Regulation of ornithine decarboxylase mRNA translation by ...

‘THEJOURNAL OP BIOLOOICAL CHEMISTRY

Vol. 263. No. 7, Issue of March 5. pp. 352&3533,19&3 Printed in U.S. A.

0 1988 by The American Society for Biochemistry and Moleculsr Biology, Inc.

Regulation of Ornithine Decarboxylase mRNA Translation by Polyamines STUDIES USING A CELL-FREESYSTEM DECARBOXYLASE GENE*

AND A CELL LINEWITH

AN AMPLIFIED ORNITHINE

(Received for publication, July 9, 1987)

Lo PerssonS From the Department of Physwlogy, University of Lund, S-223 62 Lund, Sweden

Ingvar Holm and Olle Heby From the Department of Zoophyswlogy, University of Lund, S-223 62 Lund, Sweden

The translational control of ornithine decarboxylase (ODCase) by polyamines has been studied using a cellular as well as a cell-free system. A mutant L1210 cell line, in which ODCase represents 4-5% of all soluble protein synthesized, was isolated by stepwise selection for resistancetothe ODCase inhibitor Z-difluoromethylornithine (DFMO). The exceptionally high expression of ODCase in these cells was due to amplification of the ODCasegene.When the cells were grown in the absence of DFMO, dramatic increases in cellularputrescineandspermidine levels occurred. These increases wereaccompanied by a rapid decrease in ODCase synthesis. The change in ODCase synthesis was not associated with an alteration in theamount of ODCase mRNA,demonstrating a translational control in these cells. The effects of polyamines on ODCase mRNA translation were also studied in rabbit reticulocyte lysates using mRNA isolated from the DFMOresistant cells. Lowconcentrations of spermidine stimulated synthesis of ODCase and that of total protein, when added to gel-filtered lysates. Notably, optimal stimulation of ODCase synthesis was achieved at a spermidine concentration lower than that required for an optimal rate of total protein synthesis. Higher concentrations of spermidine were inhibitory, and their effects onODCase synthesis were stronger than on protein synthesis in general, resulting in a decrease in thefraction of proteinsynthesis accounted for by ODCase. The present results demonstratethat at least part of the feedback regulation of ODCase exerted by the polyamines is due to direct inhibition of ODCase mRNA translation.

The polyamines putrescine, spermidine, and spermine are essential for cell growth and differentiation (1-4).The importance of these amines is reflected in the extensive regulation * This research was supported by Grant 04X-02212from the Swedish Medical Research Council and NaturalScience Research Council Grant B-BU 4086/114, the Medical Faculty (University of Lund), and theAke Wiberg and Magnus Bergvall Foundations. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed.

of their biosynthesis. ODCase,’ which catalyzes the first step in polyamine synthesis, has been shown to be subject to transcriptional (5-11),translational (8, 9, 12-14), as well as posttranslational (15-20) control. Induction of ODCase by various growth stimuli is usually associated with an increase in the cellular content of ODCase mRNA (5-11). Short term regulation, on the other hand, appears to be achieved by changes in the efficiency of ODCase mRNAtranslation (8,9, 12-14) and in the degradation rate of the enzyme (17-20). An excess of polyamines, either added to or synthesized by the cells, rapidly turns off the ODCase activity. This result is accomplished by two different mechanisms. One isthe induction of a special protein, named antizyme, which has an extremely strong affinity towards ODCase and which inhibits its activity (15-21). The binding of antizyme to ODCase is also believed to render the enzyme more susceptible to degradation, thus decreasing its already short half-life (17-19). The other mechanism of ODCase control exerted by the polyamines is translational.When depleting the cellular polyamine content, by using specific inhibitors of ODCase, we observed an increased synthesis of ODCase without any change in the amount of ODCase mRNA (8, 13). If, on the other hand, the cellular polyamine content was increased by providing polyamines in the culture medium, a decrease in ODCase synthesis occurred (12-14). Again, there wasno change in ODCasemessagelevel. Since the turnover of ODCase is the fastest among eukaryotic enzymes any change in the rate of ODCase synthesis will be rapidly transmitted and thus to polyto the amount of enzyme protein (22, 23), amine synthesis. Hence, it appears that the polyamines, by means of various feedback mechanisms, control their own cellular levels. The exact mechanism behind the translational control of ODCase by the polyamines is still unknown. However, it is known that ODCase mRNA has an exceptionally long noncoding 5’ leader which is believed to play an important role in the translational regulation of ODCase expression (24).It is conceivable that thepolyamines, which have strong affinity for nucleic acids (25),affect the secondary structure of the 5’ leader in a way that inhibits initiation of ODCase synthesis. Interestingly, ODCase mRNA has been reported to be poorly translated in rabbit reticulocyte lysate systems (6,26) which The abbreviations used are:ODCase, ornithine decarboxylase; DFMO, 2-difluoromethylornithine;SDS-PAGE, sodium dodecyl SUIfate-polyacrylamide gel electrophoresis; Hepes, 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid; EGTA, (ethylenebis(oxyethy1enenitri1o)ltetraacetic acid.

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Regulation of ODCase m R N A Translation usually contain large amounts of spermidine (27). Studies on the regulation of ODCase synthesis have been hampered because synthesis of ODCase represents only a minor fraction of total protein synthesis. This fraction may be as low as 1:10,000, even when ODCase synthesis is fully induced (28). To overcome the problem we have isolated a mutant cell line which overproduces ODCase. This overproducer was selected for by growing L1210cells in thepresence of increasing amounts of the ODCase inhibitor DFMO. Cells resistant to 20 mM DFMO contained an amplified ODCase gene and large amounts of ODCase message. This cell line was used to further analyze the translational control of cellular ODCase synthesis. In addition, the direct effect of polyamines on the translation of ODCase mRNA, isolated from the ODCase overproducer, was studied in acell-free system. EXPERIMENTAL PROCEDURES

Materials-Cell culture media components were from Gibco Europe. [36S]Methionine and [32P]dCTP were purchased from Amersham Corp. ~-[l-'~C]Ornithine and L%-[~,~-~H]DFMO were obtained from Du Pont-New EnglandNuclear. DFMO was generously provided by the Merrell Dow Research Institute (Strasbourg, France) and cDNA (pODC 9341, encoding mouse kidney ODCase, was a kind gift from Dr. Franklin G. Berger, Department of Biology, University of South Carolina (7). Oligo(dT)-cellulose was purchased from Pharmacia LKB Biotechnology Inc. Rabbit reticulocyte lysates were obtained from Amersham Corp., Du Pont-New England Nuclear, Promega Biotec (Madison, WI), andBethesda Research Laboratories. Cell Culture"L1210 cells weregrown in RPMI 1640medium containing 10% fetal calf serum and 50 pM 2-mercaptoethanol. They were subcultured every 4 days. DFMO-resistant cells (L1210-DFMO') were selected for by growth in medium containing increasing concentrations of DFMO (0.5, 1, 5, 10, and 20mM). After a period of approximately 2 months a cell line had been obtained which in the presence of 20 mM DFMO exhibited a growth rate that was almost the same as that of the wild-type. These cells were routinely grown in the presence of 20 mM DFMO. For analysis, cells were seeded at a density of 1.0 X IO6 cells/ml and harvested 24 h later. Determination of ODCase Activity-Cells (5 X 10') were sonicated in 1.0 ml of ice-cold 0.1 M Tris-HCI, pH 7.5, containing 0.1 mM EDTA and 0.5mM dithiothreitol (buffer A). After centrifugation at 20,000 X g for 20 min, ODCase activity was determined in aliquots of the supernatant by measuring the release of"CO, from L-[~-'*C] ornithine in the presence of saturating levels of pyridoxal 5"phosphate (0.1 mM) and L-ornithine (0.5 mM) (29). Determination of ODCase Protein Content-The amount of ODCase protein was measured essentially as described by Seely and Pegg (30). Aliquots of the 20,000 X g supernatants were incubated at room temperature for 30 min with a monospecific antibody against mouse ODCase (31), diluted 1:256,000. Purified mouse kidney ODCase (32) labeled with D L - [ ~ , ~ - ~ H ] D Fwas M O then added, and the samples were incubated for an additional 30 min. Antibody-bound radioactivity was determined after precipitation with bacterial protein A adsorbent (60 min) and centrifugation at 12,000 X g for 2 min. Purified mouse kidney ODCase was used as a standard. Determination of ODCase Synthesis-ODCase synthesis was determined by measuring the incorporation of ~ - [ ~ ~ S ] m e t h i o ninto i n e the enzyme. The cells were reseeded (1.0 X 106/ml) in methionine-free RPMI 1640 medium (Biochrom, Berlin, FRG) containing 10% dialyzed fetal calf serum. After 10 min of preincubation at 37 "C the cultures were supplemented with [36S]methionine(10 pCi/ml), After incubation for 25 min a t 37 "C the cells were collected by centrifugation (1500 X g, 10 min) and sonicated in500 plof buffer A. Aliquots of thesupernatants (containing equal amounts of acid-insoluble radioactivity) were incubated with an excess of ODCase antiserum (31) a t room temperature for 30 min. ODCase bound to the antibody was precipitated by incubation with proteinAadsorbent for an additional 30 min. After thorough washing in 10 mM Tris-HC1, pH 7.5, containing 0.1 mM EDTA, 0.5 mM dithiothreitol, 0.1% bovine serum albumin, 0.1% Triton X-100,0.1% SDS,and 0.01% Tween 80, the precipitate was fractionated by SDS-PAGE essentially as described by Persson et al. (33). Fluorography was carried out after incubating the gels in Amplify (Amersham Corp.). Determination of ODCasemRNA-ODCaue mRNA was rtet.er-

3529

mined by Northern blot analysis. Total RNA (20 pg), isolated by the guanidinium isothiocyanate/CsCl method (34), was fractionated in formaldehyde-containing 1% agarose gels (35). After transfer to Genescreen (Du Pont-New England Nuclear), the RNA was hybridized to pODC 934 labeled with [32P]dCTPusing the oligonucleotide priming technique of Feinberg and Vogelstein (36). Analysis of Genomic DNA-DNA was isolated essentially as described by Blin and Stafford (37). Isolated DNA was digested with EcoRI, BamHI, or Sac1 restriction endonucleases, fractionated on 0.8% agarose gels, transferred to Genescreen, and hybridized with 3ZP-labeledpODC 934 (36). Isolation of Poly(A)+RNA-Polyadenylated RNA wasisolated from total RNA by chromatography on oligo(dT)-celluloseas described by Aviv and Leder (38). In Vitro Tramlation of PoZy(A)+ RNA-Polyamines were removed from rabbit reticulocyte lysates by gel filtration on Bio-Gel P-6DG (Bio-Rad) using 10 mM Hepes buffer, pH 7.2, containing 1.2 mM MgCl,, 25 mM KC1,lO mM NaC1,0.05 mM EDTA, and 0.1 mM EGTA. Translation of poly(A)+RNA (0.5 pg) wascarried out for 1h a t 30 "C in the presence of 0.1 M KCl, 1.3 mM MgCl,, 1.0 mM ATP, 0.2 m M GTP, 10 mM creatine phosphate, 1.0 mM glucose, 50 pM of an amino acid mixture without methionine, and 20 pCi of [36S]methionine.The incorporation of radioactivity into ODCasewas determined after precipitation with a monospecific ODCaseantibody (31), SDS-PAGE, and fluorography. Total protein synthesis was measured after precipitation with 5% trichloroacetic acid. Determination of Polyamines-Chromatographic separation and quantitative determination of the polyamines in the cell extracts and the lysates were carried out using an automatic amino acid analyzer (Biotronik LC 5001) with o-phthaldialdehyde as reagent. RESULTS

Isolation and Characterization of a Mutant Cell Line Ouerproducing ODCase-By exposing L1210 cells to a selective pressure of stepwise increases in DFMO concentration a DFMO-resistant cell line was isolated. These cells were able to grow with a doubling time of 15 h in the presence of 20 mM DFMO. Wild-type L1210 cells grew poorly when DFMOconcentrations exceeded 1mM. The acquired resistance to DFMO was due to a large increase in the cellular content of ODCase. As shown in Table I, the DFMO-resistant cells contained several hundred-fold more ODCaseprotein than thewild-type cells. However,as also shown in Table I, only a small fraction of the huge amount of ODCase in the DFMO-resistant cells was enzymatically active, giving rise to an ODCase activity which was less than that found in the wild-type cells. The increase in ODCase content in the DFMO-resistant cells was only partly attributable to a stabilization (%fold) of the enzyme (Fig. 1).Instead, the major cause of the elevated ODCaselevelwas an increase inODCase synthesis. Pulse labeling of the cells with [35S]methioninefollowed by immunoprecipitation of ODCase with an anti-ODCase serum revealed a dramatic increase in the rate of ODCase synthesis (Fig. 2). In the DFMO-resistant cells synthesis of ODCase represented about 4-5% of soluble protein synthesis, whereas in the wild-type cells this fraction was less than 0.05%. The elevated expression of ODCase appeared to be caused TABLE I ODCase activity and ODCaseprotein content in wild-type and DFMO-resistant cells Cells were harvested 24 h after seeding. At the time of seeding DFMO (20 mM) was added to the DFMO-resistant cells, but not to the wild-type cells.ODCase activity and ODCase protein content were determined as described under "Experimental Procedures." One unit of enzyme activity was defined as the amount of enzyme catalyzing the release of 1 nmol of CO,/h. Mean k S.E., n = 4. Cells

Wild-type DFMO-resistant

Activity

Protein

units/l@ cells

ng/I@ cells k 0.06 136 f 17

0.30 0.30 k 0.02 0.09 0.01

*

Regulation of ODCase mRNA Translation

3530

B

A

100

23.1

.

50

9.4 6.7

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c

E

20

0

’

.

4.4

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2.3 2.0

. .

0

cc

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0 8-

5

0

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30

60

90

120

Minutes after cycloheximide

FIG.1. Turnover of ODCase in wild-type and DFMO-resistant cells. Wild-type cells were grown in the absence of DFMO, whereas DFMO-resistant cells were seeded in a medium containing DFMO (20 mM). Cycloheximide (50 pg/ml) was added 24 h after seeding. Cells were analyzed for ODCase activity (wild-type, 0 ) and ODCase protein (DFMO-resistant,0).Mean f S.E., n = 3.

1

2

1

2

FIG.3. Southern and Northern blot analyses of DNA and RNA from wild-type and DFMO-resistant cells. Cellswere grown as described in Fig. 1.A, total genomic DNA was digestedwith the restriction endonuclease EcoRI, fractionated on a 0.8% agarose gel, and hybridized to radiolabeledpODC 934 aftertransferto Genescreen. Lune 1, wild-type cells; lane 2, DFMO-resistant cells. Migration of DNA molecular weight standards is indicated. B, total RNA was fractionated on a 1% agarose gel, transferred to GeneScreen, and hybridized to radiolabeled pODC 934. Lune 1, wild-type cells; lane 2, DFMO-resistant cells.

T

I

I

I

I

T

1

2

T

3

FIG.2. Synthesis of ODCase in wild-type and DFMO-reas describedin Fig. 1. ODCase sistantcells. Cellsweregrown synthesis was determined by incubation with [?3]methioninefor 25 min followed by immunoprecipitation and SDS-PAGE.Lune 1, wildtype cells (ODCase antiserum);lane 2, DFMO-resistant cells (ODCase antiserum); lane 3, DFMO-resistant cells (normalrabbitserum). Arrowhead indicates the migration of purified ODCase (32) labeled with DL-[~,~-‘H]DFMO (M,= 53,000).

by gene amplification and an increased transcription. Digestion of genomic DNA with the restrictionenzyme EcoRI gave rise to at least six different restrictionfragments. One of these fragments (6.5 kilobases)was strongly amplified inthe DFMO-resistant cells (Fig. 3A). That one of several different ODCase genes was amplified was confirmed using two other restriction endonucleases, BamHIand Sac1 (resultsnot shown). The amplification of the ODCase gene was associatedwith a marked rise in the amount of cellular ODCase mRNA as revealed by hybridization to a cDNA encoding mouse ODCase (Fig. 3B). Translational Control of ODCase in DFMO-resistant CellsRadioimmunoassay of DFMO-resistant cells grown for a couple of weeks in the absenceof DFMO showed that thesecells

0

10

20

30

Days

FIG.4. Content of ODCase protein and ODCase mRNA in DFMO-resistant cells grown in the presence or absence of DFMO. Cells were seeded in the presenceof 20 mM DFMO (day 0). After 4 days of growth in DFMO containing medium the cells were cultured without DFMO until day 28 (six passages). Thereafter they were again transferred to a medium containing DFMO. At various time points, cells were assayed for ODCase protein (mean f S.E., n = 4), usinga radioimmunoassay, andODCase mRNA, using Northern blot analysis (top).

contained considerably less ODCase than the ones grown in the presence of the drug. However, this change did not seem to be caused by a loss of genetic material containing the amplified portion because the cells still showed the same resistance to DFMO. Fig. 4 shows the changes in ODCase content of DFMO-resistant cells during growth in the absence of DFMOandafter a shift back into DFMO-containing

Regulation of ODCase mRNA Translation

3531

A

1

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3

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5

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FIG. 5. ODCase synthesis in wild-type and DFMO-resistant cells grown in the presence or absence of DFMO. DFMOresistant cells were grown as described in Fig. 4. Extracts from cells labeled with [?3]methionine were precipitated with a n ODCase antiserum and analyzed on SDS-PAGE. Lane I , DFMO-resistant cells grown in the presence of DFMO; lane 2, DFMO-resistant cells grown in the absence of DFMO for 25 days; lane 3, DFMO-resistant cells grown in the absence of DFMO for 24 days and then in the presence of DFMO for 1 day; lane 4, wild-type cells grown in the absence of DFMO; lane 5, purified mouse kidney ODCase (32) labeled with DL[3,4-'H]DFMO (M, = 53,000).

TABLE I1 Polyamine content of wild-type and DFMO-resistant cells grown in the presence or absence of DFMO The DFMO-resistant cells were seeded in a medium containing DFMO (20 mM). After one passage (4 days) in theDFMO-containing medium the cells were allowed to grow with repeated passages in a DFMO-free medium for 3-4 weeks. Then DFMO was again added to the medium. Cells were collected for polyamine analysis 24 h after each passage. Wild-type cells were grown in the absence of DFMO. Mean f S.E., n = 4. Cells DFMO Putrescine Spermidine Spermine Wild-type DFMO-resistant" DFMO-resistantb DFMO-resistant'

-

+ +

nrnol/I@ cells

0.21 f 0.06