THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistr, and Molecular ’ Biolow, Inc.

Val. 267, No. 10, Issue of April 5, pp. 6946-6951,1992 Printed in U.S.A.

Mechanism of Regulation of Ornithine Decarboxylase Gene Expression by Asparagine in a Variant Mouse Neuroblastoma Cell Line*

(Received for publication, May 17,1991)

Zong Ping Chen and Kuang Yu ChenS From the Department of Chemistry, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08903

We have developed a clonal variant, named DF-40, from the N2a mouse neuroblastoma cell line, which has the ornithine decarboxylase (L-ornithine carboxy- lyase, EC 4.1.1.17, ODC) gene amplified. When DF-40 cells were maintained in a simple salt glucose medium (e.8. Earle’s balanced salt solution), L-asparagine alone was sufficient to induce a maximal increase in ODC activity. The increase in ODC activity correlated well with an increase in the amount of ODC protein. North- ern blot analysis indicated that asparagine caused a 12-15-fold increase in ODC mRNA. The half-life of ODC mRNA induced by asparagine in DF-40 cells changed from more than 8 h to about 25 min upon removal of asparagine from the culture in the presence of actinomycin D. In contrast, asparagine had little or no effect on the rate of transcription of the ODC gene. Pulse labeling of cells for 15 min with [s6S]methionine showed a 90-140-fold increase in the synthesis of ODC protein after 4-8 h of incubation with asparagine. The removal of asparagine from the medium resulted in a rapid loss of ODC protein with a half-life as short as 12 min. The presence of asparagine increased the half- life of ODC protein by 3-5-fold when measured in the presence of cycloheximide. Taken together, our data show that asparagine induced ODC gene expression in DF-40 cells, primarily by post-transcriptional stabili- zation of ODC mRNA. In addition, asparagine specifi- cally stimulated the synthesis and suppressed the deg- radation of ODC protein.

Ornithine decarboxylase (ODC)’ is the key enzyme for the biosynthesis of polyamines (putrescine, spermidine, and spermine) in eukaryotic cells (Pegg and Williams-Ashman, 1981). The regulation of this enzyme has been shown to play an important role in the control of growth and differentiation (Tabor and Tabor, 1984; Pegg, 1988; Hayashi, 1989). ODC in mammalian cells can be induced by a wide variety of agents including growth stimuli (e.g. Kontula et al. (1984), Kahana and Nathans (1984), Persson et al. (1985), and Hovis et al. (1986)). It has been shown that ODC activity can be controlled at transcriptional (Kontula et al., 1984; Sertich and Pegg,

* This work was supported in part by Grant CA-49695 from the National Institutes of Health and by a grant from the Charles and Johanna Busch Memorial Fund. 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 accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

3 To whom correspondence should be addressed Dept. of Chem- istry, Rutgers University, P. 0. Box 939, Piscataway, NJ 08855-0939. Tel.: 908-932-3739; Fax: 908-932-5312.

The abbreviations used are: ODC, ornithine decarboxylase; DFMO, a-difluoromethyl ornithine; EBSS, Earle’s balanced salt so- ution; SDS, sodium dodecyl sulfate, PAGE, polyacrylamide gel elec- ;rophoresis.

1987), translational (Kahana and Nathans, 1985; Holtta and Pohjanpelto, 1986), and post-translational levels (Fong et al., 1976; Kahana and Nathans, 1985). These studies underscore the complexity of the regulation of ODC gene expression in biological systems. The complexity of culture media and the inherent complexity of the animal could also make it difficult to delineate the biochemical pathways involved in the regu- lation of the ODC gene. We have previously reported that mouse neuroblastoma cells can be maintained in a phosphate- buffered glucose solution for more than 2 days and that a single amino acid, asparagine, can induce maximal ODC ac- tivity in cells under this condition (Chen and Canellakis, 1977). The effect of asparagine is specific; other system A or N amino acids such as glutamine, serine, or glycine are effective, but to a much lesser extent (Chen and Canellakis, 1977; Gibbs et al., 1980; Viceps-Madore et al., 1982; Law and Fong, 1987). Interestingly, sera, growth factors, or hormones are all ineffective in inducing ODC activity when cells are maintained in the salts/glucose solution. Nevertheless, these agents can potentiate the effect of suboptimal concentrations of asparagine in inducing ODC activity, suggesting that the action of serum or growth factors in growth medium may involve the action of asparagine (Chen and Canellakis, 1977; Rinehart and Canellakis, 1985; Kanamoto et al., 1987). Since ODC is a very minor component of the total soluble protein present in animal cells, less than 0.0003% even after maximal induction (Kameji et al., 1982), the availability of ODC over- producers should offer a convenient system for studying the regulation of the ODC gene. The ODC gene in cultured mammalian cells can be amplified by drug selection (e.g. McConlogue and Coffin0 (1983)). Using this approach, we have isolated a clonal variant, named as DF-40, from the N2a mouse neuroblastoma cell line (Chen and Chen, 1989). The mode of induction of ODC activity in DF-40 cells is similar to that in the parental N2a cells, but the magnitude of induction in DF-40 cells is more than 200-fold higher than that in N2a cells. In view of the unique effect of asparagine in inducing ODC activity and the potential role of asparagine in regulating ODC under physiological conditions, we have examined the mechanism of the induction of ODC activity by asparagine in this DF-40 cell line. Our results indicate that asparagine alone could induce a 15-fold increase in the level of ODC mRNA, primarily due to post-transcriptional stabi- lization of ODC mRNA. In addition, asparagine specifically stimulated the translation of ODC mRNA and increased the half-life of ODC protein. Taken together, these three effects of asparagine could account for the 200-fold increase of ODC activity in DF-40 cells maintained in the salts/glucose solu- tion.

EXPERIMENTAL PROCEDURES

Cell Culture-Both N2a and DF-40 mouse neuroblastoma cells were grown in Dulbecco’s modified Eagle’s medium (with 4500 mg of

6946

Induction of ODC Gene Expression by Asparagine 6947

glucose per liter) supplemented with 10% fetal bovine serum at 37 "C in a Forma water-jacketed CO, incubator. The isolation and charac- terization of the clonal DF-40 cell line have been described previously (Chen and Chen, 1991). For enzyme induction, confluent cultures were serum-deprived for 20-24 h in fresh Dulbecco's medium, washed twice with Earle's balanced salt solution (EBSS), and then incubated in EBSS containing 10 mM asparagine.

Effect of Asparagine on the Utilization of Ornithine-At various time points after incubating with asparagine in EBSS, [3H]ornithine (55.0 Ci/mmol) was added to the culture to a final concentration of 2 p~ (0.5 pCi/ml). After 1 h of incubation with [3H]ornithine, the cells were harvested for polyamine quantitation (Chen et al., 1982). The amount of radioactivity recovered in each individual polyamine pool, relative to the total radioactivity taken up by the cell, was used as a measure of the degree of utilization of ornithine as catalyzed by ODC in uiuo.

Poly(A)+ RNA Preparation, Northern and Slot Blot Hybridizatwn- Total cellular RNA was isolated by the method of Chomczynski and Sacchi (1987). Poly(A)+ RNA was prepared from the total RNA with an oligo(dT)-cellulose affinity column. RNA gels and Northern blot analysis were performed by standard techniques as previously de- scribed (Chang and Chen, 1988). The membranes were hybridized with 32P-radiolabeled probes. The slot blot analysis was carried out on a Genescreen Plus membrane using the Schleicher and Schuell slot blot apparatus. The membrane was then hybridized with 3zP- radiolabeled probes. The DNA probes used in this study were the plasmid pODC54 (Kontula et al., 1984), cDNA of &actin (Cleveland et al., 1980), the plasmid pfos-1 (Curran et al., 1982), and the plasmid pMC-myc54 (Stanton et al., 1983).

Nuclear Run-off Transcription Assay-Cells were harvested at various time points after treatment with asparagine. Intact nuclei were purified and frozen in liquid N, as described by Greenberg et al. (1985). For the runoff assay, the frozen nuclear extracts (200 pl) were thawed at room temperature and mixed with 200 pl of a reaction buffer (10 mM Tris-HC1, pH 8.0, 5 mM MgCIz, 300 mM KCl, 1 mM CTP, 1 mM GTP, 6 mM dithiothreitol) containing 10 pl of [a-"P] UTP (3000 Ci/mmol) and 0.1 pmol of cold UTP. The mixture was incubated at 30 'C for 30 min with shaking. The isolation of 32P- labeled RNA and the hybridization of the RNA with various DNA probes immobilized on Genescreen Plus membrane were carried out as described by Greenberg and Ziff (1984).

Quantitation of ODC Protein-The amount of ODC protein in DF- 40 cells was measured by radiolabeling with 3H-labeled a-difluoro- methyl ornithine (DFMO) as described by Erwin et al. (1983). Briefly, cells were lysed in a hypotonic buffer consisting of 25 mM Tris-HC1 (pH 7.5), 0.1 mM EDTA, 5 mM dithiothreitol, and 50 p~ pyridoxal phosphate. Cells were homogenized by a brief sonication at 4 "C and centrifuged at 13,000 X g for 6 min. The labeling was carried out by incubating the supernatant with 3.6 p~ [3H]DFM0 (5 pCil200 pg of protein) at 37 "C for 5 h. The labeled mixture was dialyzed overnight against 50 mM Tris-HC1 (pH 7.5) containing 0.5 mM EDTA, 1 mM dithiothreitol, and 0.02% Brij 35, and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography.

Synthesis and Degradation of ODC Protein-Confluent cultures of DF-40 cells were serum-deprived for 24 h and then treated with asparagine at 10 mM in EBSS at 37 "C for different lengths of time. Cells were then pulse-labeled with [35S]methionine (176 pCi/ml) in the same medium for 15 min. Cell extracts were prepared for immu- noprecipitation and analyzed by SDS-PAGE and autoradiography as previously described (Persson et al., 1984).

Biochemical Assays-ODC activity in cell lysates was determined as previously described (Chen and Canellakis, 1977). One unit of activity is defined as the release of 1 nmol of CO, from ornithine per h a t 37 "C. Protein concentration was determined by a modified Lowry's method using bovine serum albumin as the standard (Ross and Shatz, 1973).

Materials-Tissue culture supplies were obtained from GIBCO. Biochemicals were purchased from Sigma. EN3HANCE, L-[~- '~C] ornithine (54.3 mCi/mmol), [EI-~HIDFMO (39.2 Ci/mmol), and Genescreen Plus membrane were purchased from Du Pont-New England Nuclear; SDS, agarose, [a-32P]dCTP (3000 Ci/mmol), [a- 32P]UTP (3000 Ci/mmol), and Tran~~~S-label (1000 Ci/mmol) were from ICN Chemical Radioisotope Division, Irvine, CA. Restriction enzymes, RNase, proteinase K, and a nick translational kit were obtained from Bethesda Research Laboratories.

RESULTS

Induction of ODC Actiuity by Asparagine-To ensure that the mode of regulation of ODC gene expression in the variant DF-40 cells is the same as that in its parental N2a cells, we compared the induction pattern of ODC activity in these two cell lines. Fig. 1 shows that the mode of induction of ODC activity in both cell lines was similar under the two different conditions examined. A growth stimulatory condition with fetal bovine serum caused a 30-100-fold increase in ODC activity in both N2a and DF-40 cells after 3-5 h of incubation in Dulbecco's medium. When cells were incubated in a salts/ glucose solution such as EBSS, asparagine alone was suffi- cient to cause a similar or greater increase in ODC activity in both the N2a and DF-40 cells. The maximal ODC activity in DF-40 cells, whether induced by serum or by asparagine, was 2 orders of magnitude greater than that in N2a cells. The maximal ODC activity in DF-40 cells generally represents a 200-fold increase over the basal level activity. It can be noted that the basal level of ODC activity varies from 5 to 20 units/ mg protein from experiment to experiment, depending on the length of serum deprivation and general conditions of cell culture. ODC induced by asparagine was metabolically active in uiuo as shown in Table I. When ODC activity was maxi- mally induced by asparagine, almost all the [3H]ornithine taken up by DF-40 cells was converted into putrescine within a time period of 1 h. No such metabolic conversion occurred if the cells were maintained in EBSS in the absence of asparagine. This experiment also showed that the presence of

0 10 20 0 10 TIME ( hr)

20 TIME ( h r )

FIG. 1. Comparison of the time course of the induction of ODC activity in N2a and DF-40 mouse neuroblastoma cells. Confluent cultures of mouse neuroblastoma cells were serum-deprived for 22 h in Dulbecco's medium. The medium was replenished with either fresh Dulbecco's medium containing 10% fetal bovine serum (0) or EBSS containing 10 mM asparagine (e) at time zero. Cells were then harvested at indicated times for the assay of ODC activity.

TABLE I Effect of asparagine on the metabolic conversion of ornithine to

polyamines in DF-40 cells maintained in EBSS Confluent cultures of DF-40 cells were incubated with 10 mM

asparagine for 8 h and E~[~H]ornithine was added to the cultures to a final concentration of 2 p~ (0.5 pCi/ml) and incubated for another hour. Cells were then harvested and processed for polyamine analysis by dansylation and thin layer chromatographic separation as previ- ously described (Chen et al., 1982). Radioactivity recovered in each cellular polyamine pool was calculated as the percentage of total radioactivity derived from [3H]ornithine taken up by cells. The data represent means f S.D. for triplicate experiments.

Percent of total radioactivity Polyamines recovered in cellular polyamines

EBSS EBSS + Asn Putrescine 0 Spermidine 4 + 2 1 f 0.5 Spermine 1 f 0.4 1 +- 0.4

85 -C 4

6948 Induction of ODC Gene Expression by Asparagine

asparagine did not stimulate the further conversion of putres- cine to either spermidine or spermine, suggesting that among all the polyamine biosynthetic enzymes asparagine affects only the key enzyme ODC.

The Effect of Asparagine on the Steady State Level of ODC mRNA and Protein-The ODC mRNA in DF-40 cells con- sisted of a major species of about 2.2 kilobases and a minor one of 2.6 kilobases (Fig. 2A), similar to that observed in other mouse cell lines (e.g. Kontula et al. (1984)). The time course of the increase in the level of ODC mRNA was exam- ined by Northern blot analysis (Fig. M), and the increase was further quantitated with slot blot analysis as shown in Fig. 2B. A 12-15-fold increase in the steady state levels of ODC mRNA was consistently observed 8 h after the addition of asparagine. To ensure an internal control, the same blot (Fig. 2 A ) was rehybridized with a @-actin probe (Fig. 2C). The effect of asparagine on the induction of ODC mRNA is quite dramatic considering the fact that growth stimulation by serum only caused a t most a 5-7-fold increase in ODC mRNA in DF-40 cells (Fig. 3). The time course of the induc-

TIMEchr) 0 2 4 8 16 TIME(hr)O 1 -. 2 -__. 4 8 ., 16

TIME(hr)

0 2

2.2kbA "' 4 8 16

(B) ODC

:4 t2 .0kb

( A ) ODC

(C) p-actin

FIG. 2. Effect of asparagine on the time course of the expression of ODC mRNA in DF-40 cells. A, Northern blot analysis. Confluent cultures of DF-40 cells were serum-deprived for 24 h. The medium was then changed to EBSS containing 10 mM asparagine. Cells were harvested at indicated times for RNA prepa- ration; 10 pg of total RNA was fractionated on a 1% agarose- formaldehyde gel and transferred onto a Genescreen Plus membrane. The membrane was hybridized with a 32P-labeled pODC54 plasmid. The size of ODC mRNA was determined using the 0.24-9.5-kilobase RNA ladder (Bethesda Research Laboratories). B, slot blot analysis of ODC mRNA in DF-40 cells induced by asparagine. Each slot contained 1.5 pg of poly(A)' RNA. C, internal control. The same membrane used for Northern blot analysis was washed and rehybrid- ized with the 32P-labeled @-actin probe.

Time(hr) FBS Asn Time(hr)

0

"., ". ". " r - w r - . 0

3

6

9

16

2

4

0

16

FIG. 3. Comparison of the effects of asparagine and growth stimulation on the induction of ODC mRNA in DF-40 cells. Confluent cultures of DF-40 cells were serum-deprived for 24 h, washed once with EBSS, and reincubated in EBSS containing 10 mM asparagine (Asn) or in Dulbecco's medium containing 10% fetal bovine serum (FBS) for various times as indicated. Cells were harv- ested at the indicated time for RNA preparation and slot blot analysis as described under "Experimental Procedures." Each slot contained 8 pg of total RNA.

tion of ODC mRNA by asparagine was different from that induced by serum. Nevertheless, in either serum- or as- paragine-treated cultures, the increase in the ODC mRNA levels correlated well with the increase in ODC activity (Fig. 3 uersus Fig. 1). The effect of asparagine on the amount of active ODC protein was examined by the affinity labeling technique developed by Pegg and colleagues (Erwin et al., 1983). Fig. 4 shows that on SDS-PAGE, ['HIDFMO radiola- beled a protein band with an apparent molecular mass of 53,000 daltons corresponding to the size of the monomer of ODC. Based on the labeling intensity of this band, we esti- mated that the amount of ODC protein was maximally in- duced by 160-240-fold.

The Effect of Asparagine on the Transcription of ODC Gene-Nuclear runoff transcription assay (Greenberg and Ziff, 1984; Clayton and Darnell, 1983) was employed to com- pare the level of the transcriptional activity of the ODC gene with other growth-dependent genes in DF-40 cells. Fig. 5 shows that asparagine did not significantly affect the tran- scription of ODC gene in DF-40 cells maintained in EBSS. At the time when ODC activity was maximal, the transcrip- tion of the gene was only 20-30% higher than that seen in the control. Interestingly, c-myc gene transcription was also observable at all time points and appeared to be slightly enhanced by asparagine.

Stability of ODC mRNA in DF-40 Cells-Since asparagine has little effect on the transcription of the ODC gene, the increase in the steady state level of ODC mRNA in the presence of asparagine is likely to be regulated post-transcrip- tionally. If this is the case, one may expect that (i) the stability of the induced ODC mRNA will be enhanced by asparagine

EBss+ Asn EBSS * TIME(hr) 0 2 4 8 12 16 24 8 " I .

t O D C

FIG. 4. Effect of asparagine on the time course of the in- crease in the amount of ODC protein. Confluent cultures of DF- 40 cells were serum-deprived for 24 h, washed with EBSS once, and then incubated in the EBSS containing 10 mM asparagine. Cells were harvested at various times for the preparation of cytosolic lysates and affinity-labeled by [3H]DFM0. The labeled proteins were analyzed by SDS-PAGE and fluorography. Each lane contained 200 pg of cytosolic proteins.

TIME(hr) 0 2 8 . . ~ . " . -~~ ." . ". . . I .

p-actin ODC

c-myc c-f0S

PBR 322

FIG. 5. Nuclear run-off assay of the effect of asparagine on the transcription of ODC and other genes in DF-40 cells. DF- 40 cells a t 90% confluence were serum-deprived for 24 h and then treated with 10 mM asparagine in EBSS. The nuclei of treated cells were isolated at various times indicated. Nuclear transcription assays were performed with 32P-labeled transcripts using ODC, c-myc, c-fos, @-actin, and pBR322 probes. The membrane was processed for auto- radiography, and the degree of relative transcriptional activity was estimated by densitometric tracing.

Induction of ODC Gene Expression by Asparagine 6949

and (ii) this stabilizing effect of asparagine will not be influ- enced by the inhibition of RNA synthesis. To test these possibilities, we have compared the stability of the induced ODC mRNA in the presence and in the absence of asparagine, with or without blocking of RNA synthesis. Fig. 6A shows that the induced ODC mRNA in DF-40 cells appeared to be very stable in the presence of asparagine, and the stability was not affected by the addition of actinomycin D (5 pg/ml). Actinomycin D at this concentration was effective in blocking RNA synthesis in DF-40 cells. Thus, after an incubation of 8 h with freshly added asparagine, the steady state levels of the induced ODC mRNA decreased by only 15-20% whether measured in the presence or absence of actinomycin D (Fig. 6A, lanes 1-6 versus lanes 6-11 ). Assuming that the decay of ODC mRNA after blocking RNA synthesis follows a first order kinetics, the half-life of ODC mRNA in the asparagine- treated cells, estimated by extrapolation, can be as long as 16-22 h. In contrast, Fig. 623, shows that after ODC mRNA was induced by asparagine ( l a n e 2 versus lane 1 ), the removal of asparagine from the culture resulted in a rapid decay of induced ODC mRNA (Fig. 623, lanes 3 and 6 versus lane 2). This rapid decay occurred both in the presence and in the absence of actinomycin D. The half-life of induced ODC mRNA in DF-40 cells treated with actinomycin D alone was estimated to be less than 25 min (Fig. 623, lanes 2-5). Taken together, Fig. 6 represents two parallel experiments which compared the effect of asparagine on the stability of ODC mRNA in the presence and in the absence of actinomycin D. These data indicate that asparagine caused an increase in the half-life of ODC mRNA by a t least 20-fold, from 25 min to more than 8 h, suggesting that asparagine is involved in post- transcriptional stabilization of ODC mRNA. That the stabi- lizing effect of asparagine on ODC mRNA was found to be independent of RNA synthesis is also consistent with the finding that asparagine did not significantly affect the tran- scription of ODC gene (Fig. 5).

The Effect of Asparagine on the Synthesis and Degradation of ODC Protein-Since asparagine induce a 15-fold increase

( A ) tAsn (B) -Asn

Act D -Act D +Act D -Act Q

T I M E ( hr) 8-_4-2_L5vO-%12-48- -7 0 1 3 5 1 3 5 " "

~"

L 2 k b -

1 2 3 4 5 6 7 8 91011 1 2 3 4 5 6 7 0

FIG. 6. A, effect of asparagine on the stability of induced ODC mRNA in the presence or absence of actinomycin D (Act D). Con- fluent DF-40 cells were serum-deprived for 20 h followed by an incubation in EBSS containing 10 mM asparagine. After 6.5 h of incubation, designated as time zero (lane 6), cultures were washed with EBSS and then incubated in fresh EBSS containing 10 mM asparagine. Actinomycin D was added to half of the cultures to a final concentration of 5 pg/ml (lanes 1-5) and the rest used as a control (lanes 7-1 I ). After further incubation for various times as indicated, the cells were harvested for RNA preparation and Northern blot analysis. Each lane contained 12 pg of total RNA. B, effect of removal of asparagine on the stability of induced ODC mRNA. Confluent DF- 40 cells were serum-deprived for 24 h (lune I ) , washed once with EBSS, and incubated in fresh EBSS containing 10 mM asparagine for 7 h (lane 2). Cells were then washed twice to remove asparagine and incubated in EBSS in the presence (lanes 3-5) or absence (lanes 6-8) of actinomycin D (5 pg/ml) for various times as indicated. At each time point, cells were harvested for RNA isolation and Northern blot analysis. Each lane contained 15 pg of total RNA.

in the ODC mRNA but a 200-fold increase in the amount of ODC protein, it is likely that asparagine also affected the regulation of ODC gene expression at translational and/or post-translational levels. Fig. 7 shows that at 4 or 8 h after incubation with asparagine, the 15-min incorporation of [3sS] methionine into ODC protein was 90-140-fold higher than that seen in the control. Such an increase correlated well with the increase in the total amount of active ODC protein. This increase appeared to be quite specific for ODC because as- paragine did not affect general protein synthesis in cultured cells (data not shown). Although the pulse-labeling period was as short as 15 min, it is still possible that the labeling of the 53,000-dalton band may represent a steady state level if the half-life of ODC is about 15 min or shorter. To examine this possibility we measured the decay rate of ODC protein in the presence and absence of asparagine in cells maintained in salts/glucose solution. Fig. 8A shows that within 1 h after the withdrawal of asparagine from the culture, more than 95% of the active ODC protein disappeared with a half-life estimated to be about 12 min ( l a n e 2 uersus lane I). In contrast, the steady state amount of ODC protein remained

TIME( hr) 0 4 8 0 4 8

-53K

FIG. 7. Effect of asparagine on the incorporation of [36S] methionine into newly synthesized ODC protein. Confluent DF- 40 cells were serum-deprived for 24 h, washed with EBSS twice, and then incubated in fresh EBSS containing 10 mM asparagine. At the designated time after incubation cells were pulse-labeled with [35S] methionine (176 pCi per ml) for 15 min, followed by washing with EBSS containing 2 mM methionine. Cells were harvested in a hypo- tonic buffer, and cytosolic extracts were prepared as described. The cytosolic lysates (100-pg proteins) were processed for immunoprecip- itation, SDS-PAGE analysis, and autoradiography. Results from two separate experiments are shown.

(A) (6)

" -Asn + A m

TIME(hr) 0 1 3 0 3 T I M E ( b ) 0 1 3 5

-97K - 6 6 K -43K

t 53K - 31K

- 21 5 K -14K

1 2 3 4 5 6 7 FIG. 8. A, effect of asparagine on the stability of ODC protein.

Confluent cultures of DF-40 cells were treated with 10 mM asparagine in EBSS for 7 h (lanes 1 and 4 ) . Cell cultures were then washed twice with EBSS and then incubated in fresh EBSS without (lunes 2 and 3) or with (lanes 5-7) 10 mM asparagine present for additional times as indicated. At the designated time cells were harvested, an equal

with ["HJDFMO and analyzed by SDS-PAGE and fluorography. B, amount of cytosolic proteins (50 pg) was processed for affinity labeling

effect of cycloheximide on the stability of ODC protein in asparagine- treated DF-40 cells. Confluent cultures were serum-deprived for 20 h and treated with 10 mM asparagine in fresh EBSS for 4 h. Cyclohex- imide was then added to the cultures (final concentration, 50 pglml), and cells were harvested at various times thereafter for ODC quant- itation. An equal amount of cytosolic proteins (40 pg) was processed for labeling and analyzed on a 12% mini SDS-polyacrylamide gel.

6950 Induction of ODC Gene Expression by Asparagine

constant for several hours if the culture was replenished with fresh asparagine (lanes 4-7). Since the half-life of ODC in DF-40 cells measured in the presence of 50 pg/ml cyclohexi- mide was found to be enhanced by 3-4-fold with asparagine (Fig. 8B), we concluded that asparagine not only stimulated the synthesis but also suppressed the rapid degradation of ornithine decarboxylase protein.

DISCUSSION

We have previously shown that a single amino acid, as- paragine, is necessary and sufficient to induce maximal ODC activity in various tumor cells maintained in a salts/glucose solution (Chen and Canellakis, 1977; Viceps-Madore et al., 1982; Chen and Liu, 1983). We and others also showed that in primary cultures such as hepatocytes or differentiated mouse neuroblastoma cells maintained in the salts/glucose solution, the maximal induction of ODC activity requires the presence of both asparagine and serum, or growth hormones (Chen, 1980; Kanamoto et al., 1987). In all cells examined, serum or growth factors alone could not induce ODC activity if cells are maintained in salts/glucose solution. The effect of asparagine is rather specific; other A and N system amino acids are only marginally effective (Chen and Canellakis, 1977; Viceps-Madore et al., 1982). It is possible that the induction of ODC activity by various growth factors or hor- mones under physiological conditions may involve the action of asparagine. In light of this possibility it is of interest to investigate the molecular mechanism by which asparagine induces maximal ODC activity.

Our study demonstrates that the mode of ODC induction by asparagine in DF-40 mouse neuroblastoma cells is similar to that in N2a cells, except that the magnitude of induction is much greater in DF-40 cells. Among various steps involved in gene expression, the post-transcriptional stabilization of ODC mRNA appears to be the key factor for the increase in the levels of ODC mRNA in DF-40 cells induced by as- paragine. The presence of asparagine in a salt/glucose solution causes an increase of the half-life of the induced ODC mRNA by at least 20-fold with or without actinomycin D present (Fig. 6) . Although asparagine does not significantly stimulate the transcription of the ODC gene (Fig. 5), its stabilizing effect is more than enough to explain the increase in the level of ODC mRNA. The mechanism of mRNA turnover in general is poorly understood. There is evidence, however, that desta- bilizing sequences lie in the 3'-untranslated region (Shaw and Kamen, 1986; Brewer and Ross, 1988). Many transiently expressed genes such as c-fos, c-sis, and c-myc contain AT- rich regions at their 3'-end with a core sequence ATTTA (Shaw and Kamen, 1986). Interestingly, murine ODC gene also contains the ATTTA motif within its 3'-end (Kahana and Nathans, 1985). Whether the action of asparagine in- volves the sequence of the 3"untranslated region of ODC mRNA remains to be studied.

The increase in the amount of ODC protein (Fig. 4) corre- lates well with the increase in ODC activity (Fig. l), support- ing the notion that ODC activity is regulated by changes in protein amount (Pegg, 1989). The 150-240-fold increase in ODC protein stimulated by asparagine cannot be accounted for solely by the 15-fold increase in the level of ODC mRNA, suggesting that asparagine also affects translational and/or post-translational steps involved in ODC gene expression. Although the pulse-labeling data (Fig. 7 ) suggest that as- paragine stimulates the synthesis of ODC by 6-9-fold, this value is probably an overestimate because (i) ODC protein has a very short half-life, as short as 12 min in DF-40 cells when maintained in a salts/glucose solution (Fig. 8 A ) and (ii)

asparagine causes a 3-5-fold increase in the half-life of ODC protein (Fig. 8, B uersus A ) . Based on these considerations, we estimated that asparagine stimulates the rate of synthesis of ODC protein by only 3-4-fold. Taken together, our data indicate that the 200-fold increase of ODC activity induced by asparagine could be accounted for by (i) post-transcrip- tional stabilization of ODC mRNA (-15-fold), (ii) post-trans- lational stabilization of ODC protein (-4-fold), and (iii) a 4- fold increase in translation of the ODC message.

Using DFMO-resistant L1210 cells, Poulin and Pegg (1990) have recently found that hypotonic shock dramatically in- creases the rate of ODC synthesis by up to 36-fold and extends the half-life of ODC activity by 6-fold without detectable change in ODC mRNA levels. In light of this study, it is possible that the effect of asparagine on the stabilization of ODC mRNA may not be related to the osmoregulatory volume change generally associated with the Na+-dependent amino acid transport (Hudson and Schultz, 1988).

Kanamoto et al. (1987) have studied the regulation of the ODC gene in the primary cultures of rat hepatocytes. They found that when hepatocytes are incubated in the saltsf glucose medium, ODC activity is induced about 100-fold with asparagine and glucagon added together. Glucagon alone is ineffective and asparagine alone is only 30% as effective as the combination of asparagine and glucagon. Furthermore they found that asparagine alone has little or no effect on the level of ODC mRNA whereas glucagon alone increases the amount of ODC mRNA by 3-4-fold. They concluded that asparagine stimulates translation by 6-fold and glucagon in- creases ODC mRNA by %fold. Our findings differ from what they have reported in the effect of asparagine on the induction of ODC mRNA. Such a discrepancy is likely due to a differ- ence in cell type used in these two studies. Ours is a tumor cell line whereas theirs is a normal, primary cell strain. Indeed, we have recently found that asparagine caused a large increase in the ODC mRNA level in v-Ha-ras-transformed NIH 3T3 cells but not in NIH 3T3 cells when both cells were main- tained in a saltsfglucose solution? It is possible that the asparagine-mediated post-transcriptional stabilization of ODC mRNA may represent a component of an altered growth regulatory program associated with cell transformation.

Acknowledgments-We are grateful to Drs. 0. Janne, Rockefeller University, K. B. Marcu, SUNY at Stony Brook, and I. M. Verma, The Salk Institute, for providing us, respectively, with pODC54, pMC-myc, and pfos-1 plasmids. We also acknowledge Dr. P. P. McCann, Merrel Dow Research Institute, for giving us DFMO.

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