Molecular and CellularBiochemistry 103: 149--154,1991. © 1991KluwerAcademic Publishers. Printedin the Netherlands.
Effect of dexamethasone and cycloheximide on the expression of amplified EGF-receptor gene in human A431 carcinoma cell line Mohan L. Gope 1, Rajalakshmi Gope 1 and Tanya Zarucki 2 Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030, USA; Present address: 1Creighton Cancer Center, Creighton University, 24th at California Street, Omaha, NE 68178; 2 Cancer and Developmental Biology, Syntex Research, R7-275, Palo Alto, CA 94304, USA Received7 August 1990;accepted4 December1990
Key words: EGF-receptor gene, gene expression, cycloheximide, dexamethasone and A431 carcinoma cell line Abstract
Human A431 carcinoma cell line is known to have 30 fold amplified epidermal growth factor receptor (EGF-R) gene. We have studied the effect of steroid hormone dexamethasone (DEX) and protein synthesis inhibitor cycloheximide (CHX) on the expression of EGF-R gene in this cell line. DEX treatment and protein synthesis inhibition by CHX treatment cause a rapid 3 to 4 fold increase in the level of EGF-R mRNA and combined treatment of the above two agents have less than additive effect. It appears that mRNA for EGF-R accumulate within the cell during protein synthesis inhibition and upon removal of CHX, gets translated into EGF-R specific protein as judged by immuno-dot assay. We did not observe the phenomenon of 'super induction' nor much of an additive effect under condition of combined DEX and CHX treatment.
Abbreviations: EGF-R - Epidermal Growth Factor Receptor, DEX - Dexamethasone, CHX - Cycloheximide
Introduction
The cloning and sequencing of human epidermal growth factor receptor (EGF-R) cDNA by us [1] and others [2, 3] has shown that it is homologous to the avian erythroblastosis virus erb B oncogene. The human A431 epidermoid carcinoma cell line has a very large number of EGF-receptors. A431 cells contain three prominent species of EGF-R mRNA (10.5, 5.8 and 2.9kb). The 2.9kb RNA is not found in other cell line expressing moderate levels of EGF receptor and it diverges from 10.5 and 5.8 kb EGF receptor R N A from other cells at its extreme 3' end [4-7]. EGF-R gene is 30 fold
amplified in A431 cells [4, 5] and has characteristics typical of oncogenes in these tumor cells. It has been reported that transfected human IFN-B gene in chinese hamster ovary retains transcriptional inducibility by cycloheximide even when the gene is amplified 25-50 fold by selection [8]. In this report, we have investigated the effect of dexamethasone (DEX) and cycloheximide (CHX) on the expression of EGF-R mRNA in A431 cells. The results indicate that DEX treatment and the inhibition of protein synthesis by CHX treatment independently cause three to four fold increase in the level of EGF-R mRNA and combined treatment of these two agents have less than additive effect.
150 Materials and methods
Cell culture Human A431 carcinoma cells were obtained from Dr. Graham Carpenter (Vanderbilt University). Cells were maintained in Dulbecco's Modified Eagle's Medium (GIBCO) supplemented with 10% Fetal Calf Serum, 20 mM Hepes, penicillin (100 u/ ml) and Streptomycin (100/zg/ml) under a 1 0 % CO2: 90% air atmosphere at 37 ° C and media were changed every two days. When cells reached 6080% confluency, each monolayer was washed with Hank's solution and fed with fresh medium containing serum and treated with D E X (10 -7 M) and/ or CHX (10/zg/ml). This concentration of CHX inhibited protein synthesis by 95% (data not shown). RNA isolation Total R N A was extracted from A431 cells as described in Chirgwin et al. [9]. Cell monolayers were washed in PBS and lysed in 4 M guanidine thiocyanate, containing 0.1 M 2-mercaptoethanol. The cell lysate was layered on a cushion of 5.7M CsC1, in 0.1 M EDTA, and R N A was pelleted by ultracentrifugation in SW27 rotor for 18 hr at 25,000 rpm and 20 ° C. RNA dot hybridization Total cell RNA was denatured by heating for 5 rain at 60°C in 50% formamide (deionized), 6% formaldehyde, 20 mM phosphate buffer. After quenching in icewater, an equal volume of 20 x SSC (1 x SSC is 0.15 M NaCI plus 0.015 M sodium citrate, pH 7.0) was added and sample was diluted to an appropriate volume with cold 10 x SSC so that 0.2ml portion contained 2-4/zg RNA. The R N A samples were dotted onto Biodyne A membranes according to the protocol of Schloss et al. [10] using a dotblot apparatus (Schleicher and Schuell). Equal amounts of yeast tRNA were included in some experiments as controls for background hybridization. Northern analysis Twenty microgram of the total RNAs were analyzed on a formaldehyde-agarose gel [11] and hybridized with 32P-labeled EGF-R cDNA probe [1].
The filters were washed, and the bands were visualized by autoradiography as described below.
Radioactive probe and hybridization The purified insert (880 bp) of a cDNA clone isolated by us [1] was used as hybridization probe. 32p labeling of the insert by nick translation was done as previously described [1]. This insert encodes the carboxyterminal end of the human EGF-R protein and does not extend 5' into the sequences encoding the kinase domain [1]. Filters containing immobilized R N A were prehybridized for 6 to 12 hr at 42 ° C in 50% formamide (deionized), 50 mM sodium phosphate buffer (pH 6.5), 5 x SSC, 5 x Denhardt Solution, 0.1% SDS, 100 tzg/ml sonicated-denatured calf thymus D N A and 50/zg/ml denatured yeast t/RNA. They were hybridized in the same solution for 24 hr at 42°C with denatured cDNA probe at 107cpm/ml for northern blots and dot blots. Filters were washed twice (20rain each) in 2 x SSC plus 0.1% SDS at 37°C and twice (20 rain each) in 0.1 x SSC plus 0.1% SDS at 65°C. lmmuno-dot assay of EGF-receptor-specific protein Cell monolayers were washed three times with cold phosphate buffered saline, solubilized for 20 min at room temperature in RIPA buffer (10raM TrisHC1 pH 8.5, 0.15 M NaC1, 5 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing 0.1% phenylmethyl-sulfonyl fluoride (PMSF) and 200 KIU/ml aprotinin. The lysate was centrifuged at 140,000 xg for 1 h at 4 ° C. The supernatants were immediately subjected to dot-blot experiment for immunoassay. The protein dot-blots were immunoassayed with rabbit polyclonal antibody (IgG; #986) raised to affinity purified EGF-R protein from A431 cells [12, 13] in combination with nSI-labelled S. aureus protein A according to the procedures of Hawkes [14].
Results
Total RNA from control, CHX, DEX and combination of CHX and D E X treated A431 cells were
151
Fig. 2. Northern blot analysisof A431 RNA. Twenty microgram
of total RNA were loaded in each lane. Lane A - control; B 2 hr DEX + CHX treatment; C - 2 hr CHX treatment; and D 2 hr DEX treatment. Number on the right indicate sizes (in kb) of mRNA bands.
Fig. 1. Time course of the effects of CHX, DEX or both on
EGF-R specificmRNA level. Cells were exposed continuously for 3, 6, 9 and 24 hr to CHX (10/xg/ml), DEX (10-7 M) or both. Total RNA was isolated and dot-blotted onto bio-dyne membrane, hybridized to nick translated probe and analysed by autoradiography. For each panel, the upper dots contain 2t~g and the lower dots contain 1/xg of total RNA. A - Control; B CHX; C - DEX; D - CHX + DEX. quantitated for EGF-R-specific m R N A level by dot-blot hybridization technique using 32p-labelled E G F - R c D N A probe. Figure 1 shows the autoradiogram of the dot blot. The EGF-R-specific m R N A levels were higher in C H X (Fig. 1B), D E X (Fig. 1C) and combined C H X and D E X (Fig. 1D) treated samples at all time points from 3 hr to 24 hr c o m p a r e d with those of the corresponding control samples (Fig. 1A). In order to quantitate the E G F receptor m R N A in each dot, the autoradiograms were scanned. Results extrapolated from the scanning data show that C H X and D E X individually can increase the level of EGF-R-specific m R N A 3
to 4 folds respectively. The time course of induction in response to D E X , C H X and both showed that the effect is in fact considerably protracted and remain higher throughout the course of exposure to these agents compared to the controls. It is interesting to note that C H X does not seem to act synergistically with D E X . The fold increase in message level by combined treatment with D E X and CH_X is significantly higher at all time points than the individual treatments but it is not additive. Northern analysis was p e r f o r m e d using total R N A isolated from A431 cells treated with the above agents to determine the fold induction of the two m R N A species (10.5 kb and 5.8 kb) encoding the E G F - R . The northern blots were p r o b e d with the c D N A segment derived f r o m the cytoplasmic domain and the 3' untranslated region of the E G F receptor gene [1]. Figure 2 shows the expected two different sizes (10.5 and 5.8 kb) of m R N A hybridized to the probe. The intensity of the 10.5 kb band remains essentially the same but there is 2 to 3 fold increase in the intensity of 5 . 8 k b band in D E X , C H X and D E X plus C H X treated lanes. The increased E G F - R specific m R N A level was maintained in continuous presence of C H X , D E X or both for 24 hr. We wanted to investigate how
152
Fig. 4. Effect of CHX treatment on the level of EGF-R specific protein. B: Lane - 1 - Cells treated with CHX for 1 hr. Lane - 2, 3, 4 - CHX removed by washing and cells were allowed to grow in fresh medium for 1, 3 and 5 hr respectively. A: Lanes 2, 3, 4 Cells grown without any treatment for 1, 3 and 5 hr respectively. The cell lysates were analysed for EGF-R specific protein as described in materials and methods. For each panel, the upper dots contain 2 ~g and lower dots contain 4 ~g protein. Fig. 3. EGF-R specific mRNA level after 1 hr treatment with CHX and DEX. 70--80% confluent cell culture were exposed for lhr with CHX or DEX. The treatment was terminated by washing the cells thoroughly with PBS. Cells were grown in fresh medium for different time periods. Total RNA was isolated, dot-blotted onto bio-dyne membrane, hybridized to nick translated probe and analysed by autoradiography. A - Control samples for the corresponding time points as in B and C; B C H X treatment; C - DEX treatment; lane 1 - 1 hr treatment; lanes 2, 3 and 4 - treatment is stopped by washing cells with PBS and allowing them to grow in fresh medium for 1, 2 and 5 hr respectively. The upper dots contain 2/zg and the lower dots contain 1/~g of total RNA.
l o n g t h e i n c r e a s e d m R N A level is m a i n t a i n e d a f t e r t h e r e m o v a l o f t h e s e t r e a t m e n t s . T h e results in Fig. 3 shows t h a t t h e t r e a t m e n t o f A431 cells w i t h D E X o r C H X for 1 h r is e n o u g h to i n c r e a s e t h e E G F - R specific m R N A level 3 to 4 fold. This i n c r e a s e d l e v e l is m a i n t a i n e d for 2 h r a n d d e c r e a s e d to a l m o s t b a s a l level b y 3 h r a f t e r t h e r e m o v a l o f t h e s e a g e n t s b y w a s h i n g t h e ceils with P B S . T h e cell l y s a t e s f r o m c o n t r o l , C H X a n d D E X t r e a t e d A431 cells w e r e a n a l y s e d b y t h e p r o t e i n d o t - b l o t s a n d t h e level o f E G F - R - s p e c i f i c p r o t e i n
w e r e i m m u n o a s s a y e d with r a b b i t p o l y c l o n a l antib o d y ( l g G ; # 9 8 6 ) r a i s e d to affinity p u r i f i e d E G F - R p r o t e i n f r o m A431 cells (Fig. 4). T h e C H X t r e a t m e n t for 1 hr d e c r e a s e d t h e level o f E G F rec e p t o r specific p r o t e i n slightly f r o m the c o n t r o l level b u t r e m o v a l o f C H X by w a s h i n g r a p i d l y inc r e a s e d t h e p r o t e i n level 3 to 4 fold o v e r t h e c o n t r o l (Fig. 4B, lanes 2 a n d 3) a n d a l m o s t e q u a l to D E X t r e a t e d level ( d a t a n o t shown), This i n c r e a s e d level in t h e a b s e n c e o f C H X r e m a i n s stable for 3 h r a n d b e g i n s to d e c r e a s e b y 5 hr. I n t h e p r e s e n c e o f D E X , t h e level o f r e c e p t o r specific p r o t e i n is 3 to 4 fold higher than the control (data not shown).
Discussion It has b e e n s h o w n t h a t E G F - R g e n e c o p y n u m b e r c o r r e l a t e s e x t r e m e l y well with E G F - R levels in a v a r i e t y o f A 4 3 1 - r e l a t e d cells [4]. O t h e r cell lines f o u n d to c o n t a i n a s u b s t a n t i a l a m o u n t o f E G F - R p r o t e i n p r o b a b l y utilize r e g u l a t o r y m e c h a n i s m s
153 other than gene amplification to overproduce EGF receptor R N A and protein. The observed rapidity with which EGF receptor m R N A accumulates in A431 cells in response to DEX, CHX and both, suggest that the increase in the level of EGF receptor mRNA is an early event. Whether D E X and/or CHX treatment increase the rate of transcription or stability of EGF-R m R N A can be answered by in vitro transcription experiment with isolated nuclei. Since the increase in the level of mRNA is only 3 to 4 fold, it will be experimentally difficult to show convincingly any transcriptional increase by in vitro transcription experiment with isolated nuclei in this system. The A431 cells are known to synthesize a 2.9 kb mRNA and is only detected by probes derived from sequences coding for the 5' untranslated region and the extracellular domain [5]. The probe used in this study encodes the carboxy terminal end and the 3' untranslated region of the cDNA and thus it can not hybridize to the 2.9 kb mRNA present only in A431 cells. Thus our observation of only two larger sizes of EGF receptor mRNA (10.5 kb and 5.8 kb) is consistent with the observations of Ullrich et al. [5]. The 2.9 kb mRNA is known to encode a 70 KD truncated receptor protein that is secreted. Thus the observed increase in the level of EGF-R protein and mRNA in A431 cells detected by immunoblot of whole cell lysate and 3' end cDNA probe does not include the 70 KD protein or 2.9 kb mRNA. Our data show that the 10.5kb m R N A level remains almost constant whereas the level of 5.8 kb m R N A increases three to four fold by the D E X and CHX treatment (Fig. 2). It has been suggested that the 10.5 kb mRNA is a precursor of 5.8 kb mRNA [5]. This raises the possibility that these treatments (DEX and/or CHX) could affect the R N A processing steps involved. We do not observe much of an additive effect when both CHX and DEX are present. It appears most likely that DEX acts through a positive regulator and CHX through a negative regulator and in presence of both CHX not only destroys the negative regulator but also the positive regulator. Thus one observes mostly the effects of only CHX when both CHX and D E X are present.
The effect of CHX is maintained in A431 cells containing 30 fold amplification of this gene. The transfected human IFN-B gene in chinese hamster ovary can be induced by CHX even when the gene is amplified 25-50 fold [8]. Thus if a repressor controls the EGF-R gene expression, it must be present in excess of what is minimally required to regulate this gene as suggested for transfected human IFN-B gene in chinese hamster ovary [8]. Thus it appears that other highly amplified genes are also likely to be regulated in similar fashion. The data in Fig. 4 shows that when A431 cells are placed in fresh media containing CHX for 1 hr, the EGF-R protein level, as measured by immuno-dot assay, remains almost equal to control level. It has been reported that fresh sera act as inhibitors of protein degradation in cultured cells [15]. As a result though CHX treatment inhibits protein synthesis, the level of EGF-R-specific protein remained essentially unaltered (Fig. 4). When CHX is removed by washing the cells thoroughly and allowing them to grow in fresh media, the level of EGF-R protein as detected by immuno-dot assay increases rapidly (Fig. 4). Similarly, the CHX mediated increase in the EGF receptor mRNA level is maintained for 2 hr after removal of CHX (Fig. 3). These results indicate that during protein synthesis inhibition, mRNA for EGF receptors accumulate within the cell and gets translated when reinitiation of protein synthesis is allowed by washing away CHX. Since CHX is known not to block mRNA synthesis [8, 16], it could be possible that the mRNA for EGF-R is transcribed during protein synthesis inhibition, stored and translated when protein synthesis is allowed to reinitiate. It is worth noting that the presence of CHX does not lead to the synthesis of aberrant mRNA and/or protein for EGF-R as judged by immuno-assay. It has been shown that interferon mRNA isolated from human fibroblast in presence and absence of CHX can synthesize interferon equally well [17, 18]. Further, accumulation of mRNA during protein synthesis inhibition by inhibitors have been reported in other systems [19-21]. Whether the increased levels of EGF-R mRNA in the presence of D E X or CHX can be explained entirely by the increase in half-life
154 of the EGF-R m R N A or the rate of transcription is altered as well remains to be determined.
Acknowledgements We express our deep appreciation to Dr. Bert W. O'Malley for his encouragement and support in carrying out this work in his laboratory. We are grateful to Dr. G. Carpenter for kindly providing us the EGF-receptor antibody.
References 1. Simmen FA, Gope ML, Schulz TZ, Wright DA, Carpenter G, O'Malley BW: Isolation of an evolutionarily conserved epidermal growth factor receptor cDNA from human A431 carcinoma cells. Biochem Biophys Res Commun 124: 125132, 1984 2. Downward J, Yarden Y, Mayes E, Scrace G, Totty N, Stockwell P, Ullrich A, Schlessinger J, Waterfield MD: Close similarity of epidermal growth factor receptor and v-erb-B oncogene protein sequences. Nature (London) 307: 521-527, 1984 3. Yamarnoto T, Nishida T, Miyajima N, Kawai S, Ooi T, Toyoshima K: The erbB gene of avian erythroblastosis virus is a member of the src gene family. Cell 35: 71-78, 1983 4. Lin CR, Chen WS, Kruiger W, Stolarsky LS, Weber W, Evans RM, Verma IM, Gill GN, Rosenfeld MG: Expression cloning of human EGF receptor complementary DNA: gene amplification and three related messenger RNA products in A431 cells. Science 224: 843-848, 1984 5. Ullrich A, Coussens L, Hayrick JS, Dull TJ, Gray A, Tam AW, Lee J, Yarden Y, Libermann TA, Schlessinger J, Downward J, Mayes ELV, Whittle N, Waterfield MD, Seeburg PH: Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma ceils. Nature (London) 309: 418-425, 1984 6. Merlino GT, Xu Y-H, Ishii S, Clark A J, Semba K, Toyoshima K, Yamamoto T, Pastan I: Amplification and enhanced expression of the epidermal growth factor receptor gene in A431 human carcinoma cells: Science 224: 417-419, 1984 7. Xu Y-H, Ishii S, Clark AJ, Sullivan M, Wilson RK, Ma DP, Roe BA, Merlino GT, Pastan I: Human epidermal growth factor receptor cDNA is homologous to a variety of RNAs overproduced in A431 carcinoma cells. Nature (London) 309: 806-810, 1984
8. Ringold GM, Dieckmann B, Vannice JL, Trahey M, McCormick F: Inhibition of protein synthesis stimulates the transcription of human beta interferon genes in chinese hamster ovary cells. Proc Natl Acad Sci USA 81: 39643968, 1984 9. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ: Isolation of biologically active RNA from sources enriched in RNAse. Biochemistry 18: 5294--5299, 1979 10. Schloss JA, Silflow CD, Rosenbaum JL: mRNA abundance changes during flagellar regeneration in Chlamydomonas reinhardtii. Mol Cell Biol 4: 424-434, 1984 11. Maniatis T, Tritsch EF, Sambrook J: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982 12. Stoscheck CM, Carpenter G: Characteristics of antibodies to the epidermal growth factor receptor-kinase. Arch Biochem Biophys 227: 457-468, 1983 13. Stoscheck CM, Carpenter G: Characteristics and uses of antibodies to the EGF-receptor kinase. Cell Biol Int Rep 7: 529-530, 1983 14. Hawkes R: The dot immunobinding assay. In: Langone JJ, Van Vunakis H (ed) Methods in Enzymology 121: 484-491. Academic Press, New York, 1986, 121: pp 484-491 15. Poole B, Wibo M: Protein degradation in cultured cells. The effect of fresh medium, fluoride, and iodoacetate on the digestion of cellular protein of rat fibroblasts. J Biol Chem 248: 6221-6226, 1973 16. Isreal DI, Estolano MG, Galeazzi DR, Whitlock JP Jr: Superinduction of cytochrome P1-450 gene transcription by inhibition of protein synthesis in wild type and variant mouse hepatoma cells. J Biol Chem 260: 5648-5653, 1985 17. Sehgal PB, Lyles DS, Tam I: Superinduction of human fibroblast interferon production: Further evidence for increased stability of interferon messenger RNA. Virology 89: 186--198, 1978 18. Raj NBK, Pitha PM: Two levels of regulation of beta interferon gene expression in human cells. Proc Natl Acad Sci USA 80: 3923-3927, 1983 19. Warrington RC, Wratten N, Hechtman R: L-Histidinol inhibits specifically and reversibly protein and ribosomal RNA synthesis in mouse L cells. J Biol Chem 252: 52515257, 1977 20. Johnson LF, Meister R: Rapid increase in poly ( A ) + mRNA content following inhibition of protein synthesis. J Cell Physiol 92: 57-64, 1977 21. Ernest MJ, DeLap L, Feigelson P: Induction of hepatic tyrosine aminotransferase mRNA by protein synthesis inhibitors. J Biol Chem 253: 2895-2897, 1978
Address for offprints: M.L. Gope, Creighton Cancer Center, Creighton University, Criss III, Rm # 358, 24th at California Street, Omaha, NE 68178, USA
Effect of dexamethasone and cycloheximide on the expression of amplified EGF-receptor gene in human A431 carcinoma cell line Mohan L. Gope 1, Rajalakshmi Gope 1 and Tanya Zarucki 2 Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030, USA; Present address: 1Creighton Cancer Center, Creighton University, 24th at California Street, Omaha, NE 68178; 2 Cancer and Developmental Biology, Syntex Research, R7-275, Palo Alto, CA 94304, USA Received7 August 1990;accepted4 December1990
Key words: EGF-receptor gene, gene expression, cycloheximide, dexamethasone and A431 carcinoma cell line Abstract
Human A431 carcinoma cell line is known to have 30 fold amplified epidermal growth factor receptor (EGF-R) gene. We have studied the effect of steroid hormone dexamethasone (DEX) and protein synthesis inhibitor cycloheximide (CHX) on the expression of EGF-R gene in this cell line. DEX treatment and protein synthesis inhibition by CHX treatment cause a rapid 3 to 4 fold increase in the level of EGF-R mRNA and combined treatment of the above two agents have less than additive effect. It appears that mRNA for EGF-R accumulate within the cell during protein synthesis inhibition and upon removal of CHX, gets translated into EGF-R specific protein as judged by immuno-dot assay. We did not observe the phenomenon of 'super induction' nor much of an additive effect under condition of combined DEX and CHX treatment.
Abbreviations: EGF-R - Epidermal Growth Factor Receptor, DEX - Dexamethasone, CHX - Cycloheximide
Introduction
The cloning and sequencing of human epidermal growth factor receptor (EGF-R) cDNA by us [1] and others [2, 3] has shown that it is homologous to the avian erythroblastosis virus erb B oncogene. The human A431 epidermoid carcinoma cell line has a very large number of EGF-receptors. A431 cells contain three prominent species of EGF-R mRNA (10.5, 5.8 and 2.9kb). The 2.9kb RNA is not found in other cell line expressing moderate levels of EGF receptor and it diverges from 10.5 and 5.8 kb EGF receptor R N A from other cells at its extreme 3' end [4-7]. EGF-R gene is 30 fold
amplified in A431 cells [4, 5] and has characteristics typical of oncogenes in these tumor cells. It has been reported that transfected human IFN-B gene in chinese hamster ovary retains transcriptional inducibility by cycloheximide even when the gene is amplified 25-50 fold by selection [8]. In this report, we have investigated the effect of dexamethasone (DEX) and cycloheximide (CHX) on the expression of EGF-R mRNA in A431 cells. The results indicate that DEX treatment and the inhibition of protein synthesis by CHX treatment independently cause three to four fold increase in the level of EGF-R mRNA and combined treatment of these two agents have less than additive effect.
150 Materials and methods
Cell culture Human A431 carcinoma cells were obtained from Dr. Graham Carpenter (Vanderbilt University). Cells were maintained in Dulbecco's Modified Eagle's Medium (GIBCO) supplemented with 10% Fetal Calf Serum, 20 mM Hepes, penicillin (100 u/ ml) and Streptomycin (100/zg/ml) under a 1 0 % CO2: 90% air atmosphere at 37 ° C and media were changed every two days. When cells reached 6080% confluency, each monolayer was washed with Hank's solution and fed with fresh medium containing serum and treated with D E X (10 -7 M) and/ or CHX (10/zg/ml). This concentration of CHX inhibited protein synthesis by 95% (data not shown). RNA isolation Total R N A was extracted from A431 cells as described in Chirgwin et al. [9]. Cell monolayers were washed in PBS and lysed in 4 M guanidine thiocyanate, containing 0.1 M 2-mercaptoethanol. The cell lysate was layered on a cushion of 5.7M CsC1, in 0.1 M EDTA, and R N A was pelleted by ultracentrifugation in SW27 rotor for 18 hr at 25,000 rpm and 20 ° C. RNA dot hybridization Total cell RNA was denatured by heating for 5 rain at 60°C in 50% formamide (deionized), 6% formaldehyde, 20 mM phosphate buffer. After quenching in icewater, an equal volume of 20 x SSC (1 x SSC is 0.15 M NaCI plus 0.015 M sodium citrate, pH 7.0) was added and sample was diluted to an appropriate volume with cold 10 x SSC so that 0.2ml portion contained 2-4/zg RNA. The R N A samples were dotted onto Biodyne A membranes according to the protocol of Schloss et al. [10] using a dotblot apparatus (Schleicher and Schuell). Equal amounts of yeast tRNA were included in some experiments as controls for background hybridization. Northern analysis Twenty microgram of the total RNAs were analyzed on a formaldehyde-agarose gel [11] and hybridized with 32P-labeled EGF-R cDNA probe [1].
The filters were washed, and the bands were visualized by autoradiography as described below.
Radioactive probe and hybridization The purified insert (880 bp) of a cDNA clone isolated by us [1] was used as hybridization probe. 32p labeling of the insert by nick translation was done as previously described [1]. This insert encodes the carboxyterminal end of the human EGF-R protein and does not extend 5' into the sequences encoding the kinase domain [1]. Filters containing immobilized R N A were prehybridized for 6 to 12 hr at 42 ° C in 50% formamide (deionized), 50 mM sodium phosphate buffer (pH 6.5), 5 x SSC, 5 x Denhardt Solution, 0.1% SDS, 100 tzg/ml sonicated-denatured calf thymus D N A and 50/zg/ml denatured yeast t/RNA. They were hybridized in the same solution for 24 hr at 42°C with denatured cDNA probe at 107cpm/ml for northern blots and dot blots. Filters were washed twice (20rain each) in 2 x SSC plus 0.1% SDS at 37°C and twice (20 rain each) in 0.1 x SSC plus 0.1% SDS at 65°C. lmmuno-dot assay of EGF-receptor-specific protein Cell monolayers were washed three times with cold phosphate buffered saline, solubilized for 20 min at room temperature in RIPA buffer (10raM TrisHC1 pH 8.5, 0.15 M NaC1, 5 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing 0.1% phenylmethyl-sulfonyl fluoride (PMSF) and 200 KIU/ml aprotinin. The lysate was centrifuged at 140,000 xg for 1 h at 4 ° C. The supernatants were immediately subjected to dot-blot experiment for immunoassay. The protein dot-blots were immunoassayed with rabbit polyclonal antibody (IgG; #986) raised to affinity purified EGF-R protein from A431 cells [12, 13] in combination with nSI-labelled S. aureus protein A according to the procedures of Hawkes [14].
Results
Total RNA from control, CHX, DEX and combination of CHX and D E X treated A431 cells were
151
Fig. 2. Northern blot analysisof A431 RNA. Twenty microgram
of total RNA were loaded in each lane. Lane A - control; B 2 hr DEX + CHX treatment; C - 2 hr CHX treatment; and D 2 hr DEX treatment. Number on the right indicate sizes (in kb) of mRNA bands.
Fig. 1. Time course of the effects of CHX, DEX or both on
EGF-R specificmRNA level. Cells were exposed continuously for 3, 6, 9 and 24 hr to CHX (10/xg/ml), DEX (10-7 M) or both. Total RNA was isolated and dot-blotted onto bio-dyne membrane, hybridized to nick translated probe and analysed by autoradiography. For each panel, the upper dots contain 2t~g and the lower dots contain 1/xg of total RNA. A - Control; B CHX; C - DEX; D - CHX + DEX. quantitated for EGF-R-specific m R N A level by dot-blot hybridization technique using 32p-labelled E G F - R c D N A probe. Figure 1 shows the autoradiogram of the dot blot. The EGF-R-specific m R N A levels were higher in C H X (Fig. 1B), D E X (Fig. 1C) and combined C H X and D E X (Fig. 1D) treated samples at all time points from 3 hr to 24 hr c o m p a r e d with those of the corresponding control samples (Fig. 1A). In order to quantitate the E G F receptor m R N A in each dot, the autoradiograms were scanned. Results extrapolated from the scanning data show that C H X and D E X individually can increase the level of EGF-R-specific m R N A 3
to 4 folds respectively. The time course of induction in response to D E X , C H X and both showed that the effect is in fact considerably protracted and remain higher throughout the course of exposure to these agents compared to the controls. It is interesting to note that C H X does not seem to act synergistically with D E X . The fold increase in message level by combined treatment with D E X and CH_X is significantly higher at all time points than the individual treatments but it is not additive. Northern analysis was p e r f o r m e d using total R N A isolated from A431 cells treated with the above agents to determine the fold induction of the two m R N A species (10.5 kb and 5.8 kb) encoding the E G F - R . The northern blots were p r o b e d with the c D N A segment derived f r o m the cytoplasmic domain and the 3' untranslated region of the E G F receptor gene [1]. Figure 2 shows the expected two different sizes (10.5 and 5.8 kb) of m R N A hybridized to the probe. The intensity of the 10.5 kb band remains essentially the same but there is 2 to 3 fold increase in the intensity of 5 . 8 k b band in D E X , C H X and D E X plus C H X treated lanes. The increased E G F - R specific m R N A level was maintained in continuous presence of C H X , D E X or both for 24 hr. We wanted to investigate how
152
Fig. 4. Effect of CHX treatment on the level of EGF-R specific protein. B: Lane - 1 - Cells treated with CHX for 1 hr. Lane - 2, 3, 4 - CHX removed by washing and cells were allowed to grow in fresh medium for 1, 3 and 5 hr respectively. A: Lanes 2, 3, 4 Cells grown without any treatment for 1, 3 and 5 hr respectively. The cell lysates were analysed for EGF-R specific protein as described in materials and methods. For each panel, the upper dots contain 2 ~g and lower dots contain 4 ~g protein. Fig. 3. EGF-R specific mRNA level after 1 hr treatment with CHX and DEX. 70--80% confluent cell culture were exposed for lhr with CHX or DEX. The treatment was terminated by washing the cells thoroughly with PBS. Cells were grown in fresh medium for different time periods. Total RNA was isolated, dot-blotted onto bio-dyne membrane, hybridized to nick translated probe and analysed by autoradiography. A - Control samples for the corresponding time points as in B and C; B C H X treatment; C - DEX treatment; lane 1 - 1 hr treatment; lanes 2, 3 and 4 - treatment is stopped by washing cells with PBS and allowing them to grow in fresh medium for 1, 2 and 5 hr respectively. The upper dots contain 2/zg and the lower dots contain 1/~g of total RNA.
l o n g t h e i n c r e a s e d m R N A level is m a i n t a i n e d a f t e r t h e r e m o v a l o f t h e s e t r e a t m e n t s . T h e results in Fig. 3 shows t h a t t h e t r e a t m e n t o f A431 cells w i t h D E X o r C H X for 1 h r is e n o u g h to i n c r e a s e t h e E G F - R specific m R N A level 3 to 4 fold. This i n c r e a s e d l e v e l is m a i n t a i n e d for 2 h r a n d d e c r e a s e d to a l m o s t b a s a l level b y 3 h r a f t e r t h e r e m o v a l o f t h e s e a g e n t s b y w a s h i n g t h e ceils with P B S . T h e cell l y s a t e s f r o m c o n t r o l , C H X a n d D E X t r e a t e d A431 cells w e r e a n a l y s e d b y t h e p r o t e i n d o t - b l o t s a n d t h e level o f E G F - R - s p e c i f i c p r o t e i n
w e r e i m m u n o a s s a y e d with r a b b i t p o l y c l o n a l antib o d y ( l g G ; # 9 8 6 ) r a i s e d to affinity p u r i f i e d E G F - R p r o t e i n f r o m A431 cells (Fig. 4). T h e C H X t r e a t m e n t for 1 hr d e c r e a s e d t h e level o f E G F rec e p t o r specific p r o t e i n slightly f r o m the c o n t r o l level b u t r e m o v a l o f C H X by w a s h i n g r a p i d l y inc r e a s e d t h e p r o t e i n level 3 to 4 fold o v e r t h e c o n t r o l (Fig. 4B, lanes 2 a n d 3) a n d a l m o s t e q u a l to D E X t r e a t e d level ( d a t a n o t shown), This i n c r e a s e d level in t h e a b s e n c e o f C H X r e m a i n s stable for 3 h r a n d b e g i n s to d e c r e a s e b y 5 hr. I n t h e p r e s e n c e o f D E X , t h e level o f r e c e p t o r specific p r o t e i n is 3 to 4 fold higher than the control (data not shown).
Discussion It has b e e n s h o w n t h a t E G F - R g e n e c o p y n u m b e r c o r r e l a t e s e x t r e m e l y well with E G F - R levels in a v a r i e t y o f A 4 3 1 - r e l a t e d cells [4]. O t h e r cell lines f o u n d to c o n t a i n a s u b s t a n t i a l a m o u n t o f E G F - R p r o t e i n p r o b a b l y utilize r e g u l a t o r y m e c h a n i s m s
153 other than gene amplification to overproduce EGF receptor R N A and protein. The observed rapidity with which EGF receptor m R N A accumulates in A431 cells in response to DEX, CHX and both, suggest that the increase in the level of EGF receptor mRNA is an early event. Whether D E X and/or CHX treatment increase the rate of transcription or stability of EGF-R m R N A can be answered by in vitro transcription experiment with isolated nuclei. Since the increase in the level of mRNA is only 3 to 4 fold, it will be experimentally difficult to show convincingly any transcriptional increase by in vitro transcription experiment with isolated nuclei in this system. The A431 cells are known to synthesize a 2.9 kb mRNA and is only detected by probes derived from sequences coding for the 5' untranslated region and the extracellular domain [5]. The probe used in this study encodes the carboxy terminal end and the 3' untranslated region of the cDNA and thus it can not hybridize to the 2.9 kb mRNA present only in A431 cells. Thus our observation of only two larger sizes of EGF receptor mRNA (10.5 kb and 5.8 kb) is consistent with the observations of Ullrich et al. [5]. The 2.9 kb mRNA is known to encode a 70 KD truncated receptor protein that is secreted. Thus the observed increase in the level of EGF-R protein and mRNA in A431 cells detected by immunoblot of whole cell lysate and 3' end cDNA probe does not include the 70 KD protein or 2.9 kb mRNA. Our data show that the 10.5kb m R N A level remains almost constant whereas the level of 5.8 kb m R N A increases three to four fold by the D E X and CHX treatment (Fig. 2). It has been suggested that the 10.5 kb mRNA is a precursor of 5.8 kb mRNA [5]. This raises the possibility that these treatments (DEX and/or CHX) could affect the R N A processing steps involved. We do not observe much of an additive effect when both CHX and DEX are present. It appears most likely that DEX acts through a positive regulator and CHX through a negative regulator and in presence of both CHX not only destroys the negative regulator but also the positive regulator. Thus one observes mostly the effects of only CHX when both CHX and D E X are present.
The effect of CHX is maintained in A431 cells containing 30 fold amplification of this gene. The transfected human IFN-B gene in chinese hamster ovary can be induced by CHX even when the gene is amplified 25-50 fold [8]. Thus if a repressor controls the EGF-R gene expression, it must be present in excess of what is minimally required to regulate this gene as suggested for transfected human IFN-B gene in chinese hamster ovary [8]. Thus it appears that other highly amplified genes are also likely to be regulated in similar fashion. The data in Fig. 4 shows that when A431 cells are placed in fresh media containing CHX for 1 hr, the EGF-R protein level, as measured by immuno-dot assay, remains almost equal to control level. It has been reported that fresh sera act as inhibitors of protein degradation in cultured cells [15]. As a result though CHX treatment inhibits protein synthesis, the level of EGF-R-specific protein remained essentially unaltered (Fig. 4). When CHX is removed by washing the cells thoroughly and allowing them to grow in fresh media, the level of EGF-R protein as detected by immuno-dot assay increases rapidly (Fig. 4). Similarly, the CHX mediated increase in the EGF receptor mRNA level is maintained for 2 hr after removal of CHX (Fig. 3). These results indicate that during protein synthesis inhibition, mRNA for EGF receptors accumulate within the cell and gets translated when reinitiation of protein synthesis is allowed by washing away CHX. Since CHX is known not to block mRNA synthesis [8, 16], it could be possible that the mRNA for EGF-R is transcribed during protein synthesis inhibition, stored and translated when protein synthesis is allowed to reinitiate. It is worth noting that the presence of CHX does not lead to the synthesis of aberrant mRNA and/or protein for EGF-R as judged by immuno-assay. It has been shown that interferon mRNA isolated from human fibroblast in presence and absence of CHX can synthesize interferon equally well [17, 18]. Further, accumulation of mRNA during protein synthesis inhibition by inhibitors have been reported in other systems [19-21]. Whether the increased levels of EGF-R mRNA in the presence of D E X or CHX can be explained entirely by the increase in half-life
154 of the EGF-R m R N A or the rate of transcription is altered as well remains to be determined.
Acknowledgements We express our deep appreciation to Dr. Bert W. O'Malley for his encouragement and support in carrying out this work in his laboratory. We are grateful to Dr. G. Carpenter for kindly providing us the EGF-receptor antibody.
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Address for offprints: M.L. Gope, Creighton Cancer Center, Creighton University, Criss III, Rm # 358, 24th at California Street, Omaha, NE 68178, USA
