 2). Similar pattern of results were obtained in two independentexperiments. To determine whether BC exerted a similar effect on another cell cycle related gene the filters were stripped and probed again for c-Ha-ras mRNA. As illustrated in Fig. 3, the expression of the I .4 kb transcript for c-Ha-ras increasedto the sameextent in regenerating livers of both control (Group C) and BC treated rats (Group /3). The densitometric measurementsat these time points also indicated no significant differences in cHa-ras mRNA levels between the control and experimental groups (data not shown). The fact that there is no difference in the Ha-ras transcripts levels indicates that the changes observed in the HMG-CoA reductase mRNA levels in Fig. I are not due to loading artifacts. This was also confirmed by hybridizing the

c-Ha-ras

RL

8h

Fig. 3. Effect of BC on tbe level of c-Ha-t-as mBNA

18h

transcripts. probe. A single band at I .4 kb was seen in the autoradiograms.

22h

2Ch

The blot shown in Fig. I was stripped

26h

and mhybridized

36h

with a v-Ha-t-as

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RL

C

P

Fig. 4. Effect of BC on the transcription of HMG-CoA mductase gene. Nuclei prepared from livers of BC (B) or corn oil (C) treated animals subjected to 2/3 PH and killed 8 h later and from livers of control rats taken at the time of PH (RL) were used in a nuclear run-off assay. One and 3pg of linearized, alkali denaturated HMG-CoA reductase and GAPD plasmids were spotted in slots as indicated. Equal amounts of radioactivity were allowed to hybridize to the immobilized DNA on each filter for 72 h at 42°C. Absence of unlabelled nucleotides or a-amanitin (1 &ml of incubation mixture) abolished transcription. Further details are given in the text.

same filter with the probe for GAPD (data not shown). These results suggest that the inhibitory effect of BC on the level of HMG-CoA reductase transcripts is not a reflection of a general inhibition of mRNA synthesis. It is of interest to note that even though BC had no significant effect on the levels of mRNA transcripts of c-Ha-ras, it could still affect the ras-product by inhibiting the post-translational farnesylation of ras-proteins [24,25]. 3.2. Effect of BC on HMG-CoA reductasegene transcription

In order to determine whether the observed decrease in transcript levels of HMG CoA reductase in

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BC treated liver is a reflection of decreased transcription and/or decreased stability of the mRNA, the effect of BC on the rate of transcription of HMGCoA reductase gene was determined using a nuclear run-off assay. Liver nuclei were isolated from both control animals and rats treated with BC for 3 consecutive weeks and sacrificed 8 h after PH. When compared to nuclei from resting livers, similar increases of HMG-CoA reductase gene transcription were observed in nuclei of regenerating livers of both control and BC treated animals sacrificed 8 h after PH (Fig. 4). As shown in Fig. 5, these results were confirmed by densitometric analysis of the autoradiograms. Transcription of GAPD, which served as control, was also unaffected under these experimental conditions. Inclusion of a-amanitin or omitting one of the unlabelled nucleotides abolished incorporation of label into RNA indicating the reaction was RNA polymerase II dependent. Thus, although administration of BC partially lowered reductase mRNA transcript levels, the carotenoid had little effect on the transcription rates of HMG-CoA reductase gene sug-

03

ac 091

0 Resting Liver

Corn Oil

Be&aCarotene

Fig. 5. Effect of BC on the transcription of HMG-CoA reductase gene. The figure represents quantitative analysis obtained by laser densitometry of the autoradiogmph shown in Fig. 4. The area of the band is expressed in arbitrary units. The bars represent the transcription of the reductase gene in nuclei from resting livers and regenerating livers 8 h after 2/3 PH from rats treated with corn oil or BC. Each bar represents the results of one experiment. The entire experiment was repeated twice with a similar pattern of msults.

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gesting that the carotenoid regulates the expression of HMG-CoA reductase by a post-transcriptional mechanism. HMG-CoA reductase is a highly regulated enzyme controlled by several feedback-regulation mechanisms. Accordingly, many sterol and non-s&rot isoprenoid products of mevalonic acid modulate the reductase at transcriptional and post-transcriptional levels 121. Changes in splicing, polyadenylation, transport to the cytoplasm or changes in HMG-CoA reductase mRNA stability are some of the possibilities that could account for the observed effects. The mechanism mediating the decrease in reductase mRNA levels by the carotenoid could be the activation of a specific ribonuclease, as has been suggested for glucocorticoids [26,27]. Moreover, it has been shown that the HM$G-CoA reductase gene lacks the classic TATA-sequence box and initiates transcription from multiple sites. Each of the initiation sites uses a different splice donor site for an intron in the S-untranslated region, thus producing multiple mRNAs with different S-untranslated regions, all of them subject to coordinate control [28]. Therefore, BC could induce the synthesis of a less stable mRNA due to differential splicing. Detailed studies are obviously required to further elucidate the molecular mechanisms involved in these effects of BC. Extensive data have been accumulated demonstrating the importance of cholesterol and mevalonate-derived prw for cell proliferation and malignant transform&on [7,29,30]. Based on these findings, HMG-CoA reductase inhibitors, commonly used as plasma cho&rterol-lowering drugs, have received increased at&&on as potential pharmacological agents to intiyibit the growth of tumors [31. 321.

In recent years, BC has also been implicated as a cancer chemopreventive agent [lO-121. However, the mechanisms by which BC influences cancer development are not yet completely understood. Since BC is metabolized to retinoids, it is possible that some of its chemopreventive effects are related to these metabolites. However, it has been demonstrated that BC is absorbed and taken up by several tissues in rodents and human [33-353 and suggested that some of the effects of BC are independent of its provitamin A properties [9,36,37]. For example, it has been sug-

gested that BC-induced upregulation of the gap junctional protein connexin 43 is independent of its provitamin A properties [36]. A change in the cell to cell communication induced by BC could be one mechanism by which BC can exert its chemopreventive effect. BC could also inhibit tumor development by being an antioxidant [38]. The observation made in this study that BC regulates HMG~ CoA reductase expression suggests that the described cancer chemopreventive effect of the BC might also be attributed to its interference with the isoprenoid metabolism. It may be noted that these effects of BC are not mutually exclusive and it is likely that all of these effects of BC contribute to its cancer chemopreventive effects. Acknowledgements

We wish to thank Ms. Lori Cutler for her excellent secretarial assistance. The technical assistance of Sar Hsu is gratefully acknowledged. This work was supported in part by the National Cancer Institute of Canada with funds from the Canadian Cancer Society. F.S.M. is the recipient of a CAPES postdoctoral fellowship. References

Ul Brown, M.S. and Goldstein, J.L. (1980) Multivalent feed-

back regulation of HMG-CoA reductase, a control mechanism coordinating issprenoid synthesis and cell growth. J. Lipid Res., 21, 505-517. I21 Goldsteiri, J.L. and Brown, MS. (1990) Regulation of the mevalonate pathway. Nature (London), 343,425-430. L31 Nakanishi, M., Goldstein, J.L. and Brown. MS. (1988). Multivalent control of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Mevalonate-derived product inhibits translation of mRNA and accelerates degradation of enzyme. J. Biol. Chem., 263, 89298937. [41 Rosser, D.S.E., Aehby, M.N., Ellis, J.L. and Edwards, P.A. (1989) Coordinate regulation of 3-hydroxy-3methylglutaryl-coenzyme A reductase, and prenyltransferase synthesis but not degradation in HepG2 cells. J. Biol. Chem., 264, 12653-l 2656. I51 Giron, M.D., Havel, CM. and Watson, J.A. (19%) Isopcntenoid synthesis in eukaryotic cells. An initiating role for post-translational control of 3-hydroxy-3methylgtutaryl coenzyme A reductase. Arch. Biochem. Biophys., 302, 265271. WI Siperstein, M.D. (1984) Role of cholesterogenesis and isoprenoid synthesis in DNA replication and cell growth. J. Lipid Res.. 25, 1462-1468.

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[7] Soma, M.R., Corsini, A. and Paoletti. R. (1992) Cholesterol and mevalonic acid modulation in cell metabolism and multiplication. Toxicol. Lett., 64/65, l-15. [8] Quesney-Huneeus, V., Galick, H.A., Siperstein, M.D., Erickson, S.K., Spencer, T.A. and Nelson, J.A. (1983) The dual role of mevalonate in the cell cycle. J. Biol. Chem., 258.378-385. [9] Moreno, F.S., Rizzi, M.B.S.L., Dagli, M.L.Z. and Penteado, M.V.C. (1991) Inhibitory effects of b-carotene on preneoplastic lesions induced in Wistar rats by the resistant hepatocyte model. Carcinogenesis (London), 12, 1817-1822. [lo] Byers, T. and Perry, G. (1992) Dietary carotenes, vitamin C, and vitamin E as protective antioxidants in human cancers, Annu. Rev. Nutr., 12,139-159. [ll] Gerster, H. (1993) Anticarcinogenic effect of common carotenoids. Int. J. Vit. Nutr. Res., 63.93-121. [12] Ziegler, R.G. (1989) A review of epidemiological evidence that carotenoids reduce the risk of cancer. J. Nun., 119, 116-122. [13] Garg, V.K. and Douglas, T.J. (1983) Hydroxymethylglutaryl CoA reductase in plants. In: ydroxy-methylglutaryl Coenzyme A Reductase, pp. 29-37. Editor: J.R. Sabine. CRC Press, Boca Raton, FL. [14] Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry, 18, 5294--5299. [15] Aviv, H. and Leder, P. (1972) Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid cellulose. Proc. Natl. Acad. Sci. USA, 69, 1408-1412. [16] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) In: Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Ha&or Laboratory Press, Cold Spring Harbor, NY. [17] Feinberg, A.P. and Vogelstein, B. (1984) Addendum: a technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem., 137, 266-267. [ 181 Chin, D.J., Gil, G., Russell, D.W., Llscum, L., Luskey, K.L., Basu, S.K., Okayama, H., Berg, P., Goldstein, J.L. and Brown, M.S. (1984) Nucleotide sequence of 3-hydroxy-3methylglutaryl coenzyme A mductase, a glycoprotein of endoplasmic reticulum. Nature (London), 308.613-617. [19] Schibler, U., Hagenbtlchle, 0.. Wellauer, P.K. and Pittet, A.C. (1983) Two promoters of different strengths control the transcription of the mouse alpha-amylase Amy-l in the parotid gland and the liver. Cell, 33, 501-508. [20] McKnight, G.S. and Palmiter, R.D. (1979) Transcriptional regulation of the ovalbumin and conalbumin genes by steroid hormones in chick oviduct. J. Biol. Chem., 254, 90509058. [21] Greenberg, M.E. and Ziff, E.B. (1984) Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene. Nature (London), 311.433438. [22] Trentalance, A., Leoni, S., Mangiantini, M.T., Spagniolo, S., Feingold, K., Hughes-Fulford, M., Siperstein. M., Cooper, A.D. and Erickson, S.K. (1984) Regulation of 3-hydroxy-3methylglutaryl-coenzyme A reductase and cholesterol syn-

Letters

[23] [24] [25] [26]

[27]

[28]

[29]

[30]

[31]

[32]

[33] [34]

[35]

[36]

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thesis and esterification during the first cell cycle of liver regeneration. Biochim. Biophys. Acta, 794, 142-151. Quesney-Huneeus, V., Wiley, M.H. and Siperstein, M.D. (1979) Essential role for mevalonate synthesis in DNA replication. Proc. Natl. Acad. Sci. USA, 76, 5056-5060. Hancock, J.F., Magee, AI., Childs, J.E. and Marshall, C.J. (1989) All ras proteins are polyisoprenylated but only some are palmitoylated. Cell, 57, 1167-l 177. Casey,P.J., Solski, P.A., Der, C.J. and Buss, J.E. (1989) P21 ras is modified by a farnesyl isoprenoid. Proc. Natl. Acad. Sci. USA, 86.8323-8327. Simonet, W.S. and Ness, G.C. (1989) Post-transcriptional regulation of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA in rat liver. Glucocorticoids block the stabilization caused by thyroid hormones. J. Biol. Chem., 264.569-573. Gessani, S., McCandless, S. and Baglioni, C. (1988) The glucocorticoid dexamethasone inhibits synthesis of interferon by decreasing the level of its mRNA. J. Biol. Chem., 263,7454-7457. Reynolds, G.A., Goldstein, J.L. and Brown, MS. (1985) Multiple mRNAs for 3-hydroxy-3-methylglutaryl coenzyme A reductase determined by multiple transcription initiation sites and intron splicing sites in 5’-untranslated region. J. Biol. Chem., 260,10369-10377. Brown, M.S. and Goldstein, J.L. (1974) Suppression of 3hydroxy-3-methylglutaryl coenzyme A reductase activity and inhibition of growth of human flbroblasts by ‘I-ketocholesterol. J. Biol. Chem., 249,7306-7312. Bennis, F., Favre, G., Le Gaillard, F. and Soula, G. (1993) Importance of mevalonate-derived products in the control of HMG-CoA reductase activity and growth of human lung adenocarcinoma cell line A549. Int. J. Cancer, 55,640-645. Maltese, W.A., Defendini, R., Green, R.A., Sheridan, K.M. and Donley, D.K. (1985) Suppression of mmine neuroblastoma growth in vivobynu%inolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. J. Clin. Invest., 76, 1748-1754. Sumi, S., Beauchamp, D., Townsend, Jr., CM., Uchida, T., Murakami, M., Rajararnan, S., Ishizuka, J. and Thompson, J.C. (1992) Inhibition of pancreatic adenocarcinoma cell growth by lovastatin. Gastroenterology, 103,982-989. Mathews-Roth, M. (1977) The carotenoid content of various organs of animals administered large amounts of betacarotene. Nutr. Rep. Int., 16,419-423. Jones, C.S., Sly, L., Chen, L.C., Ben, T., Brugh-Collins, M., Lichti, U. and de Luca, L.M. (1994) Retinoid and betacarotene concentrations in skin, papillomas and carcinomas, liver and serum of mice fed retinoic acid or beta-carotene to suppress skin tumor formation. Nutr. Cancer, 21,83-93. Mobarhan, S., Shiau, A., Grande, A., Kolli, S., StacewiezSapuntzakis, M., Oldham, T., Liao, Y.. Bowen, P., Dyanvanapalli, M., Kazi, N., McNeal, K. and Frommel, T. (1994) /?Carotene supplementation results in an increased serum and colonic mucosal concentration of /l-carotene and a decrease in a-tocopherol concentration in patients with colonic neoplasia. Cancer Epidemiol. Biomarkers Prev., 3.501-505. Zhang, L.X., Cooney, R.V. and Bertram, J.S. (1992) Carotenoids up-regulate Connexin-43 gene expression independ-

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ent of their provitamin A or antioxidant properties. Cancer Res., 52.5107-57 12. [37] Edes, T.E., Thornton, Jr., W. and Shah, J. (1989) Betacarotene and aryl hydrocarbon hydroxylase in the rat: an effect of beta-carotene independent of vitamin A activity. J. Nutr., 119, 796-799.

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1381 Canfield, L.M., Forage, J.W. and Valenzoela, J.G. (1992) Carotenoids as celhdar antioxidants. Proc. Sot. Exp. Biol. Med., 200.260-265.