Expression of c-Myc in glucocorticoid-treated fibroblastic cells

ft. Steroid Biochem. Molec. Biol. Vol. 50, No. 3/4, pp. 109-119, 1994 Copyright © 1994 Elsevier Science Ltd 0960-0760(94)E0075-V Printed in Great Britain. All rights reserved 0960-0760/94 $7.00 + 0.00

Pergamon

E x p r e s s i o n of c-Myc in G l u c o c o r t i c o i d t r e a t e d F i b r o b l a s t i c Cells Gloria H. Frost, Kunsoo Rhee, Tianlin Ma and E. Aubrey Thompson* Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, T X 77550, U.S.A. G l u c o c o r t i c o i d s i n h i b i t p r o l i f e r a t i o n o f L929 fibroblastic cells in culture. I n h i b i t i o n o f p r o l i f e r a t i o n is reversible and is n o t a s s o c i a t e d w i t h c h a n g e s in the plating efficiency o f the cells. Flow c y t o m e t r i c analysis indicates t h a t g l u c o c o r t i c o i d - t r e a t e d cells exhibit a decrease in the p e r c e n t a g e o f cells w i t h D N A c o n t e n t > 2 N . T h y m i d i n e kinase e x p r e s s i o n is i n h i b i t e d as cells w i t h 2 N D N A c o n t e n t a c c u m u l a t e . These o b s e r v a t i o n s i n d i c a t e that g l u c o c o r t i c o i d s arrest p r o l i f e r a t i o n o f L929 cells in the G1 p h a s e o f the cell cycle. The a b u n d a n c e o f c - M y c m R N A does n o t decrease in g l u c o c o r t i c o i d t r e a t e d cells, and c-Myc p r o t e i n c o n t e n t in d e x a m e t h a s o n e - t r e a t e d cells is a p p r o x i m a t e l y the s a m e as that d e t e c t e d in m i d - l o g phase cells. N u c l e a r r u n - o n t r a n s c r i p t i o n o f c-Myc is n o t i n h i b i t e d by g l u c o c o r t i c o i d s . These o b s e r v a t i o n s i n d i c a t e that g l u c o c o r t i c o i d r e g u l a t i o n o f fibroblastic cell p r o l i f e r a t i o n does not i n v o l v e i n h i b i t i o n o f c-Myc t r a n s c r i p t i o n . A l t h o u g h r e g u l a t i o n o f c-Myc e x p r e s s i o n is central to the m e c h a n i s m w h e r e b y g l u c o c o r t i c o i d s r e g u l a t e p r o l i f e r a t i o n o f l y m p h o i d cells, it is clear t h a t different m e c h a n i s m s m u s t be i n v o l v e d in g l u c o c o r t i c o i d r e g u l a t i o n of fibroblastic cell p r o l i f e r a t i o n .

J. Steroid Biochem. Molec. Biol., Vol. 50, No. 3/4, pp. 109-119, 1994

INTRODUCTION

tiation is linked to an increase in the frequency of premature termination and a corresponding decrease in c-Myc expression [15]. On the other hand, Burkitt's lymphoma cells do not exhibit attenuation, and the resultant increase in c-Myc expression is thought to contribute to the malignant phenotype of these cells [16]. Regulation of transcriptional elongation is by no means the only mechanism that governs c-Myc expression (for review see [7]). However, attenuation of c-Myc transcription is almost universal and appears to play a significant role in cell proliferation and differentiation, particularly in hematopoietic cells. Glucocorticoid regulation of c-~yc expression has been studied extensively in lymphoid cells. Malignant T cells of both murine [17-19] and human [20, 21] origin exhibit glucocorticoid-mediated inhibition of c-Myc expression. T h e abundance of c-Myc m R N A decreases rapidly in glucocorticoid-treated T lymphoma and leukemia cells, and statistically significant changes are detected within 10 min in some cases [19]. In murine lymphoma cells, glucocorticoids rapidly inhibit nuclear run-on transcription of c-Myc [18]. However, regulation of m R N A stability may also govern c-Myc expression in human leukemia cells [21]. Antisense knockout of c-Myc can recapitulate the effects of dexamethasone in CCRF C E M - C 7 human T

Regulation of c-Myc expression has been studied in detail in a n u m b e r of cell lines under a variety of conditions [1-7]. In almost all circumstances, proliferating cells express c-Myc m R N A in abundance, whereas non-proliferating cells express the gene to a substantially reduced extent. T h e r e are few cell types in which inhibition of proliferation is not associated with inhibition of c-Myc expression. Observations of this sort argue that there is a relationship between the capacity for cell proliferation and the ability to express c-Myc. For example, inhibition of c-Myc expression in H L 6 0 leukemic cells is associated with withdrawal from the cell cycle and differentiation [2, 8-10]. Regulation of c-Myc in H L 6 0 cells can be affected by a n u m b e r of agents that cause G1 arrest and inhibit c-Myc transcription. T h e initial effect appears to be due to an increase in premature termination or attenuation of transcription [2, 11, 12]. T h e low level of c-Myc expression in quiescent T lymphocytes is maintained by premature termination, which is relieved upon mitogen stimulation [13, 14]. Mouse erythroleukemia cells resemble H L 6 0 cells in that differen*Correspondence to E. A. T h o m p s o n Received 17 Jan. 1994; accepted 15 Mar. 1994 109

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leukemia cells, and transient expression of c-Myc from chimaeric expression vectors can attenuate the effects of dexamethasone in the same cell line [22]. T h e s e observations indicate that glucocorticoid regulation of c-Myc expression may be a c o m m o n mechanism to account for the m a n y and varied effects of glucocorticoids on cell proliferation. Glucocorticoids play an important physiological and pharmacological role in regulating proliferation of fibroblastic cells [23, 24]. However, glucocorticoid regulation of fibroblast proliferation has not been studied as thoroughly as has glucocorticoid effects on lymphoid cells. Both lymphoid and fibroblastic cells are of connective tissue origin, and it is not unreasonable to suppose that the manner in which these cells respond to glucocorticoids may share c o m m o n features. For example, glucocorticoids inhibit proliferation of papovavirus-transformed C129 mouse fibroblasts; and glucocorticoids inhibit expression of c-Myc in papovavirus-transformed C129 cells [24]. On the other hand, glucocorticoid regulation of c-Myc in untransformed C129 fibroblastic cells was difficult to d e m o n strate. This manuscript describes a series of experiments that were designed to examine glucocorticoid regulation of L929 mouse fibroblastic cell proliferation and to determine the role of c-Myc in this process. L929 fibroblasts were selected because the proliferation of these cells is known to be inhibited by natural and synthetic glucocorticoids [25, 26]. T h e response is mediated by the glucocorticoid receptor [26, 27]. Preliminary data indicated that L929 cells are m u c h more sensitive to glucocorticoids than are C129 cells, and it was hoped that a more robust response might facilitate analysis of gene regulation. F u r t h e r m o r e , there is a preliminary report that glucocorticoids may regulate expression of c-Myc in L929 cells [28]. T h e initial experiments were undertaken to define the growth response to glucocorticoids and to test the hypothesis that glucocorticoid inhibition of L929 cell proliferation is attributable to inhibition of expression of c-Myc. T h e data are not consistent with this hypothesis. It has been determined that the abundance of c-Myc m R N A does not decrease as L929 cells withdraw from the cell cycle. F u r t h e r m o r e , attenuation of c-Myc transcription does not occur in L929 ceils. T h e data suggest that these cells are defective in an important regulatory mechanism, which m a y account for high basal expression and failure to regulate c-Myc. M A T E R I A L S AND M E T H O D S

Reagents Restriction endonucleases and enzymes for modification of D N A were purchased from B R L (Gaithersburg, M D ) or N e w England Biolabs (Beverly, MA) and were used according to the manufacturer's suggested procedures. Proteinase K was purchased from Boeh-

ringer M a n n h e i m (Indianapolis, IN). All [32p]-labeled nucleoside triphosphates were obtained from D u P o n t N e w England Nuclear (Wilmington, DE). Fetal bovine serum was obtained from Flow Laboratories (McLean, VA). All other chemicals were purchased from U.S. Biochemical Corp. (Cleveland, OH), Sigma Chemical Co. (St Louis, M O ) or Fisher Scientific (Houston, T X ) . Nitrocellulose (BA85, 0.45 # M ) was manufactured by Schleicher and Schuell (Keene, N H ) .

Cell cycle analysis L929.06 cell line is a subclone of the mouse L cell ( N C T C 929) selected for sensitivity to dexamethasone. L929.06 cells were grown in Dulbecco's modified Eagle's ( D M E ) m e d i u m supplemented with 5% fetal bovine serum (FBS) and subcultured by treatment with trypsin. L929.06 cells were plated at a density of 8000 cells/cm 2. After 24-48 h, when in mid-logarithmic growth, the cells were treated with either vehicle (ethanol to a final concentration 0.1%) or dexamethasone (to a final concentration, 0.1/~M). T h e cells were harvested with trypsin and washed with phosphatebuffered saline containing 1 m M MgC12. T h e cells were suspended in 1.0 ml of phosphate-buffered saline plus 3 ml of 95% ethanol, sedimented by centrifugation at 200 g for 5 min and suspended in a modified Krishan's propidium iodide solution containing 0.005% propidium iodide, 0.002% R N a s e A, 0.3% Nonidet P40, and 0.1% sodium citrate [29]. T h e cells were lysed by a 30 min incubation at 4°C, and the resulting nuclei were collected by centrifugation at 200 g for 5 min. Nuclei were suspended in fresh modified Krishan's p r o p i d i u m iodide solution and analyzed on a Coulter Epics V Flow Cytometer (Coulter Electronics, Hialeah, F L ) equipped with an argon laser source. Dye excitation was at 488 n m with fluorescence emission measured through a 515 n m long pass interference, a 515 n m long pass absorption and a 590 nm dichroic and 590 n m long pass absorption filters. D N A histograms were derived from an analysis of 25,000 cells.

Northern blot analysis Total R N A from control and dexamethasone-treated cells was extracted with proteinase K and sodium dodecyl sulfate as previously described [30]. Total R N A was denatured with 6% formaldehyde and 50% formamide, fractionated on a 1% agarose gel containing 2.2 M formaldehyde and blotted onto nitrocellulose m e m b r a n e s according to T h o m a s [31]. T h e size of the m R N A s was estimated by comparison with ethidium bromide-stained 28S and 18S r R N A . Nitrocellulose filters were hybridized overnight at 55°C in the presence of 10% dextran sulfate, 50% formamide, 5 × SSC, 5 x D e n h a r d t ' s solution and 100 # g / m l calf thymus D N A . All hybridization filters were washed three times at 65°C for 1 h (each wash), as described previously [32]. Plasmid p5B, corresponding to a fragment of mouse 18 S r R N A [33], was used as an internal

c-Myc Regulation in Fibroblasts control for N o r t h e r n analysis of m R N A . Full length mouse c-Myc clone pMc-Myc54 was obtained from Kenneth Marcu [34].

Thymidine kinase expression T h e abundance of thymidine kinase m R N A was estimated by N o r t h e r n blot analysis, as described above. T h e probe was a nick-translated restriction fragment from the mouse Tk-1 c D N A clone p M T K 4 [35]. T h y m i d i n e kinase enzyme activity was estimated by measuring the rate of phosphorylation of T M P in extracts from L929 cells, as described elsewhere [32].

Ribonuclease protection assay Total R N A from control and dexamethasone-treated cells was extracted, as described above, and 1 0 p g of sample R N A was dissolved in 30 pt hybridization buffer containing 5 x 105 ~2p cpm of probe RNA. T h e mixture was incubated for 3-7 min at 85-90°C to denature RNA and incubated at 60°C overnight. T h e n 160 #1 of RNase digestion buffer, containing 10 m M Tris-HC1 (pH 7.5), 0 . 3 M NaC1, 5 m M E D T A , 1 5 # g RNase A, and 40-50 U RNase T1 was added and incubated at 30°C for 20-60 min. Proteinase K (200 #g/ml) and sodium dodecyl sulfate (0.5% w/v) were added and incubated for 15 min at 37°C. T h e reaction was extracted with phenol:chloroform, carrier t R N A (10 #g) was added to the aqueous phase which was diluted by addition of 1 vol of H20, and nucleic acids were precipitated with ethanol. T h e R N A pellet was dissolved in 4 M urea, 90 m M Tris, 90 m M boric acid, 0.05% (w/v) sodium dodecyl sulfate, 0.025 (w/v) xylene cyanol, 0.025 (w/v) bromphenol blue. T h e double-stranded R N A : R N A hybrids were resolved on 5% polyacrylamide by electrophoresis in T B E buffer (100 m M Tris, 2 m M E D T A and 100 m M boric acid). T h e gels were dried and exposed to X-ray film. T h e relative amount of each transcript was quantified by densitometry using an Applied Imaging BV14000 digital image analysis system. T h e c-Myc probe was a 547 nucleotide antisense probe prepared by T 7 R N A polymerase transcription of a c-Myc subclone containing a ScaI/NotI fragment. This is expected to protect fragments of 342 and 178 nucleotides, corresponding to transcripts initiated at the P1 or P2 c-Myc promoters respectively. T h e internal standard is a 136 nucleotide-sense probe prepared by T 7 R N A polymerase transcription of a subclone containing an HindlII/PvuH fragment that lies between P1 and P2 ( + 24 to + 139 relative to P1). T h e expected protected fragment is 121 nucleotides and was used to control for efficiency of sample recovery and hybridization. Experimental details are described elsewhere [19].

Measurement of c-Myc protein T h e protocol is described in detail in Antibodies: ,4 Laboratory Manual [36]. Cells were harvested by scraping and washed by centrifugation in phosphate-

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buffered saline. T h e cell pellet was lysed in 100 #1 of R I P A lysis buffer ( 1 5 0 m M NaC1, 1.0% NP-40, 0.5% deoxycholate, 0.1% SDS, 5 0 m M Tris, p H S . 0 ) containing aprotinin (2/~g/ml), leupeptin (0.Spg/ml), P M S F (35#g/ml) and pepstatin A (0.7pg/ml) for 30 min. Cell debris were sedimented by centrifugation (10 min in an E p e n d o r f microcentrifuge) and 40/~g of protein was resolved by electrophoresis on 7.5% polyacrylamide gel containing SDS. Proteins were transferred to nitrocellulose by electroblotting. Filters were probed with a c-Myc monoclonal antibody (Monoclonal C33, catalog # S C 4 2 , Santa Cruz Biotechnology) diluted 1:75. A horseradish peroxidaseconjugated goat anti-mouse secondary antibody (Chemicon) was used at a dilution of 1:4000 and detection was achieved using the Amersham E C L kit.

Nuclear run-on transcription assay Nuclear run-on transcription was carried out as described by Mahajan and T h o m p s o n [37]. Briefly, nuclei were isolated from control and dexamethasonetreated cells and transcription was carried out in the presence of [~32-P]UTP. R N A was isolated after digestion with DNase plus proteinase K in the presence of CaC12. Labeled R N A was washed by precipitation with trichloroacetic acid. Aliquots were withdrawn after transcription and before hybridization to calculate the recovery of labeled RNA, which was 30-60%. T o compare control and dexamethasone-treated cells, equal nuclear equivalents of labeled R N A were hybridized to 3 # g of the appropriate D N A probes, immobilized on nitrocellulose. T h e c-Myc probes used in the run-on experiments were M13 subclones derived from pMc-Myc54 as previously described [18]. T h e probe for exon I was a 379 bp HindlII-XhoI fragment and the probe for exon II was a PstI fragment of 4 1 7 b p subcloned into M13mp8. T h e M13 recombinant containing the RNAlike strand was used to control symmetric transcription and nonspecific hybridization to the R N A complementary strand. T h e Syrian hamster 5S gene [38] was used as an internal control for hybridization efficiency in nuclear run-on transcription experiments [18, 32, 37]. RESULTS

Growth of L929.06 cells in the presence of glucocorticoids L929 cells were obtained from A T C C and 11 subclones were isolated. T h e data presented below summarize the observations derived from a n u m b e r of experiments undertaken to characterize the manner in which dexamethasone influences the proliferation of the L929.06 subclone. T h e behavior of L929.06 is indistinguishable from that of the other clones or the uncloned parental population. Proliferation of these cells was severely inhibited by addition of 0.1 # M dexamethasone, as shown in Fig. 1. Less than one doubling was observed within 96 h after addition of the

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Gloria H. Frost et al.

steroid. Over the course of the experiment shown in Fig. 1, control cultures doubled every 34 h on average, whereas the glucocorticoid-treated cultures doubled every 113 h. In six independent experiments the mean population doubling time (over a period of 96 h) of control cultures was calculated to be 35(___ 10)h (range 32-46 h). T h e population doubling time of parallel, dexamethasone-treated cultures was estimated to be 106(___ 54)h (range 7 4 - 2 8 2 h, P < 0.001 by unpaired t). (Standard deviations are given in parentheses.) T h e ability of L929.06 cells to adhere to the plates and form colonies was not affected, as shown in Fig. 2. Plating efficiency was not reduced when dexamethasone was added at the time of plating or 24 h thereafter (cross-hatched bars). However, a significant reduction in the n u m b e r of cells/colony was observed (open bars). T h e effects of glucocorticoids are reversible. Cells were exposed to glucocorticoids for various periods of time, and the plating efficiency was measured thereafter. As shown in Fig. 3, the plating efficiency was not reduced when L929 cells were exposed to dexamethasone for up to 4 days. In a separate experiment, the rates of proliferation were determined for cells that had been rescued after 96 h in dexamethasone. T h e p o p u lation doubling times of such cultures was 34( _ 4)h and was not significantly different from that of naive cultures. T h e s e data indicate that glucocorticoids do not kill L929 cells and do not cause irreversible differentiation. Glucocorticoids and G1 arrest o f L 9 2 9 . 0 6 cells

Nuclei from L929 cells were stained with propidium iodide and analyzed for D N A content by flow cytometry. Representative data are shown in Fig. 4. Glucocorticoids caused a decrease in the n u m b e r of cells having D N A content > 2 N . Quantitative data 70-

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Fig. 2. T h e effect o f d e x a m e t h a s o n e o n t h e p l a t i n g efficiency o f L929.06 cells. A s u s p e n s i o n o f cells w a s p r e p a r e d a n d u s e d to i n o c u l a t e 3 c m t i s s u e c u l t u r e p l a t e s w i t h 102(4-3) cells in 3 m l o f c o m p l e t e m e d i u m . O n e set o f p l a t e s (Dex) r e c e i v e d 0.1/JM d e x a m e t h a s o n e . C o n t r o l p l a t e s r e c e i v e d vehicle alone. A t h i r d set o f p l a t e s w a s m a i n t a i n e d in c o m p l e t e m e d i u m f o r 24 h p r i o r to a d d i t i o n o f 0.1 p M d e x a m e t h a s o n e ( D e x p 24 h). A f t e r 6 days, t h e m e d i u m w a s d e c a n t e d a n d t h e p l a t e s w e r e w a s h e d w i t h p h o s p h a t e - b u f f e r e d saline. T h e cells w e r e fixed w i t h 1%o g l u t a r a l d e h y d e a n d s t a i n e d w i t h 1% c y t o d i a c h r o m e . The n u m b e r of colonies/plate was counted u n d e r a dissecting m i c r o s c o p e and the n u m b e r of cells/colony u n d e r an inverted m i c r o s c o p e . T h e e r r o r b a r s r e p r e s e n t one s t a n d a r d d e v i a t i o n a n d t h e n u m b e r o f p l a t e s o r c o l o n i e s c o u n t e d is e x p r e s s e d in p a r e n t h e s e s below each bar.

are presented in T a b l e 1. T h e data indicate that glucocorticoids caused a decrease in the percentage of cells in S, G2, and M phases of the cell cycle. A corresponding increase in G1 phase cells was observed. This situation is reversible and the percentage of cells in S, G2 and M increased following removal of dexamethasone from arrested cultures. T h e s e data are consistent with the conclusion that L929 cells accumulate in G1 in the presence of glucocorticoids. Glucocorticoid-treated L929 cells exhibit decreased incorporation of [3H]thymidine [39]. T h i s is due, in

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Fig. 1. T h e effect o f d e x a m e t h a s o n e o n p r o l i f e r a t i o n o f L929.06 cells. A s e r i e s o f 25 c m 2 T flasks w e r e i n o c u l a t e d w i t h 1.0( + 0.07) x 10 s cells in 5 m l o f c o m p l e t e m e d i u m . O n e s e r i e s r e c e i v e d 0.1 p M d e x a m e t h a s o n e a n d t h e s e c o n d a n e q u i v a l e n t v o l u m e o f v e h i c l e (70% e t h a n o l ) . Cells w e r e h a r v e s t e d by t r e a t m e n t w i t h t r y p s i n at 2 4 h i n t e r v a l s . Cell n u m b e r a n d v i a b i l i t y w e r e d e t e r m i n e d as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . V i a b i l i t y in all c a s e s w a s > 9 5 % . E a c h t i m e p o i n t w a s a s s a y e d in t r i p l i c a t e d a n d t h e e r r o r b a r s r e p r e s e n t one s t a n d a r d deviation.

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Fig. 3. Viability o f d e x a m e t h a s o n e - t r e a t e d cells. L929 cells w e r e e x p o s e d to 0.1/~M d e x a m e t h a s o n e f o r p e r i o d s o f t i m e c o r r e s p o n d i n g to t h o s e s h o w n b e l o w e a c h b a r . T h e r e a f t e r , t h e cells w e r e d i s a g g r e g a t e d a n d w a s h e d in p h o s p h a t e - b u f f e r e d s a l i n e to r e m o v e r e s i d u a l d e x a m e t h a s o n e . A l i q u o t s o f 50 cells in 3 m l o f c o m p l e t e m e d i u m w e r e u s e d to i n o c u l a t e 3 c m p l a t e s . C o l o n i e s w e r e c o u n t e d (as d e s c r i b e d in the l e g e n d to Fig. 2) 1 week a f t e r i n o c u l a t i o n . T h e e r r o r b a r s r e p r e s e n t one s t a n d a r d deviation f r o m the m e a n of 5 or m o r e plates.

c-Myc Regulation in Fibroblasts

isolated 12 and 24 h after rescue from glucocorticoids. In parallel, extracts were prepared from cultures, treated as described above. T h e s e extracts were assayed for thymidine kinase activity. T h e data are summarized in Fig. 5. Lane C (lane 1) contains R N A from control cells. Lane D (lane 2) contains R N A from dexamethasone-treated cells. Lanes R12 and R24 (lanes 3 and 4) contain R N A from cells that had been rescued for 12 and 24 h, respectively. Quantitative data from T K m R N A analysis and T K enzymatic activity in the corresponding samples are given below the autoradiogram. T h e abundance of T K m R N A in glucocorticoidtreated cells was < 5 % of that observed in control cells, with a corresponding decrease in thymidine kinase activity ( T K Act.). T h e effects of dexamethasone are reversible, and both T K m R N A and activity increased rapidly after removal of dexamethasone. Both activity and m R N A abundance at 24 h after withdrawal of dexamethasone were 5-10 times higher than control. T h i s observation indicates that when L929 cells are rescued from dexamethasone, they undergo a synchronous entry into S phase within 24 h after removal of the steroid. T h y m i d i n e kinase expression is known to be expressed in late G1/early S phase and repressed in cells that are arrested in G1 by serum starvation [40] and glucocorticoids [32, 39]. T h e data shown in Fig. 5 are consistent with the conclusion that glucocorticoids cause G1 arrest of L929 cells.

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Fig. 4. F l u o r e s c e n t c y t o m e t r y of L929 cells. Cytofluorographic analysis of the DNA content from L929.06 cells e x p o s e d to solvent (C) or 0.1/~M dexamethasone (D) for 24 h. Quantitative data are given in Table 1.

part, to inhibition of D N A synthesis, as shown in T a b l e 1. In addition, glucocorticoid-treated L929 express very low levels of thymidine kinase [39]. Mid-log phase L929 cultures were treated with dexamethasone for 24 h and R N A was extracted and assayed for the abundance of thymidine kinase ( T K ) m R N A . In addition, cultures were treated with dexamethasone for 24 h. T h e r e a f t e r the hormone-containing m e d i u m was removed and replaced with fresh m e d i u m . R N A was

Table 1. Cell cycle distribution of glucocorticoid-treated cells Cell cycle phase: DNA content:

G1 2N

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S > 2N < 4N

G2 + M 4N TotaP Number

Percent of cells Control 68.5 24 h Dexamethasone 93.0 24 h Dexll~24 h rescue 47.0 aTotal number of particles counted

5.6 0.6 7.8

17.2 2.8 16.6

18,816 23.608 18,464

Like thymidine kinase, c-Myc expression is also reduced in cells that are arrested in G1 [reviewed in 4-7]. Expression of c-Myc was analyzed using an R N a s e protection assay to quantify the transcripts arising from the two major c-Myc promoters, P1 and P2. T h e results of a representative study are shown in Fig. 6. T h e R N A that was analyzed in this experiment was that obtained in the experiment described in Fig. 5. Lane C contains control R N A ; lane D, R N A from dexamethasone-treated cells; and lanes R12 and R24 contain R N A from cells that had been rescued for 12 and 24h. T h e r e was no significant change in the abundance of the P1 or P2 transcripts in glucocorticoid-treated cells and the abundance of neither P1 nor P2 m R N A changed as cells re-entered the cell cycle following removal of dexamethasone. T h e data shown in Fig. 6(B) indicate that the P2 transcript is about 80 times m o r e abundant than the transcript arising from P1. (See the legend to Fig. 6 for an explanation of the calculations involved in comparing the abundance of the two m R N A s . ) I m m u n o b l o t t i n g ("Western blotting") protocols were used to measure expression of c-Myc protein in L929 cells. T h e results of a typical experiment are shown in Fig. 7. T h e c-Myc antibody used in these experiments recognizes primarily a protein of about 64 K. T h e abundance of this protein did not decrease in glucocorticoid-treated L929 cells (compare lanes 3

Gloria H. Frost

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et al.

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460

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2688

Fig. 5. Glucocorticoid regulation of thymidine kinase (Tk-1) expression in L929 cells. L929 cells were treated for 24 h with 0.1/IM dexamethasone and RNA was extracted as described in Materials and Methods. Parallel cultures were treated for 24 h. Thereafter, the dexamethasone-containing medium was removed and replaced with warm (37°C) medium. This m e d i u m was removed after 30 min and replaced with fresh, warm medium. RNA was extracted 12 h and 24h after removal of glucocorticoids. Total RNA (10 ltg) was resolved by electrophoreses, blotted onto nitrocellulose, and hybridized with nick-translated TK cDNA probes. The filters were exposed overnight. The intensities of the autoradiographic signals were determined as described in Materials and Methods. The integrated results of quantification are shown below each lane (TK mRNA) in units of m m 2 x OD. In parallel, cytoplasmic extracts were prepared from control, treated and rescued cultures. These extracts were assayed for thymidine kinase activity, as described in Materials and Methods. The activity data (TK Act.) given below each lane are expressed as initial velocity of thymidine phosphorylated in units of cpm of [3HlTMP/min/mg protein.

a nd 4 o f Fig. 7). E x p r e s s i o n o f c-Myc is i n h i b i t e d in g l u c o c o r t i c o i d - t r e a t e d P1798 T lymphoma cells (18, 19). T h e c-Myc a n t i b o d y r e c o g n i z e s a P1798 p r o tein o f t h e a p p r o p r i a t e size, a n d the a b u n d a n c e o f this p r o t e i n was r e d u c e d in g l u c o c o r t i c o i d - t r e a t e d P1798 cells ( c o m p a r e s lanes 1 and 2). T h e c-Myc m R N A in P 179 8 l y m p h o m a is 3 - 5 t i m e s m o r e a b u n d a n t t h a n in L 9 2 9 cells (not shown). T h i s is c o n s i s t e n t w i t h t h e

o b s e r v a t i o n that t h e p r e s u m p t i v e c-Myc p r o t e i n is also m o r e a b u n d a n t in extracts f r o m P1798 cells ( c o m p a r e lanes 1 a n d 3). T h e o b s e r v a t i o n that the a b u n d a n c e o f c-Myc m R N A does n o t c h a n g e in g l u c o c o r t i c o i d - t r e a t e d cells suggests that t r a n s c r i p t i o n o f c-Alyc persists in G 1 a r r e s t e d L 9 2 9 cells. R u n - o n t r a n s c r i p t i o n o f c-Myc was m e a s u r e d in n u c l e i isolated f r o m L 9 2 9 cells. As s h o w n

(A) IS

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NIIIH

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Fig. 6. A b u n d a n c e o f c-Myc m R N A in L929 cells. P a n e l A c o n t a i n s t h e r e s u l t s of a n R N a s e p r o t e c t i o n a s s a y . L a n e M c o n t a i n s 32P-labeled r e s t r i c t i o n f r a g m e n t s d e r i v e d by d i g e s t i o n of b a c t e r i o p h a g e phiX174 w i t h HaeIIl. L a n e P c o n t a i n s t h e 3ZP-labeled a n t i s e n s e R N A p r o b e . T h i s w a s m i x e d w i t h a n u n l a b e l e d c-Myc i n t e r n a l s t a n d a r d m R N A ( t r a n s c r i b e d f r o m p M I S as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s ) . T h e 117nt p r o t e c t e d f r a g m e n t d e r i v e d f r o m h y b r i d i z a t i o n o f t h e p r o b e a n d t h e i n t e r n a l s t a n d a r d R N A is s h o w n in l a n e IS. T o t a l R N A w a s i s o l a t e d f r o m m i d - l o g p h a s e cells (lane C) as well as cells t h a t h a d b e e n t r e a t e d w i t h 0.1 p M d e x a m e t h a s o n e for 24 h (lane D). In a d d i t i o n , cells w e r e t r e a t e d w i t h d e x a m e t h a s o n e for 24 h. T h e a g o n i s t w a s r e m o v e d a n d R N A w a s i s o l a t e d 12 h a n d 24 h a f t e r r e s c u e (lanes R12 a n d R24, r e s p e c t i v e l y ) . T h e p o s i t i o n s of t h e f r a g m e n t s p r o t e c t e d by t h e P1 a n d P2 t r a n s c r i p t s a r e as i n d i c a t e d . P a n e l B c o n t a i n s q u a n t i t a t i v e d a t a d e r i v e d by d e n s i t o m e t r i c a n a l y s i s o f P a n e l A. Note t h a t t h e scales f o r P1 a n d P2 differ by 40-fold (P2 × 10 -4 = P1 × 0.004). T h e p r o t e c t e d f r a g m e n t s differ in [32PLUMP c o n t e n t by a f a c t o r o f 1.9. C o n s e q u e n t l y , if t h e a u t o r a d i o g r a p h i c i n t e n s i t y o f t h e P2 f r a g m e n t is 40 t i m e s t h a t o f t h e P1, t h e a b u n d a n c e o f t h e P2 f r a g m e n t is a l m o s t 80 t i m e s g r e a t e r .

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70.6

D

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Fig. 7. Glucocorticoid r e g u l a t i o n o f c-Myc p r o t e i n in L929 and P1798 cells. E x t r a c t s were p r e p a r e d f r o m m i d - l o g p h a s e and g l u c o c o r t i c o i d - t r e a t e d c u l t u r e s o f P1798 T l y m p h o m a cells (lanes 1 a n d 2, respectively) and f r o m L929 cells (lanes 3 a n d 4, respectively). P r o t e i n s were resolved on S D S - p o l y a c r y l a m i d e gels a n d b l o t t e d on nitrocellulose filters. The filters were p r o b e d with a m o n o c l o n a l a n t i b o d y against c-Myc p r o t e i n , a n d i m m u n o r e a c t i o n was visualized by e n h a n c e d c h e m i l u m i n e s c e n c e . Technical details are given in M a t e r i a l s a n d Methods.

in Fig. 8(A), several single-stranded probes were used to assess transcription of the entire c-Myc gene [pMcMyc 54 and c-Myc ( + ) ] as well as exon I and exon II. T h e probes that correspond to exons I and I I are approximately the same size (379 and 417 bp, respectively) and both transcribed ( + ) and RNA-like ( - ) strands are included to assess a s y m m e t r y of transcription. Several interesting p h e n o m e n a are observed. Initially, glucocorticoids had no discernible effect upon transcription, as one would predict from the data shown above. Furthermore, transcription of exon I was equivalent to that of exon II, as illustrated by the quantitative data shown in Fig. 8(B). ( T h e data shown in Fig. 8(B) are corrected for length of the probes and for U M P content.) T h e s e data suggest that attenuation of transcription does not prevail, to any significant extent, in mid-log phase L929 cells or in cells that have been arrested in G1 by addition of dexamethasone. Figure 8(B) also contains the results of nuclear run-on experiments carried out with P1798 l y m p h o m a cells, which are known to exhibit attenuation [18]. T h e transcriptional activity of exon I I in P1798 cells was only about 20% of that of exon I. This observation indicates that only one R N A polymerase molecule in five is capable of reading through the first exon in P1798 T l y m p h o m a cells. T h e extent of attenuation is L929 cells is strikingly reduced. DISCUSSION Regulation of c-Myc is central to proliferation of hematopoietic cells. Cell cycle arrest of H L 6 0 cells is thought to be due to a decrease in the abundance of c-Myc m R N A [2, 8-11, 41, 42]. Proliferation of erythroleukemia cells is also directly related to c-Myc

expression [15, 43, 44]. Activated splenic lymphocytes express high levels of c-Myc m R N A [45, 46] and glucocorticoids inhibit c-Myc expression and block mitogen stimulation of such cells (unpublished data from this laboratory). Glucocorticoids inhibit expression of c-Myc in h u m a n C C R F - C E M leukemic cells [20, 21, 28] as well as mouse T l y m p h o m a $49 [17, 28] and P1798 cells [18, 19]. These data indicate that mitotic activity of hematopoietic cells is dependent upon c-Myc expression and that glucocorticoid inhibition of lymphoid cell proliferation involves regulation of c-Myc. As in hematopoietic cells, regulation of c-Myc is also tightly linked to fibroblast proliferation (reviewed in 7). S e r u m starvation imposes a G O arrest on untransformed fibroblasts, and c-Myc m R N A abundance decreases. T h e abundance of c-Myc m R N A increases rapidly following serum stimulation of quiescent fibroblasts. It has been reported that activation of a Mycestrogen receptor chimaeric protein is sufficient to drive serum-starved fibroblasts into S phase [47]. Glucocorticoids inhibit fibroblast proliferation in culture and in vivo [23, 24], although the mechanism(s) have not been studied in great detail. L929 fibroblasts undergo G 1 arrest in the presence of glucocorticoids. It is not known if G1 arrest occurs in glucocorticoidtreated papovavirus-transformed C129 fibroblasts, but the proliferation of such cells is strikingly inhibited by glucocorticoids [24]. G r o w t h arrest of transformed C129 cells is associated with a decrease in c-Myc m R N A [24]. However, untransformed fibroblasts exhibit a different response. T h e abundance of c-Myc m R N A and c-Myc protein does not decrease as glucocorticoid-treated L929 cells cease to proliferate. Glucocorticoids also inhibit proliferation of untransformed

c-Myc R e g u l a t i o n in F i b r o b l a s t s

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Fig. 8. N u c l e a r r u n - o n t r a n s c r i p t i o n o f c-Myc i n L929 cells. N u c l e i w e r e i s o l a t e d f r o m c o n t r o l cells a n d f r o m t h o s e t h a t w e r e e x p o s e d to 0.1 ItM d e x a m e t h a s o n e f o r 24 h. N u c l e a r r u n - o n t r a n s c r i p t i o n w a s c a r r i e d o u t w i t h 1 0 7 n u c l e i a n d t h e p r o d u c t s o f t r a n s c r i p t i o n w e r e i s o l a t e d a n d h y b r i d i z e d to p r o b e s c o r r e s p o n d i n g to t h e 5S R N A g e n e ( p T H 1 ) , v e c t o r ( p U C 9 ) , o r v a r i o u s d e r i v a t i v e s o f c-Myc, as s h o w n in P a n e l A. T h e d e r i v a t i v e s i n c l u d e d o u b l e - s t r a n d e d , f u l l - l e n g t h e D N A (pMc-Myc54), M13 c l o n e s c o r r e s p o n d i n g to b o t h s t r a n d s o f f u l l - l e n g t h c-Myc e D N A [c-Myc (+) a n d c-Myc ( - ) ] a n d b o t h s t r a n d s o f s u b c l o n e s f r o m e x o n s I a n d II (as i n d i c a t e d ) . L a n e C contains the products of hybridization from control nuclei and lane D those from alex-treated nuclei. The i n t e n s i t y o f t h e a u t o r a d i o g r a p h i c s i g n a l s f r o m e x o n s I a n d II w e r e q u a n t i f i e d , a n d a r e p r e s e n t e d in P a n e l B, r e l a t i v e to t r a n s c r i p t i o n o f t h e 5S R N A g e n e . All d a t a a r e n o r m a l i z e d f o r l e n g t h o f t h e s i n g l e s t r a n d e d p r o b e a n d U M P c o n t e n t . T h e d a t a s h o w n in t h i s f i g u r e a r e t h e a v e r a g e o f t w o e x p e r i m e n t s . R u n - o n t r a n s c r i p t i o n o f e x o n s I a n d II w a s a l s o a n a l y z e d in n u c l e i i s o l a t e d f r o m P1798 l y m p h o m a cells.

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C129 ceils, with very little effect o n the a b u n d a n c e of c - M y c m R N A [24]. T h e a b u n d a n c e of c - M y c p r o t e i n was n o t assayed in g l u c o c o r t i c o i d - t r e a t e d C129 cells, so one c a n n o t say for certain that g l u c o c o r t i c o i d - a r r e s t e d , u n t r a n s f o r m e d C129 cells c o n t i n u e to express c - M y c . E x a m p l e s of G l - a r r e s t e d cells that express c - M y c are very rare [for review see 7]; and, w h e n one considers the evidence that links glucocorticoid r e g u l a t i o n of l y m p h o i d cell p r o l i f e r a t i o n to i n h i b i t i o n of c - M y c , it is u n a n t i c i p a t e d that g l u c o c o r t i c o i d - i n h i b i t e d , u n t r a n s f o r m e d fibroblasts fail to t u r n off c - M y c . It is also s u r p r i s i n g that L929 cells do n o t e x h i b i t a t t e n u a t i o n of c - M y c t r a n s c r i p t i o n . A l m o s t all cells that have b e e n e x a m i n e d e x h i b i t significant a t t e n u a t i o n of c - M y c [2, 12, 44, 48-50]. T h e process is best o b s e r v e d i n n u c l e a r r u n - o n t r a n s c r i p t i o n assays, w h i c h reveal that the R N A p o l y m e r a s e 1I p a c k i n g d e n s i t y of c - M y c exon I is often > 1 0 times that of exon I I . Loss of a t t e n u a t i o n c o n t r i b u t e s to activation of c - M y c in B u r k i t t ' s l y m p h o m a cells [16, 48]. It has b e e n p r o p o s e d that failure of a t t e n u a t i o n is d u e to the p r e d o m i n a n t u t i l i z a t i o n of the P1 p r o m o t e r i n B u r k i t t ' s l y m p h o m a cells [16, 48]. A c c o r d i n g to this hypothesis, only those t r a n s c r i p t s initiated at P2 are susceptible to p r e m a t u r e t e r m i n a t i o n , w h i c h m i g h t therefore be avoided b y a p r o m o t e r switch. T h i s s i t u a t i o n c a n n o t a c c o u n t for failure of a t t e n u a t i o n in L929 cells, w h i c h appear to use the P2 p r o m o t e r almost exclusively. T h e lack of a t t e n u a t i o n in L 9 2 9 cells could reflect some change in the s e q u e n c e or c o n t e x t of the c - M y c gene. A l t e r n a t i v e l y , L929 cells m a y h a r b o r a defect in the expression of one or m o r e s u b s t a n c e s that affect a t t e n u a t i o n in trans. T h e i n f o r m a t i o n that is c u r r e n t l y available is insufficient to d i s c r i m i n a t e b e t w e e n these alternative hypotheses. F i b r o b l a s t i c a n d l y m p h o i d cells share a c o m m o n ontological origin, a n d b o t h types of cells exhibit g r o w t h i n h i b i t i o n in the presence of glucocorticoids. O n e m i g h t be i n c l i n e d to speculate that g l u c o c o r t i c o i d m e d i a t e d g r o w t h arrest in closely related cell types m i g h t reflect similar, if n o t identical, m e c h a n i s m s . T h i s is clearly n o t the case. I n h i b i t i o n of c - M y c e x p r e s s i o n is critical to the a n t i - p r o l i f e r a t i v e a n d lytic responses that are triggered b y glucocorticoids in l y m p h o i d cells of t h y m i c origin [17-22]. T h e response is rapid [19], due to i n h i b i t i o n of t r a n s c r i p t i o n [18], a n d i n s e n s i t i v e to i n h i b i t i o n of p r o t e i n synthesis (T. M a a n d E. A. T h o m p s o n , u n p u b l i s h e d observation). A l t h o u g h it has n o t b e e n directly d e m o n s t r a t e d , the data s t r o n g l y suggest that the glucocorticoid r e c e p t o r interacts with the c - M y c p r o m o t e r so as to i n h i b i t t r a n s c r i p t i o n . O n e is i n c l i n e d to speculate that r e g u l a t i o n of c - M y c m i g h t be a c o m m o n m e c h a n i s m w h e r e b y glucocorticoids influence p r o l i f e r a t i o n of cells of m a n y different types, b u t the data p r e s e n t e d above are i n c o n s i s t e n t with this c o n c l u s i o n . R e g u l a t i o n of c - M y c does n o t a c c o u n t for glucocorticoid i n h i b i t i o n of p r o l i f e r a t i o n of the best characterized, glucocorticoid sensitive fibroblastic cell line, L929. A l t e r n a t i v e m e c h a n i s m s m u s t be c o n s i d e r e d

tO a c c o u n t for glucocorticoid effects o n fibroblast p r o liferation. Acknowledgements--This work was supported in part by grants from the National Cancer Institute CA24347 and the National Institute o1 Diabetes, Digestive, and Kidney Diseases DK42788.

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