8-Br-cAMP inhibits the transient expression of firefly luciferase

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8-Br-cAMP inhibits the transient expression of firefly luciferase G . T . C o k e r , iii, L. V i n n e d g e a n d K . L . O ' M a l l e y Department of Anatomy and Neurobiology, Washington University Medical School, St. Louis, MO 63110, USA Received 3 April 1989 The genes for firefly luciferase and chloramphenicol acetyltransferase (CAT) were used as reporter genes to explore the activation of heterologous promoters by 8-Br-cAMP. Cells were transfected with a CAT gene/tyrosine hydroxylase promoter, which contains a cAMP response element. Extracts from cells treated with 8-Br-cAMP had 340% more enzyme activity than untreated cells. In contrast, treated cells transfected with a tyrosine hydroxylase/luciferase construct had 30% less activity than control cells. Simian virus and rous sarcoma virus promoters/luciferase constructs also had lower activities in cells treated with 8-Br-cAMP than untreated cells. The inhibition of luciferase enzyme activity by cAMP appears to be posttranscriptional since both luciferase and CAT RNA levels were similarly increased in cells treated with 8-Br-cAMP or l-methyl-3-isobutylmethylxanthine.The lower level of luciferase activity was not due to simple allosteric inhibition. We conclude that constructs using the firefly luciferase as a reporter gene are unsuitable for studying the effects of cAMP on the regulation of promoters. Luciferase; Transfection; Bromo cyclic AMP, 8-; Reporter gene; Methyl-3-isobutylmethylxanthine, 1-

1. I N T R O D U C T I O N

The firely luciferase gene is being increasingly used as a reporter gene to explore the regulation of heterologous promoters (e.g. [1,2]). As such it offers a number of advantages over other reporter genes in that the luciferase enzyme assay is easy, sensitive, does not require radioactive material, and is amenable to automation [3]. However, the luciferase reporter gene has not been tested in as many situations as the most commonly used reporter gene, chloramphenicol acetyltransferase (CAT). We have tested the effect of a cAMP derivative on heterologous promoters fused to the luciferase reporter gene. A number of genes have been identified as being regulated at the transcriptional level by cAMP [4]. Two such genes are tyrosine hydroxylase (TH) and somatostatin. In this report we show that treatCorrespondence address: G.T. Coker, iii, Box 8108, Department of Anatomy, Washington University Medical School, St. Louis, MO 63110, USA Abbreviations:

CAT, chloramphenicol acetyltransferase; IBMX, l-methyl-3-isobutylxanthine; TH, tyrosine hydroxylase

ment of transiently transfected cells by 8-Br-cAMP reduces the activity of various promoter/luciferase fusion genes. 2. M A T E R I A L S

AND METHODS

2.1. Plasmids The somatostatin promoter linked to the CAT reporter gene was provided (pD(-71)CAT) by Dr M. Montminy (UCSD); pRSVCAT (rous sarcoma virus promoter/CAT) was provided by Dr B. Howard (NIH); pRSVL (rous sarcoma virus promoter/luciferase reporter gene), pSV232AL (an enhancerless simian virus promoter/luciferase) and pSVOAL (luciferase reporter gene) were gifts from Dr J. deWet (UCSD). To facilitate cloning we constructed a plasmid that contained an EcoRV site flanked by HindlII sites. An AluI fragment (27 to 276) from the rat TH promoter was inserted into the EcoRV site [5]. This fragment was excised using HindlII and inserted in the proper orientation at the HindlII site of pSVOAL [3] creating prTHluc and into pSVOCAT, creating prTHCAT. -

2.2. Transfection Rat fibroblast cells (NIH 3T3) were grown in Dulbecco's modified Eagle's media supplemented with 10070 fetal calf serum. Rat pheochromocytoma cells (PCG2) were grown in the same media supplemented with 10°7o fetal calf and 5°70 horse serum. Cells (5 x l0 s) were transferred to 10-cm plates and transfected the next day using calcium phosphate-mediated transfection [6]. Cells were treated either with a mixture of test

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© 1989 Federation of European Biochemical Societies

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plasmids (101tg) or with the test plasmid (20/zg) plus pRSVCAT or pRSVL (2/zg) as an internal control. 6 h later the cells were treated with 25% glycerol in media without serum for 1 min, rinsed once and fresh complete media added. Isobutylmethylxanthine (IBMX; Sigma) and freshly prepared 8-Br-cAMP (Sigma) were added to final concentrations of 1 raM. The ceils were harvested 24 h later and assayed for luciferase [3] and CAT [7] enzyme ~activityor for RNA levels [8]. Luciferase activity was computed by measuring the height of the peak immediately after injection of ATP. 3. R E S U L T S E x t r a c t s o f P C G 2 cells t r a n s f e c t e d with prTHCAT and pD(-71)CAT (somatostatin prom o t e r ) h a d 3 . 4 - 5 . 5 - f o l d higher levels o f C A T act i v i t y o n t r e a t m e n t o f cells with 1 m M 8 - B r - c A M P (fig. 1). In c o n t r a s t , extracts o f cell t r a n s f e c t e d with the s a m e T H p r o m o t e r f r a g m e n t p l a c e d in f r o n t o f the luciferase r e p o r t e r gene ( p r T H l u c ) t r e a t e d with 8 - B r - c A M P h a d lower activities t h a n u n t r e a t e d cells. T o d e t e r m i n e if the r e d u c t i o n in e n z y m e activity was intrinsic to the T H p r o m o t e r , we also exa m i n e d t w o viral p r o m o t e r s , s i m i a n virus ( p S V 2 3 2 A L d e l t a 5 ' ) a n d rous s a r c o m a virus ( p R S V L ) , a t t a c h e d to luciferase gene. T r e a t m e n t w i t h 8 - B r - c A M P o f cells t r a n s f e c t e d with b o t h o f these p l a s m i d s also resulted in l o w e r e n z y m e activities t h a n u n t r e a t e d cells. S i m i l a r results were o b t a i n e d when we used N I H 3T3 cells (not shown). I n o r d e r to c o n f i r m a n d extend these results, P C G 2 cells were c o t r a n s f e c t e d with p r T H l u c a n d ~BI oontrol R e

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p r T H C A T a n d t r e a t e d with the p h o s p h o d i e s t e r a s e i n h i b i t o r , I B M X , a n d / o r 8 - B r - c A M P (fig.2). T r e a t m e n t with 8 - B r - c A M P o r with I B M X resulted in 6- a n d 8-fold e n h a n c e m e n t o f C A T activity over t h a t o f c o n t r o l . A d d i t i o n o f I B M X a n d 8-Brc A M P resulted in higher levels t h a n when either c o m p o n e n t was a d d e d s e p a r a t e l y . In c o n t r a s t , luciferase activity was n o t s t i m u l a t e d b u t was either r e d u c e d o r r e m a i n e d the s a m e w h e n either I B M X or 8 - B r - c A M P was a d d e d . T o d e t e r m i n e if the i n h i b i t i o n o f e n z y m e activity was t r a n s c r i p t i o n a l o r p o s t t r a n s c r i p t i o n a l , we a s s a y e d R N A f r o m c o n t r o l a n d t r e a t e d cells. In c o n t r a s t to e n z y m e activities, the levels o f luciferase a n d C A T R N A s were i n c r e a s e d to a s i m i l a r degree b y t r e a t m e n t with either I B M X o r 8 - B r - c A M P (fig.2). T o test w h e t h e r the d i m i n i s h e d activity was the result o f i n h i b i t i o n o f luciferase e n z y m e activity b y either 8 - B r - c A M P o r 8 - B r - A M P , we a s s a y e d c o m m e r c i a l luciferase (Sigma) in the presence o f several d i f f e r e n t c o n c e n t r a t i o n s o f these reagents. F i r e f l y luciferase has t w o active c a t a l y t i c sites [9]; o n e site, Io, r e s p o n d s to a d d i t i o n o f A T P b y a r a p i d pulse o f activity a n d a r a p i d d e c a y (within 3 0 - 6 0 s). T h e s e c o n d site, I1, is active at a b o u t 10-20°70 o f I0 at s a t u r a t i n g levels o f A T P a n d persists o v e r several minutes. B o t h A M P a n d c A M P are c o m p e t i t i v e i n h i b i t o r s o f A T P at I0 [10] with Ki values o f 0.25 a n d 0.9 m M , respectively. A s s h o w n in fig.3, 8 - B r - A M P when present at 0.25 m M in12

5

10l

BLue

~

Lu¢ RNA

~Cat

~

Cat RNA

I a

4

t i V e

3

L

IoD(-71)CAT

prTHCAT

DrTHluc

pSV232AL

pRSVL

Fig. 1. Effect of 8-Br-cAMP on luciferase and CAT activity. PCG2 cells were transfected with a mixture of test plasmid (20/~g) and the control plasmids, pRSVcat or pRSVL. After 23 h incubation with 1 mM 8-Br-cAMP, cells were extracted and enzyme activities determined. Activities were normalized to respective control activities. Values are averages of 3 replicates (SE < 10O7o).

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control

IBMX

cAMP

IBMX÷cAMP

Fig.2. Effect of IBMX and 8-Br-cAMP on luciferase and CAT activity and RNA levels. PCG2 cells were transfected with a mixture of prTHCAT and prTHluc (10 jtg each). After 23 h incubation with 1 mM 8-Br-cAMP and/or 1 mM IBMX, cells were extracted and enzyme activities and RNA levels determined. Values are averages of 3 replicates (SE < 10%).

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Fig.3. Effect of AMP derivatives on luciferase activity. Luciferase (30/zU) was assayed in the presence of various concentrations of 8-Br-AMP. ATP (final concentration 4.0 raM) initiated the reaction. Squares, I0 activity; circles, I1 activity. hibited I0 activity by 42% and had no effect on I1 activity. In contrast, 8 - B r - c A M P at a final concentration o f 1 m M did not inhibit either site (not shown). T o determine if cell extracts contained a c o m p o u n d that inhibited luciferase activity, c o m m e r cial luciferase was preincubated for 5 min with extracts o f ceils that had been treated or untreated with 8 - B r - c A M P . There was no effect on exogenous luciferase activity demonstrating the absence o f any inhibitory c o m p o u n d s present in cell extracts. In contrast, we did find that certain preparations o f A T P contained inhibitory comp o u n d s , presumably A M P . To minimize degradation, stock solutions o f A T P were rapidly adjusted to p H 7.0 and quick frozen. 4. D I S C U S S I O N We and others have shown that c A M P derivatives increase the transcription o f T H and s o m a t o s t a t i n / C A T reporter genes in transient expression assays [11,12]. This increase in transcriptional activity is mediated by the presence o f a c A M P response element 5' to the T A T A box in b o t h o f these genes [4]. Surprisingly, when an identical T H p r o m o t e r fragment is fused to luciferase and C A T reporter genes very different responses are elicited by the presence o f 8 - B r - c A M P (fig. 1). In the same experimental" paradigm, C A T activity was increased in extracts f r o m treated cells whereas luciferase activity was decreased in c o m p a r i s o n to

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c o n t r o l values (fig.2). Therefore, the reduction o f luciferase activity u p o n addition o f 8 - B r - A M P does not appear to be due to the nature o f the attached p r o m o t e r or the presence o f a c A M P response element as neither p S V 2 3 2 A L delta 5' n o r p R S V L contain this sequence (fig. 1). A l t h o u g h luciferase can be inhibited by A M P derivatives (fig.3; [10]) our mixing experiments ruled out simple inhibition o f enzyme activity by an allosteric inhibitor such as 8 - B r - A M P or A M P . Because R N A for b o t h C A T and luciferase were induced to a similar degree u p o n the addition o f either 8 - B r - c A M P or I B M X (fig.3), the observed decrease in luciferase activity presumably involves its translational efficiency a n d / o r protein stability in the presence o f c A M P . As firefly luciferase is intimately involved in A T P metabolism, it is not unreasonable to suggest that c A M P m a y regulate luciferase protein levels via these mechanisms. W h a t e v e r the means o f inhibition, our data d e m o n s t r a t e that luciferase should not be used in examining the response o f heterologous p r o m o t e r s to c A M P .

Acknowledgement: This work was supported in part by a grant from Monsanto Company (St. Louis, MO). REFERENCES [1] Van Zonneveld, A.J., Curriden, S.A. and Loskutoff, D.J. (1988) Proc. Natl. Acad. Sci. USA 85, 5525-5529. [2] Waterman, M.L., Adler, S., Nelson, C., Greene, G.L., Evans, R.M. and Rosenfeld, M.G. (1988) Mol. Endocrinol. 2, 14-21. [3] DeWet, J.R., Wood, K.V., DeLuca, M., Helinski, D.R. and Subramani, S. (1987) Mol. Cell. Biol. 7, 725-737. [4] Lin, Y.-S. and Green, M.R. (1988) Proc. Natl. Acad. Sci. USA 85, 3396-3400. [5] Coker, G.T., iii, Vinnedge, L. and O'Malley, K.L. (1988) Biochem. Biophys. Res. Commun. 157, 1341-1347. [6] Chen, C. and Okayama, H. (1987) Mol. Cell. Biol. 7, 2745-2752. [7] Burzio, L.O., Zarraga, A.M. and Siddiqui, M.A.Q. (1986) Gene Anal. Tech. 3, 93-95. [8] Ley, T.J., Connolly, N.L., Katamine, S., Cheah, M.S.C., Senior, R.M. and Robbins, K.C. (1989) Mol. Cell. Biol. 9, 92-99. [9] DeLuca, M. and McElroy, W.D. (1984) Biochem. Biophys. Res. Commun. 123, 764-770. [10] Lee, R.T., Denburg, J.L. and McElroy, W.D. (1970) Arch. Biochem. Biophys. 141, 38-52. [ll] Lewis, E.J., Tank, A.W., Weiner, N. and Chikarishi, D.M. (1983) J. Biol. Chem. 258, 14632-14637. [12] Montminy, M.R., Sevarino, K.A., Wagner, J.A., Mandel, G. and Goodman, R.H. (1986) Proc. Natl. Acad. Sci. USA 83, 6682-6686. 185