enhancement of expression of exogenous genes by 2-aminopurine

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc

Vol . 266, No. 2, Issue of January 15. Prmted m U. S. A. pp: 873-879,1991

Enhancement of Expression of Exogenous Genes by 2-Aminopurine REGULATION AT THE POST-TRANSCRIPTIONAL LEVEL*

(Received for publication, July 20, 1990)

Dhananjaya V. R. Kalvakolanu, Sudip K. Bandyopadhyay, Raj K. TiwariS, and Ganes C. Sen8 From the Department of Molecular Biology, The Cleveland Clinic Foundation, Cleveland, Ohio 44195

In human and mouse cell lines, expression of exogenous genes was enhanced by treatment with 2-aminopurine (2-AP). Chloramphenicol acetyltransferase (CAT) and neomycin phosphotransferase activities were increased by up to SO-fold upon 2-AP treatment of cells permanently transfected with genes encoding these enzymes. Neomycin phosphotransferase activity was also increased by this treatment in cells infected with a retroviral vector carrying the neomycin phosphotransferase gene. 2-AP-mediated increase in CAT activity was observed in various cell lines which had been permanently transfected with chimeric CAT genes containing transcriptional regulatory elements of the interferon-inducible genes 6-16 and 561, SV40 early genes, mouse mammary tumor viral gene, or metallothionein I1 gene. The increase in the cellular CAT enzymatic activity was due to an elevated level of CAT protein. The 2-AP-mediated enhancement of CAT expression was operative at the translational level; the rate of transcription of CAT mRNA or its steady-state level was affected only marginally. The translational up-regulation by 2-AP was restricted to the genes introduced from outside; there was no gross change in the rate of synthesis of other cellular proteins.

We have been studying the regulation of expression of interferon (1FN)’-a inducible genes. This regulation in the HeLaM cells is atypical in the sense that the transcriptional induction of many genes by IFN-a, in this cell line, requires ongoing protein synthesis (1,2). This need for ongoing protein synthesis can be obviated by pretreating the cells with IFN- y. We proposed that both IFN-y and IFN-a can elicit a signal, signal 1, which leads to the synthesis of a putative protein. This protein by itself, however, is not sufficient for transcriptional induction of the genes that we tested. Signal 2, generated by IFN-a but not by IFN-y, is also needed for this process. We showed that some of these genes, e.g. 561 and 6- 16, can also be induced directly by double-stranded (ds) RNA

* This work was supported in part by United States Public Health Service Grant AI-22510 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

NY 10021. 4 Current address: Memorial Sloan-Kettering Center, New York,

0636. I To whom all correspondence should be addressed. Tel.: 216-444-

‘The abbreviations used are: IFN, interferon; dsRNA, double- stranded RNA, 2-AP, 2-aminopurine; CAT, chloramphenicol acetyltransferase; ELISA, enzyme-linked immunosorbent assay; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; eIF-2, eukary- otic initiation factor-2.

(3). But unlike IFN-a, although dsRNA can produce a signal functionally equivalent to signal 2, it cannot produce signal 1.

We examined whether the dsRNA-dependent protein kinase might be involved in the transduction of dsRNA-elicited signals necessary for gene induction. We and others observed that 2-aminopurine (2-AP), a known inhibitor of the dsRNA- dependent protein kinase, can selectively block the induction of genes 561 and 6-16 by dsRNA (4, 5). Similarly, induction of the IFN-P gene by dsRNA is also blocked by 2-AP (6, 7). Our experiments with HeLaM cells demonstrated that 2-AP blocks the production of functional signal 2 by dsRNA, but not by IFN-a. In contrast, signal 1 production by IFN-a or by IFN-0 is blocked by 2-AP (4).

To obtain a better understanding of the mechanism of IFN- mediated signal transduction, we generated a series of HeLaM cell lines which had been permanently transfected with chimeric genes containing the coding region of the bacterial chloramphenicol acetyltransferase (CAT) gene and IFN-responsive regulatory regions of different IFN-a inducible genes (8-12). We observed that in these transfectants, CAT enzymatic activity was induced by IFN-a and that transcriptional induction of the CAT gene had the same characteristics as that of the resident IFN-inducible genes. For example, in both cases, generation of signal 1 and signal 2 was needed. Signal 1 production and hence the transcriptional induction of the resident and the transfected gene, as analyzed by nuclear run- off transcription, was blocked by 2-aminopurine.’

During these studies, we observed that although 2-AP blocked the induction of CAT mRNA transcription by IFN, it enhanced the level of basal CAT enzymatic activity present in treated or IFN-untreated cells. In a series of experiments reported here we demonstrated that this elevation of CAT activity was not due to an elevated level of CAT mRNA but rather due to a better translatability of the CAT mRNA in 2- AP-treated cells. This phenomenon was true for another transfected gene as well. Moreover it appears to be independent of the recipient cell line and of the promoter/enhancers driving the expression of the transfected gene. Interestingly, the same 2-AP-mediated enhancement of expression was also observed for an exogenous gene which was introduced via infection with a retroviral vector.

EXPERIMENTAL PROCEDURES

Chemi~ak-[[y-~*P]ATP, [’4C]chloramphenicol, [w3’P]CTP, and [a-”PIUTP were obtained from Amersham Corp. CAT-ELISA kit was purchased from 5’ to 3’ Inc., West Chester, PA. Minimum essential medium, Dulbecco’s modified Eagle’s medium, and G418 were purchased from GIBCO, Grand Island, NY. Acetyl-coA was obtained from Boehringer Mannheim. Kanamycin sulfate and 2- aminopurine were purchased from Sigma. Sources of IFN-CY and VSV-

* D. V. R. Kalvakolanu, S. K. Bandyopadhyay, and G. C. Sen, unpublished observations.

873

a74 Translational Regulation by 2-Aminopurine

Dl411 were descrihed in earlier publications ( 3 ) . All other chemicals used were of analytical grade.

Plasmids and Cdl Clulture"CS20:XAT, a chimeric gene containing upstream sequences from the IFN-inducible human 6-16 gene placed 5' to the CAT gene was a gift from George Stark (Imperial Cancer Research Fund, London). The sequences represent the -603 to +437 part of the 6-16 gene promoter (11). pRM-CAT plasmid contained promoter sequences from -621 to +B positions of the IFN inducible 56K gene placed upstream of CAT gene ( 1 3 ) . This plasmid was a gift from Marc Wathelet (University Libre de Bruxelles, Hrussels). pHSI- CAT plasmid has CAT gene with the upstream promoter sequences of the metallothionein I 1 gene. This plasmid was a generous gift from Michael Karin (University of California at San Diego). Construction of pSV2-CAT and its structure were described earlier (14) . Plasmid pIPB-1 carried the 'heo" gene driven by the herpes simplex virus- thymidine kinase gene promoter and 3'-polyadenylylation sequences. The plasmid was provided by Richard Axel (Columhia University, New York). pSV2-neo (15) contained SV40 enhancer and promoter sequences placed upstream of the neo gene. pMAM-neo CAT was purchased from Clonetech Laboratories, Inc., Palo Alto. CA. This plasmid contains the dexamethasone-inducible murine mammary tumor virus-long term repeat promoter upstream of the CAT gene. In addition, there is a neo gene under the control of SV40 early promoter in the same plasmid. 1,929 cells and HeLaM cells were cultured in Dulhecco's modified Eagle's medium and minimal essential medium, supplement.ed with 10% fetal calf serum, respectively. All plasmids were transfected into HeLaM or 1,929 cells by the calcium phosphate precipitation method (16, 17). A dominant selection marker plasmid, either pSV2-neo or pIPB-1, was also co-transfected into the cells. Cells were selected (1 mg/ml of G418) for neomycin resistance and individual clones as well as their pools were isolated and tested for expression of CAT gene. CG1 Neo' cells contain the neo gene introduced into CGl cells by retroviral infection (18).

Enzvrne Assays-Extracts from transfected cells were prepared by 3 cycles of freeze-thaw lysis. Chloramphenicol acetyltransferase (CAT) was assayed using equal amounts of total protein from transfected cells as per previously descrihed protocols (14). The assay mixture contained 30 p l of 1 M Tris-CI, pH 7.8, 2.5 pl of ["C] chloramphenicol, 20 pI of cell extract, 12.5 pl of water, and 10 p1 of 4 mM acetyl-coA. After incubating for 1 h a t 37 "C, the samples were extracted with 1 ml of ethyl acetate, organic phases were collected, dried. and dissolved in 30 p l of ethyl acetate. The samples were spotted on Silica Gel 60 thin layer chromatographic plates (CAT No. 5748, EM Science, Cherry Hill, Nd) and the plates were developed in a solvent system comprising chloroform and methanol in a ratio of 955 (V/V). After chromatography, the plates were dried and autoradiographed. Spots corresponding to acetylated and nonacetylated chloramphenicol were excised from the chromatogram, counted in a liquid scintillation counter, and percent acetylations were calculated. In order to accurately determine the fold stimulation values, we used suitable protein concentrations for assaying different samples so that the enzyme activity remained within a linear response range.

Neomycin phosphotransferase (NPT-11) activity was determined according to the protocol described above hy Fregien and Davidson (19). Briefly, equal amounts of cytoplasmic extracts were electropho- resed through a 7.5% nondenaturing polyacrylamide gel and the gel was incuhated in the enzyme assay buffer for 45 min at 37 'C. The assay buffer contained 20 mM Tris-CI, pH 7.3, 13 mM MgCI,, 100 mM NH,CI, 0.5 mM dithiothreitol, 62.5 pg/ml of kanamycin sulfate, 1.25 mM ATP, and 20 pCi/ml of [y-:"P]ATP (3000 Ci/mmol). After the assay, the products of enzyme activity were blotted onto a phospho- cellulose paper, Whatman P81, in a Southern t-ype of transfer using distilled water as a hlotting solution. The hlots were washed with distilled water twice at 70 "C, dried. and autoradiographed.

ELISA-Sandwich enzyme-linked immunosorbent assay was used to determine the level of CAT protein in the total cytoplasmic extracts. CAT-ELISA was performed according to manufacturer's specifications. Pure CAT protein, ranging from 50 to 300 pg, was used to generate a standard plot. The absorbance values from test samples were extrapolated over a standard plot and CAT protein amounts were determined. Briefly, the extracts containing CAT protein were hound to ELISA plates for 2 h at room temperature. Plates were washed and incuhated with hiotinylated anti-CAT antibody for 1 h a t room temperature. After washing the plates extensively, strep- tavidin-conjugated alkaline phosphatase was added and incuhated for 30 min a t room temperature. The wells were washed again and incuhated with substrate solution containing p-nitrophenol phos-

phate for 30 min a t room temperature. Absorbance at 405 nm was determined in an ELISA reader. Irntransfected cell extracts were assayed for hackground activity.

RNase A I'rotwtion Assay-RNase A protection assay I I f . 201 was performed using a 250-hase long. "1'-labeled antisense CAT RNA prepared from a pSP64 vector rarrving the CAT gene. Cells were treated with various agents for 6 h and total cytoplasmic RNA was prepared (19). Equal amounts of total cytoplasmic RNAs (100 pgl were hybridized to the antisense CAT RNA prohe in hybridization huffer ( 4 0 mM HEPES, pH 6.3.400 mM NaCI. 1 mM EDTA. and Ho'; formamide) overnight at 45 "C and then digested with RNase A ( 4 0

pg/ml) for 1 h at 30 "C. After phenol extraction and ethanol precipi- tat ion, the samples were separated on a 10'; urea/polyacl?;lamitle gel. The gels were dried and autoradiographed.

Nuchar Run-off Transcriplion-l'rocedures were descrihed else- where (2). Cells were treated with different agents, lysed after 3 h of exposure, nuclei were isolated, and nascently synthesized RNA was prepared using [u-"l']UTl'. The RNAs. thus ohtained. were hybridized to CAT gene, and 6-16 gene immohilized on nitrocellulose filters. h DNA was used as negative control. Following hvhridization for 72 h at 42 "C, the filters were washed extensively with 2 X SSC. O.lrE sodium dodecyl sulfate four times at room temperature. and with 0 . 1 X SSC (1 X SSC: 15 mM sodium citrate. 150 mM NaCl (pH 7.0)), 0.1% sodium dodecyl sulfate for three times at 6.5 "C. The filters were then treated with RNase A I50 pg/ml) for 30 min at 37 "C, washed. dried, and autoradiographed. For these assays, purified CAT insert and 6-16 cDNA insert were used as immohilized probes.

RESULTS

Induction of CA T Activity by 2-AP-To gain further understanding of the mechanisms of gene induction by IFNs in HeLaM cells, we studied the characteristics of induction of CAT activity in HeLaM cells which had been permanently transfected with a chimeric gene, GS203CAT, containing the upstream regulatory region of the IFN-inducible gene 6-16 attached to the CAT coding sequence. Permanent transfectants were isolated by G418 resistance imparted by a co- transfected neo gene. Individual clonal derivatives were tested for induction of CAT activity by IFN-n. In one such clone, IFN-n strongly induced CAT activity (Fig. lA, lanes I and 21, whereas IFN-7 induced it only marginally (data not shown). We have reported previously that 6-16 mRNA transcription in HeLaM cells is induced not only by IFN-n but also by a consecutive treatment with IFN-7 followed by an infection with VSV-DI-011, which is a defective interferring particle of vesicular stomatitis virus carrying a defective dsRNA genome (3). The latter treatment also induced CAT activity (Fig. 1H. lane 6) demonstrating that the resident 6-16 gene and the transfected chimeric gene are induced by similar agents. To

(A) (8)

96 Acetylation 4 33 12.2 46 8.5 58.6 84.1 94-0

a

a - 0 I d.

1 2 3 4 5 6 7 8

FIG. 1. Effect of various agents on CAT activity in HeLaM cells transfected with CS203CAT. A, effect of IFN-cr and 2-AI' on CAT activity in HeLaM (iS2O:ICAT CAT clone 4. Imnr /, no treatment: lane 2; hlFN-cr (.500 units/ml): lane 3. 2-AP 1 1 0 mM); lanp 4, hIFN-cr (500 units/ml) and 2-AI' (10 mM). Cells were treated with these agents for 16 h and 48 pg of total protein from each sample was assayed for CAT activity. H . effects of other inducers in HeLaM GSZORCAT CAT clone 12. lane 5. no treatment; lane 6. hlFN-y (500 units/ml) for 6 h, followed hy infection with VSV-Dl-011 ( 3 1 for 12 h; lane 7, 2-AI' (10 mM): lane 8. hlFN-y ( 5 0 0 units/ml) and ?-AI' ( 1 0 mM) followed hy infection with VSV-Dl-011. Total cell extracts were prepared and 192 pg of protein was assayed for CAT activity.

Translational Regulation by 2-Aminopurine 875

compare the induction patterns of the two genes further, the effects of 2-aminopurine were examined. We have shown previously that in HeLaM cells, 2-AP blocks gene induction by IFN-a as well as that by dsRNA (4). Surprisingly, in the transfectants, induction of CAT activity by IFN-a (Fig. lA, lane 4 ) or by VSV-DI-011 (Fig. lB, lane 8) was not blocked by 2-AP although in the same transfectant the induction of the 6-16 mRNA by the same agents was completely blocked by 2-AP (data not shown). Quantitation of the CAT activity indicated that cells treated with IFN-a and 2-AP had a higher level of CAT activity as compared to the cells treated with IFN-a alone (lanes 2 and 4). Thus, it appeared that 2-AP was boosting the CAT activity in both IFN-a- and dsRNA-induced cells. Indeed this was true in untreated cells as well. Treat- ment with 2-AP alone increased the level of CAT activity (Fig. 1, lanes I, 3.5, and 7). This increase was not due to the acetic acid present in 2-AP solutions; and 2-AP also did not have any effect on CAT enzymatic activity when added directly to the assay mixture (data not shown). Moreover, an extract made from untransfected HeLaM cells treated with 2-AP did not boost CAT activity when mixed with an extract containing this enzyme, ruling out the possibility of a 2-AP metabolite being responsible for the boost in enzyme activity (data not shown).

2-AP Induction of CAT Activity Is Cell and Promoter In- dependent-In the experiments shown in Fig. 1, induction of CAT activity by 2-AP was measured in two clonal lines of GS203CAT-transfected HeLaM cells. To test the universality of this phenomenon, we repeated the experiments with a pool of HeLaM transfectants containing approximately 120 independent clones. As shown in Fig. 2 (lanes I and 2), 2-AP could induce CAT activity strongly (18-fold) in this pool indicating that the phenomenon is not restricted to specific clones. The same conclusion was also reached by testing several clones individually (data not shown). Although the absolute degree of stimulation of CAT activity by 2-AP varied among the different clones tested, the stimulation was substantial in all of them. However, there were some clones in which neither a basal CAT activity was detectable, nor could 2-AP boost the activity to a detectable level (data not shown). The observed 2-AP-mediated stimulation was not restricted to human HeLaM cells. CAT activity was stimulated 20-fold (Fig. 2, lanes 3 and 4) in a pool of permanent transfectants of mouse L929 cells in which the same chimeric CAT gene had been introduced.

In the next series of experiments we investigated whether CAT expression from other chimeric CAT genes can be stimulated by 2-AP as well. For this purpose, several chimeric CAT genes containing various promoter-regulatory elements were transfected into HeLaM cells and permanent transfectants were isolated by selection with G418. Effects of 2-AP

%Acetylation 1.3 239 1.5 30.7

a

' 0

1 2 3 4

FIG. 2. Effect of 2-AP on CAT activity in pools of clones. CAT activity was measured in HeLaM and L929 cells, after isolating the pools of cells, transfected with CS203CAT. Lanes 1 and 2, HeLaM cells; lanes 3 and 4, L929 cells. Lanes 1 and 3 were left untreated, lanes 2 and 4 were treated with 10 mM 2-AP for I5 h in growth medium. Total cell extracts were made and 5 pg of protein was used in each assay.

on CAT expression in these transfectants were tested (Fig. 3). In a clone transfected with pRMCAT (driven by the regulatory elements of the IFN-inducible I F 4 6 gene), CAT activity was stimulated 43-fold by 2-AP (Fig. 3, lanes I and 2). Similar stimulation was observed in a transfectant containing a chimeric CAT gene (a gift of P. Pitha, .Johns Hopkins University, Baltimore) (21) in which the IFN-responsive promoter was a tetramer of AATGAA (data not shown). 2-AP stimulated CAT expression from other promoters as well. In cells transfected with pSVZCAT, in which CAT expression is driven by the SV40 enhancer/promoter elements, the basal level of CAT expression was high (Fig. 3, lane 3) , but it was elevated further by 2-AP (lane 4 ) . In cells transfected with a CAT transcription unit driven by the metallothionein promoter, 2-AP had similar effects (fanes 5 and 6). All permanent transfectants expressing CAT from different transcription units which were tested for the 2-AP response were isolated by selection with G418. This selection procedure took advantage of co-transfection of the test vector with a tk-neo or a pSV2-neo gene. We wondered whether co- transfection with the two similar plasmid vectors, one containing the CAT gene, and the other containing the neo gene contributed to the 2-AP-stimulated phenomenon. To test this idea, a single expression vector containing both the CAT and the neo gene was transfected into HeLaM cells. Transcription of CAT was driven by the glucocorticoid-responsive murine mammary tumor virus promoter and the neo gene was driven by the SV40 promoter. The transfectants were selected by G418 resistance. As shown in Fig. 3, lanes 7 and 8, 2-AP stimulated CAT expression in these cells strongly. The degree of stimulation by 2-AP was very similar in another set of transfectants (lanes 9 and IO), which were co-transfected with pSV2-neo and the murine mammary tumor virus-CAT vector. These results indicate that neither the mode of selection of the transfectants nor the nature of the transcriptional regulatory elements of the chimeric CAT gene is critical for eliciting the 2-AP-mediated stimulation of CAT activity.

Dose-Response and Kinetics-To characterize the variable parameters of the 2-AP response, dose-response and kinetic analyses were done. The degree of stimulation of CAT activity increased with increasing doses of 2-AP within the range of concentrations tested (Fig. 4). The dose-response for this activity was somewhat different from that observed for inhibition of IFN-inducible gene expression (4). In all three sets of transfectants, a pool of HeLaM cells transfected with GS203CAT. a pool of L929 cells transfected with the same

XAceW8bon 1.7 72.7 7 4 31.0 1.5 24.5 0.6 11.8 0 8 17.8

m

0 - 0 0 0 0

1 2 3 4 5 6 7 8 9 1 0

FIG. 3. Effect of 2-AP on CAT gene expression driven by various promoters. CAT plasmid constructs containing various promoters or enhancers were transfected into HeLaM cells and stably transfected cell clones as well as their pcmls were isolated. The transfectants were then treated with 10 rnM 2"AP. I.:qual amounts o f protein from untreated and 2-AP-treated cell extracts were assayed for CAT activity. Lanes 1 and 2. pRM-CAT clone 11; lanes 9 and 4 . pSV2-CAT; lanes -5 and 6, pHSI-CAT; lanes 7 and 8. pMAM-neo- CAT pool-1; laws 9 and I f ) . pMAM-neo CAT pool-2. In the ca.se of pMAM-CAT-neo pool-2, cells were co-transfected with pSV2"neo. Lanes I , 3. 5. 7. and 9 were left untreated and lanes 2. 4 . 6. 8. and 10 were treated with 10 mM 2-AP for 15 h in growth medium. The amounts of proteins used were: lanes I and 2. 4 0 pg; lanes 9 and 4, 2 pg; lanes 5 and 6. 134 pg; lanes 7-10, 194 pg.

876 Translational Regulation by 2-Aminopurine

3

n w (hr)

FIG. 6. Stimulation of CAT activity by 2-AP is due to increased amounts of CAT protein. Cell extracts were prepared after different treatments and assayed for CAT protein content using a sandwich ELISA. A, the effect of 2-AP (10 mM) on the amount of CAT protein in HeLaM GS203CAT pool (13) and L929 GS203CAT pool (W). 1 and 3, untreated cell extracts; 2 and 4, 2-AP (10 mM) treated cell extracts. R, the kinetics of 2-AP treatment on HeLaM pSV2-CAT clone. Cells were treated with 2-AP (10 mM) for various lengths of time and cell extracts were assayed for CAT protein as described above.

Y

0 2 4 6 8 1 0 1 2

2-AP (mM) FIG. 4. Effect of concentration of 2-AP on CAT activity.

Stably transfected cell lines containing HeLaM-PRM-CAT clone 11 and pools containing GS203CAT in HeLaM and L929 cells treated with various concentrations of 2-AP for 14 h in growth medium. Cell extracts were prepared and 20 pg of total cell protein from each sample was assayed for CAT activity. HeLaM GS203CAT pools (0); L929 GS203CAT pool (W); and HeLaM pRM CAT clone 11 (A).

50 r C

3 ,/// IFN- a

0 0 10 20

TIME (hr)

FIG. 5. Kinetics of stimulation of CAT activity in HeLaM cells. HeLaM cell clones containing GS203CAT (0) and pSV2-CAT (0) were treated with 2-AP (10 mM) for various lengths of time and cell extracts were prepared. Equal amounts of protein from HeLaM GS203CAT clone 4 (24 pg) and pSV2-CAT (2 p g ) were assayed for CAT activity.

2-AP

FIG. 7. Effect of 2 - A P on the rate of transcription of CAT and endogenous genes. HeLaM GS203CAT clone 4 cells were treated with none ( C ) IFN-n (500 units/ml), and 2-AP (10 mM) for 3 h. Nuclei were isolated and run-off transcriptions were performed as described under "Experimental Procedures." Total nuclear RNAs were hybridized to nitrocellulose filters containing A phage DNA ( A ) . CAT gene insert (CAT), and cDNA insert of the IFN-inducible 6-16 gene (6-16).

chimeric gene, and a clone of HeLaM cells transfected with pRM-CAT, an increase in CAT activity was observed even a t the lowest dose tested (2.5 mM). The absolute levels of basal CAT activity were different in different cell lines as were the maximum levels of CAT activity observed in cells treated with 10 mM 2-AP. The maximum increase in activity in the different cell lines varied from about 10-fold to about 35-fold. Even the highest dose of 2-AP did not have any gross adverse effect on cellular metabolism. Total cellular protein synthesis and RNA synthesis were only marginally (less than 5%) decreased and the overall pattern of protein synthesis as revealed by electrophoretic analysis of [:''SS]methionine-Iabeled proteins was not affected by 10 mM 2-AP (data not shown).

The manifestation of the stimulating effect of 2-AP was a relatively slow process (Fig. 5). The kinetics of induction were measured in two cell lines. In a GS203CAT-transfected HeLaM clonal line an increase inactivity was observable 3 h after the beginning of 2-AP treatment and it continued up to the last time point tested (12 h). In the pSV2-CAT-transfected cells, the basal level of CAT activity was high, but it increased further with increasing periods of incubation with 2-AP.

Mechanism of Induction of CAT Activity by 2-AP-For understanding the molecular basis of induction of CAT enzymatic activity by 2-AP, a detailed analysis of CAT gene expression in 2-AP-treated cells was undertaken. CAT protein levels were measured by ELISA using pure bacterial CAT protein as the standard. As shown in Fig. 6A, in GS203CAT- transfected HeLaM cells and L929 cells, 2-AP treatment increased the CAT protein level by about 12- to 15-fold. In pSV2-CAT-transfected cells, the increase was about 6- to 7 -

fold (Fig. 6B). The kinetics of increase in CAT protein level mirrored the kinetics of increase in CAT enzymatic activity (Figs. 5 and 6B), suggesting a direct causal relationship be- tween them. The observed increase in CAT protein level in 2-AP-treated cells was not due to an increased stability of the protein. In both 2-AP-treated and untreated cells, CAT activity remained constant over a period of 16 h even if new protein synthesis was blocked by cycloheximide (data not shown), suggesting that CAT protein has a long half-life irrespective of 2-AP treatment.

An increased steady-state level of CAT protein in the 2- AP-treated cells without a change in iL9 turnover rate indicated an enhanced rate of its synthesis. T o investigate whether such an increase in synthesis was due to an elevated level of CAT mRNA, i t s rate of transcription and i t s cellular steady-state level were measured. Run-off transcriptions were done using nuclei isolated from untreated, IFN-a-treated, and 2-AP-treated cells and the rates of transcription of CAT mRNA and 6-16 mRNA were measured. As shown in Fig. 7 , IFN-a treatment strongly increased the rate of transcription of both CAT mRNA and 6-16 mRNA. In contrast, these rates were not increased by 2-AP after 3 h of treatment although by this time an increase in CAT enzymatic activity was already observable (Fig. 5). A longer exposure of the autora- diogram of Fig. 7 showed a low level of CAT mRNA transcription in the 2-AP-treated cells, however, the level was not higher than the basal level of transcription of this mRNA in


the untreated cells. These results indicate that unlike IFN-a, 2-AP does not induce CAT activity in these transfectants by increasing the rate of transcription of CAT mRNA. Similarly the steady-state level of CAT mRNA was also not increased appreciably in 2-AP-treated cells. The steady-state levels of CAT mRNA were measured by a RNase-protection assay. Two CAT-related RNAs were present in untreated cells (Fig. 8) and their levels were appreciably increased by IFN-a treatment. In contrast, the levels of these two RNAs were only marginally (at most 2-fold) increased in the 2-AP-treated cells. This slight increase in the CAT mRNA level in 2-AP- treated cells cannot by itself account for the dramatic increase in CAT protein level. Therefore, it appears that the 2-AP- mediated regulation must operate at the translational level.

Effects of 2-AP on the Expression of Other Transfected Genes-Does 2-AP increase the expression of transfected genes other than CAT? All our permanent transfectants expressing CAT activity were selected by co-transfection with a neomycin-resistance gene. I t was therefore convenient to investigate, in the same cells, the effect of 2-AP on the levels of neomycin phosphotransferase activity. This enzymatic activity was measured by an in situ enzymatic assay using electrophoretically separated proteins from cell extracts (19). In the experiment shown in Fig. 9, three cell lines were analyzed; one harbored a herpes virus thymidine kinase promoter-driven neo gene, whereas the other two contained SV40 promoter-driven ne0 genes. In all three lines 2-AP treatment enhanced the level of neomycin phosphotransferase activity. Similar enhancement of expression has also been observed for adenovirus E1A protein in a transfected HeLa line (data not shown). These results suggest that the 2-AP effect is not gene specific and 2-AP would enhance the expression of any transfected gene.

Effects of 2-AP on the Expression of a Gene Introduced by a Retrouiral Vector-In the next set of experiments we examined whether the mode of introduction of an exogenous gene into a cell influences the effect of 2-AP on its expression. Mouse CG1 cells were infected with a retroviral vector carrying the neo gene and clonal derivatives of the infected cells were isolated by G418 selection (18). In these cells, 2-AP stimulated the neo gene expression as measured by the in situ enzyme assay (Fig. 10). Increasing doses of 2-AP increased the level of the enzymatic activity (Fig. 1OA) as did increased time of incubation with 10 mM 2-AP (Fig. 10B). These results

M C a 2-AP

1 2 3 4

FIG. 8. Determination of cytoplasmic CAT mRNA level by RNase A protection assay. Lane 1, pBR322 HpuII markers (M); lune 2, untreated cells (C); lane 3, hIFN-a (500 units/ml) treated cells; lune 4, 2-AP (10 mM)-treated cells. Cells were treated with these agents for 6 h in growth medium before the extraction of RNA.

c u p c 2 A P c m

. - 6 - * 11)

% I 2 3 4 5 6

FIG. 9. Effect of 2-AP on neomycin phosphotransferase I1 activity. Equal amounts of total cytoplasmic proteins from HeLaM GS203CAT clone 4 (200 pg), HeLaM PRM CAT clone 11 (130 pg), and HeLaM pMAM-neo-CAT pool-1 (130 pg) were used in a in-gel assay as described under “Experimental Procedures.” Lunes I and 2, HeLaM GS203CAT clone 4 carrying IPB-1 plasmid lunes 3 and 4, HeLaM pRM-CAT clone 11 carrying pSV2-neo plasmid; lunes 5 and 6, HeLaM pMAM-neo-CAT pool-1 carrying CAT and neo genes in the same plasmid but driven by different promoters. C, extracts of untreated cells; 2-AP, 10 mM 2-AP extracts of treated cells. All treatments were done for 15 h.

(A) (6)

”” -4

1 2 3 4 5 6 7 8

FIG. 10. Effect of 2-AP on neomycin phosphotransferase I1 activity in CG1 Neo’ cells. A, the effect of different concentration of 2-AP on NPT-I1 activity. Cells were treated with the following concentrations of 2-AP in growth medium for 15 h. Lane I , 0 mM; hne 2,2.5 mM; hne 3 , 5 mM; hne 4, 10 mM. Equal amounts of protein (130 pg) were assayed for the NPT-I1 enzyme activity. B, the kinetics of stimulation of NPT-I1 activity by 2-AP. Cells were treated with 10 mM 2-AP for various lengths of time. Lane 1, 0 h; lune 2, 6 h; lune 3, 12 h; lune 4, 18 h. Equal amounts of protein (200 pg) were used from each sample.

demonstrate that 2-AP stimulates the expression of the same exogenous gene neo introduced either by transfection or by retroviral infection.

DISCUSSION

2-AP was originally described as an inhibitor of the dsRNA- dependent protein kinase (22, 23). Using 2-AP-mediated inhibition as an index, several laboratories tested the possible involvement of this protein kinase in transducing signals elicited by dsRNA leading to transcriptional induction of specific genes such as IFN-/I, 561, and 6-16 (4, 5, 7). An observed inhibition by 2-AP was interpreted as an indication of involvement of the dsRNA-dependent protein kinase in this process. This straightforward interpretation was, however, complicated by the observation that in HeLaM cells, induction of these genes by IFN-a is also blocked by 2-AP (4). This block is due to a defect in signal 1 production which has recently been traced to 2-AP-mediated inhibition of synthesis of a trans-acting factor which mediates signal 1 (24). It is unclear whether the dsRNA-dependent protein kinase is involved in this process especially because no apparent source of dsRNA was provided. I t is therefore conceivable that 2-AP exerts the effect on transcription by an entirely different and unknown route. As a means of understanding this effect further, we began testing the effect of 2-AP on IFN-induced expression of transfected chimeric test genes containing IFN- responsive transcriptional elements. The experiments re-

878 Translational Regulation by 2-Aminopurine

ported here showed that 2-AP had a profound effect on the translatability of mRNAs encoded by such transfected genes. This effect overshadowed the inhibitory effects of 2-AP on transcriptional induction by IFN. As a result, overall expression assay by measuring CAT activity could not be used for studying the effects of 2-AP on IFN-induced transcription. More direct assays such as measurements of the CAT mRNA level and the CAT mRNA transriptional rate had to be used for this purpose. This observation may apply to the studies of many other inducible gene systems as well. There are many reports in the literature in which effects of chemicals such as phorbol 12-myristate 13-acetate or CAMP on inducer-mediated signal transduction pathway are tested by measuring the activity of CAT encoded by an appropriate transfected chimeric gene, In these studies, unless shown otherwise, the possibility remains open that an observed effect on CAT activity is not due to an altered rate of transcription of the gene but due to an effect of the used chemical on the translatability of CAT mRNA.

Kaufman and Murtha (25) reported that 2-AP increases the translatability of adenosine deaminase and dihydrofolate reductase mRNAs transcribed from transfected genes. How- ever, they used transient transfection assays for these experiments and the observed selective effects of 2-AP on the expression of the transfected gene could be attributed to its episomal location. In contrast, in the experiments reported here only permanently transfected cell lines were used. In these lines, the transfected genes are integrated into the chromosomes making them physically indistinguishable from the cellular chromosomal genes. Moreover, we showed that 2- AP-mediated enhanced translation was independent of cell lines, transcription units, promoters, and coding bodies. The phenotype was stable over time and it was uniformly exhibited in all transfected clonal derivatives which had a basal level of expression of the transfected gene.

The striking observation was made that the expression of a gene introduced via a retroviral vector was also enhanced by 2-AP. DNA introduced by transfection is known to form concatamers and usually multiple tandem copies of the transfected gene integrate into the genome. In contrast, genes introduced by retroviral infection follow completely different routes. The viral RNA is reverse transcribed in the cytoplasm and the DNA product integrates in unit copies. Irrespective of the mode of introduction of the exogenous gene, the same cellular transcription machinery copies them as well as the resident genes to produce the corresponding mRNAs. Simi- larly, the mRNAs are processed similarly as well as trans- ported to the cytoplasm in a similar fashion. Any model explaining the selective effect of 2-AP on foreign genes has to define the “foreignness” in molecular terms taking our recent observations into account. Our observations make it difficult to attribute this effect to artifacts created by the transfection procedures such as production of dsRNA in a transient fashion (25, 26). I t would be hard to explain how such dsRNA will be produced from a chromosomally integrated gene introduced either by transfection or retroviral infection.

The stimulating effect of 2-AP on the translational effi- ciency of mRNAs of transfected genes is thought to be mediated by its inhibitory effect on the dsRNA-dependent protein kinase. In vitro, 2-AP inhibits the activity of this kinase but whether the same mechanism operates in uiuo remains to be shown. However, this view is supported by data obtained from a number of viral translational systems. Adenoviral VAI RNA is essential for efficient translation of late adenoviral mRNAs and numerous lines of investigation indicate that

this action is mediated by inhibiting the action of the dsRNA- dependent protein kinase (27-30). It has been shown using transient tranfection assays, that VA, like 2-AP, can enhance the translatability of mRNAs encoded by transfected genes (25, 31, 32). Thus co-transfection of VA producing plasmids with a test gene increases the level of expression of the test gene. This enhancing effect is accompanied by an inhibition of action of the dsRNA-dependent protein kinase (31, 33). Similar enhanced translation of a transfected gene was also observed when an expression vector containing the reovirus S4 gene was co-transfected (34). Polypeptide a3 encoded by this gene is known to have a strong affinity for dsRNA (35). Its action could therefore be mediated by sequestration of dsRNA and the resultant prevention of activation of the protein kinase.

Protein synthesis initiation factor eIF-2 is a known substrate of the dsRNA-dependent protein kinase and the state of its phosphorylation affects its function profoundly (36). Recent elegant genetic studies have clearly demonstrated that eIF-2 phosphorylation is the key step in dsRNA-protein kinase action (37). It was also shown that VA-mediated allevia- tion of translational block correlates with an inhibition of eIF-2 phosphorylation (32, 38). One may envisage that the cellular phosphorylation state of eIF-2 is constantly regulated by the kinase. When foreign genes are introduced, activators of the kinase such as dsRNA is produced and eIF-2 phosphorylation equilibrium is disturbed. Agents such as VA RNA, reovirus a3, or 2-AP can prevent activation of the kinase and hence phosphorylation of eIF-2. However, even if all elements of this scenario are correct, the basis of selectivity remains obscure. Why eIF-2 phosphorylation state would affect the rate of translation of a “foreign” mRNA but not of a “native” mRNA? Principles of “localized activation” of the kinase have been invoked for explaining this selectivity (25, 39). Recent results, reporting that reovirus S1 mRNA translation, but not S4 mRNA translation, is enhanced by 2-AP supports this notion because reovirus S1 mRNA, but not S4 mRNA, is a potent activator of the kinase (40). However, it is hard to imagine how both CAT and neomycin phosphotransferase transcripts studied here and other cellular mRNAs tested by others would share this property of reovirus S1 mRNA. In our attempts to understand the mechanism of this phenomenon, we should consider the possibility that eIF-2 may be directly involved in mRNA discrimination and its phosphorylation state may influence this property (41).

As discussed above, the mechanism of translational stimulation by 2-AP of permanently expressing exogenous genes remains obscure at the present time. However, this stimulating effect can be exploited for practical purposes. Expression assays of transfected genes such as CAT are widely used for promoter/enhancer analysis. The sensitivity of such assays can be markedly increased by including 2-AP in the culture medium. Our experiments suggest that the 2-AP stimulatory effect is more marked when the basal level of expression is low. This may make the analysis of weak promoter elements possible. Such analysis may reveal that transcriptional regulatory elements thought to be nonfunctional in certain cell types may indeed be active a t a low level. For example, the experiments described in this paper showed that IFN-stimulated gene transcription units are active constitutively. The constitutive expression could be enhanced up to 50-fold by 2- AP, raising it to the range of IFN-stimulated expression of the same gene. This 2-AP-enhanced expression can also be used to our advantage in analyzing promoter strength in cells which are difficult to culture and available in low numbers. Another practical advantage of this phenomenon could be in


producing a higher quantity of a protein encoded by a transfected gene. A higher level of a protein may be desirable for analyzing its function in uiuo. We have enhanced the production of the adenoviral E1A protein in a transfected cell line for this purpose (data not shown). This strategy is especially desirable if the protein whose function is being tested has deleterious effects on a cell if continually expressed in high quantity. In the same vein, if a transfected cell line is being used as a source of a scarce protein, its production can be enhanced by using 2-AP, an inexpensive chemical. Even a modest increase in the level of production can translate into a big saving of resources.

Acknowledgments-We are grateful to George Stark, Ian Kerr, Paula Pitha, Marc Wathelet, Michael Karin, and Richard Axel for generously providing various chimeric plasmids. We thank William Livingston 111 for expert technical assistance, and Margaret Leet for the preparation of this manuscript.

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