Molecular and Cellular Endocrinology, 75 (1991) 91-100 © 1991 Elsevier Scientific Publishers Ireland, Ltd. 0303-7207/91/$03.50
91
MOLCEL 02423
A comparison of transcriptional regulatory element activities in transformed and non-transformed rat anterior pituitary cells Timothy A. Coleman 1, Piotr Chomczynski 2, Lawrence A. Frohman 2 and John J. Kopchick 1 1 Department of Zoological and Biomedical Sciences, Molecular and Cellular Biology Program and Edison Animal Biotechnology Center, Ohio University, Athens, OH, U.S.A., and e Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH, U.S.A. (Received 14 September 1990; accepted 17 October 1990)
Key words: GH-3 and P3 (rat pituitary) cells; Transient transfection; Transcriptional regulatory elements; Metallothionein promoter induction
Summary Transformed (GH-3) and non-transformed (P3) rat anterior pituitary cells were compared in their ability to direct expression of plasmids containing a variety of eukaryotic transcriptional regulatory elements (TREs). These include the herpes simplex virus thymidine kinase (HSV-TK), Rous sarcoma virus long terminal repeat (RSV-LTR), simian virus 40 early (SV-40E), human cytomegalovirus immediate-early (CMV-IE) and mouse metallothionein 1 (mMT-1) TREs. Chloramphenicol acetyl transferase (CAT) gene expression served as a reporter in this study. Following transient transfection, the cell lines exhibited similar profiles of TRE utilization. In each cell fine, CMV-IE was most efficient in directing reporter gene expression, although 2-fold greater activity was observed in GH-3 versus P3 cells. RSV-LTR directed gene expression was lower than that of CMV-IE while both HSV-TK and SV-40E were inactive in each cell line. Also, the mMT-1 promoter was inducible by addition of ZnC12 to the culture media, though the level required for maximal activation differed between the two cell lines. Transfected GH-3 and P3 cells, therefore, displayed similar TRE utilization profiles yet significant differences were observed in the ability of these cell lines to respond to specific regulatory elements.
Introduction
The mammalian anterior pituitary gland contains five phenotypically distinct cell types which are defined by the hormones which they produce (Farquhar et al., 1975; Dada et al., 1984). Two
Address for correspondence: Dr. John J. Kopchick, Edison Animal Biotechnology Center, 201 Wilson Hall, Ohio University, Athens, OH 45701, U.S.A.
closely related pituitary cell types are somatotrophs and lactotrophs which secrete predominantly growth hormone (GH) and prolactin (PRL), respectively. Tashjian et al. (1968) described isolation of three clonal strains of epithelial cells from two, 7-month-old female W i s t a r / F u r t h rats bearing a pituitary tumor. One clonal line, GH-3 (ATCC CCL 82.1), was shown to secrete high levels of rat GH while maintaining the ability to respond to the hormonal effectors, thyroid hormone (T3) and
92 glucocorticoids (Martial et al., 1977). GH-3 cells have been used extensively to study hormone ( G H and PRL) gene expression (Bancroft, 1981). Recently, a non-transformed rat anterior pituitary somatomammotroph cell line (PO) has been isolated without the use of transforming agents and successfully propagated in culture for more than 2 years (Chomczynski et al., 1988). The original cell line contained epithelial-like cells that were heterogenous in their secretory properties yet maintained their ability to respond to hormonal stimulation by T3, cortisol and growth hormone releasing hormone (GRH). Clonal derivatives of PO cells exhibited markedly different ratios of G H and PRL secretion, e.g. one line (P3) secreted barely detectable amounts of PRL. In addition, PO cells unlike GH-3 cells, do not form tumors when injected into syngeneic animals (Chomczynski et al., 1988). Pituitary tumor cell lines have been used extensively as host cells for both transient and stable transfection studies (Pasleau et al., 1985; Flug et al., 1987; McCormick et al., 1988). The properties of these cell lines that make them suitable for such studies are unknown, but may relate to the fact that they are transformed. Although transformation of normal cells may lead to activation of genes involved in transcription, there have been few studies designed specifically to address this issue. The availability of the non-transformed PO cell line has now permitted an examination of whether utilization of specific eukaryotic transcriptional regulatory elements (TREs) is altered in transformed as compared to non-transformed cells from the same tissue and species. Plasmids containing the herpes simplex virus thymidine kinase (HSV-TK), Rous sarcoma virus long terminal repeat (RSV-LTR), simian virus 40 early (SV-40E), cytomegalovirus immediate-early (CMV-IE), and the mouse metallothionein 1 (mMT-1) TREs attached to the reporter gene, chloramphenicol acetyl transferase (CAT), were introduced into GH-3 and P3 cells and their ability to direct C A T gene expression evaluated. The goals of these studies were: (1) Elucidation of the relative ability of five commonly utilized TREs in directing reporter gene expression in GH-3 and P3 cells. (2) Examination of differences in TRE utilization in GH-3 and P3 cells which may be indica-
tive of differences in trans-acting transcription factors involved in rat pituitary cell transformation. Materials and methods
Cell culture and transient transfection GH-3 and P3 cell lines were maintained in culture medium consisting of Dulbecco's modified Eagle's medium (DMEM) containing 10% Nuserum (Collaborative Research, Bedford, MA, U.S.A.), 2 mM L-glutamine and 50 # g / m l gentamicin (Gibco, Grand Island, NY, U.S.A.). A modification of the DEAE-dextran mediated transfection system (Lopata et al., 1984; Pasleau et al., 1985) was used to introduce plasmid DNAs into both P3 and GH-3 cells. Between 1.0 and 1.5 )< 10 6 cells were plated onto 35-mm tissue culture plates. After 24 h incubation in culture medium, the cells were rinsed twice with serumfree DMEM. Cells were transfected with 2 /~g of p D N A diluted in 1.0 ml of serum-free D M E M containing 0.2 mg of DEAE-dextran. After incubation for 30 min at 37 ° C, the D N A / D E A E dextran solution was removed and the cells washed sequentially with phosphate buffered saline (PBS), serum-free DMEM, and finally cultured in 2.0 ml of culture medium. Medium was changed daily until day 5 post-transfection. For pulse labelling studies, culture medium was removed and cells incubated in 2 ml of methionine-free DMEM. After 1 h incubation, medium was removed and 1 ml of methionine-free D M E M containing 20/~Ci L-[35S]methionine (1163 Ci/mmol, NEG-009T, DuPont NEN, Wilmington, DE, U.S.A.) added. Following a 6 h incubation period, culture media and cell extracts were individually analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE; Laemmli, 1970). Plasmid construction Bacterial strains transformed with plasmids pRSV-CAT (RSV-LTR; ATCC-37152) and pSV2CAT (SV-40E; ATCC-37155) were purchased from American Type Culture Collection (Rockville, MD, U.S.A.) (Gorman et al., 1982a, b). Plasmids pBLCAT-3 (promoterless) and pBLCAT-2 (HSVTK) were gifts from B. Luckow and G. Schutz
93 Cla I
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o~ Fig. 1. Construction of pCMVIE-CAT and pMT1-CAT. A 1200 bp ClaI to BgllI linear fragment containing the CMV-IE TRE (top left, open box) and a 1700 bp EcoRI to BgllI linear fragment containing the mMT-1 TRE (top fight, open box) were ligated into the unique BgllI site of the multiple cloning region located 5' of the CAT reporter gene. The plasmid pBLCAT-3 contains the bacterial CAT gene (black box) linked to the SV-40 small t intron and polyadenylation signal (stippled box) in a pUC 18 vector. Open arrows denote the plasmid ampicillin (Amp) resistance gene and origin of replication (ORI). Closed arrows represent the primary CAT transcript. The translational start (A TG) and stop (TAA) codons are indicated.
94 (Institut ftir Zell- und Tumorbiologie, Heidelberg, F.R.G.). pBLCAT-3 contains the entire CAT gene as well as the SV-40 small t intron and polyadenylation signals inserted into the polylinker region of pUC18 (Luckow and Schutz, 1987). pBLCAT-2 includes a 156 bp BamHI/BgllI fragment spanning the HSV-TK promoter that was inserted into the corresponding sites of pBLCAT-3. pBLCAT-3 was used for the construction of pCMVIE-CAT and pMT1-CAT. Briefly, the vector DNA was linearized at a unique BgllI site located 5' of the CAT gene (Fig. 1). The termini of the linear DNA fragments were made flush by incubation with Klenow polymerase and dNTPs. Other DNA fragments containing TREs were derived from plasmids pCMV-BGHb and pMKMT-1. Plasmid pCMV-BGHb (Pasleau et al., 1985) was digested with ClaI and BgllI which generated three linear DNA fragments, the termini of which were made flush as described above. A 1200 bp fragment corresponding to the CMV-IE T R E was isolated by 1% agarose gel electrophoresis, electroeluted, and further purified using an Elutip-D column (Schleicher & Schuell, Keene, N H , U.S.A.). Plasmid pMK-MT-1 (Brinster et al., 1981) was digested with EcoRI and BgllI which generated three linear D N A fragments and the entire reaction treated with Klenow polymerase and dNTPs as above. A 1700 bp fragment corresponding to the mMT-1 T R E was isolated by 1% agarose gel electrophoresis and purified as described. The T R E containing fragments were mixed with the linear pBLCAT-3 vector at 30:1 molar ratios in the presence of T4 DNA ligase (New England BioLabs, Beverly, MA, U.S.A.) and the resulting ligation mixture used to transform competent Escherichia coli (MC 1061). Bacterial colonies were grown on LB containing 50 # g / m l ampicillin and resistent colonies were screened by restriction enzymatic digestion analyses. Plasmid DNAs were prepared by lysozyme-Triton X-100 lysis and cesium chloride-ethidium bromide equilibrium gradient centrifugation (Maniatis et al., 1982).
CA T assays CAT assays were performed as described (Neumann et al., 1987) with the following modifications. Five days after transfection, cells were rinsed with PBS and harvested using 1.0 ml of PBS
containing 1 mM EDTA. After centrifugation at 1300 x g, the cells were resuspended in 100/~1 of Tris-HC1 (0.1 M Tris pH 8.0) aznd lysed by the addition of 10 #1 2% Triton X-100 (Nachtigal et al., 1989). The suspension was incubated on ice for 10 min, frozen at - 7 0 ° C and thawed at 37 ° C. Insoluble material was removed by centrifugation (16,000 x g, 10 min), and the extracts inactivated by incubation at 6 8 ° C for 10 min. Denatured material was removed by centrifugation as described above. Cell extracts (100/~1) were added to 7 ml scintillation vials. A reaction mixture (150/~1) containing 2 ~tl [3H]acetyl-CoA (200.0 m C i / m m o l ; 1 #Ci per assay; NET-290L, DuPont NEN, Wilmington, DE, U.S.A.), 50 t~l of 5 m M chloramphenicol (in water), 15/~1 of 1.0 M Tris-HC1, pH 8.0 and 83/~1 of water was added to each vial. The reactions then were overlayed with 5 ml of scintillation fluid (Econo-fluor, DuPont NEN, Wilmington, DE, U.S.A.) and radioactivity determined after 1 and 2 h incubation at room temperature. CAT activity was expressed as counts per m i n u t e / p e r hour of incubation/per/xg of cell p r o t e i n / p e r #g of transfected p D N A ( c p m / h / / ~ g protein//xg pDNA).
mMT-1 induction In order to measure the inducibility of the mMT-1 T R E by heavy metals, GH-3 and P3 cells were transiently transfected with 2 /~g of pMT1CAT as described. After overnight incubation, the culture medium was removed and the cells were incubated with culture medium containing 0, 25 ~M, 50 ~tM, 75 #M, 100 # M and 150 t~M ZnC12. Media containing the various ZnC12 concentrations were replaced daily and the cells assayed for CAT activity on day 5 post-transfection. Nuclear DNA uptake and hybridization Nuclei from transfected cells were released by a hypotonic lysis procedure (Peterson, 1985) and plasmid D N A isolated as described (Hirt, 1967). Pelleted nuclei were resuspended in 250 /xl T r i s / E D T A (10 mM Tris-HCl 8.0; 10 mM EDTA), and lysed by the addition of 250/~1 lysis buffer (10 mM Tris-HC1 pH 8.0; 10 mM EDTA; 1.2% w / v SDS). NaCI was added to a final concentration of 1 M and chromosomal D N A precipitated on ice for approximately 2 h. Chromosomal D N A was
95 pelleted by centrifugation at 16,000 x g for 15 min and the aqueous phase transferred to a clean 1.5 ml Eppendorf tube. Protein was extracted with an equal volume of p h e n o l / c h l o r o f o r m / iso-amyl alcohol ( 2 5 : 2 4 : 1 ) and nuclear plasmid D N A precipitated with 2 volumes of - 2 0 ° C EtOH. The D N A was pelleted, washed 1 x with 70% EtOH ( - 20 ° C), dried briefly, and resuspended in 30/xl TE (10 mM Tris-HC1 pH 8.0; 1 mM EDTA) containing 2 0 / ~ g / m l RNAse A. D N A samples were diluted to 270/tl with TE, denatured by addition of 30 /L1 3 M N a O H and incubation at 65 ° C for 30 min, and neutralized by addition of an equal volume of 2 M NH4OAc, pH 7.4. Equal volumes (300 #l) of each sample were immediately vacuum blotted in duplicate to Nytran membranes (0.45/~m; Schleicher & Schuell, Keene, NH, U.S.A.). The membranes were hybridized to either a rRNA specific or CAT specific D N A probe and processed according to the manufacturer's directions. A 551 bp HindIII to NcoI fragment encoding 29 bp of the 5' non-translated region, translational start signal and 174 amino acids of protein coding sequence was isolated from pSV-2 CAT. This DNA fragment, common to all the CAT containing plasmids, was radiolabelled and hybridized with D N A isolated from transiently transfected cells. A synthetic oligonucleotide corresponding to nucleotides 4011-4036 of the human 28S rRNA (Barbu and Dautry, 1989) was prepared and used as a control to normalize for the amount of D N A applied to the membrane.
Results Cellular morphology A comparison of the morphology of transformed (GH-3) and non-transformed (P3) rat anterior pituitary cells is shown in Fig. 2. Both cell lines consist of epithelial-like cells. Neither cell line exhibited growth to confluency though GH-3 cells continued to grow in batches (or foci) while P3 cells exhibited density-dependent growth inhibition as described previously (Chomczynski et al., 1988). Hormone secretion To compare the secretory products of each cultured line, the cells were incubated for 6 h with methionine-free D M E M containing 20 /~Ci [3SS]methionine and the resulting culture media and cell extracts analyzed by SDS-PAGE. Two radioactive bands were found in culture media derived from GH-3 cells (Fig. 3, lane B). Their apparent molecular masses of 24 kDa and 22 kDa correspond to PRL and GH, respectively (Miller and Eberhardt, 1983; Chomczynski et al., 1988). P3 cells secreted predominantly G H (Fig. 3, lane D). T R E utilization The two cell lines were compared in their ability to direct CAT reporter gene expression after transient transfection of pDNAs. A summary of the results is shown in Fig. 4. The promoterless plasmid, pBL-CAT3, served as a negative control
© Fig. 2. Photomicrograph(200x) of cultured rat anterior pituitary tumor GH-3 cells (panel a) and non-transformed somatomammotroph P3 cells (panel b).
96
because no significant difference in C A T activity was observed between mock transfected and transfected cells (data not shown). The values represent the mean + the standard deviation of four different assay values from two separate transfection experiments. Differences between group means were compared by t-test. The C M V - I E T R E was most efficient in directing reporter gene expression in both G H - 3 and P3 cells (Fig. 4). The C M V - I E T R E was approximately 2-fold more active in G H - 3 cells than in P3 cells ( p < 0.01). The RSV-LTR directed reporter gene expression at 28% and 54% that of C M V - I E in G H - 3 and P3 cells, respectively. mMT-1 directed expression at a low (2.4% and 2.9% that of C M V - I E in G H - 3 and P3, respectively) but reproducible level in the absence of ZnC12. The H S V - T K and SV-40E T R E s did not
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Fig. 4. Comparison of T R E utilization in GH-3 and P3 cells. C A T activity was determined after transient transfection of reporter plasmids containing different T R E s into GH-3 (hatched boxes) or P3 (open boxes) cells. Values expressed as c p m / h / # g of cell protein//~g of transfected p D N A represent the m e a n ± SD of four values from two separate transfections (Materials and Methods). Differences between groups were determined by t-test ( *p < 0.01 vs. GH-3).
direct detectable levels of reporter gene expression in either G H - 3 or P3 cell lines.
PRLI~ GH I~
Fig. 3. Autoradiograph of pulse labelled cell extracts and culture media from rat anterior pituitary cells. Cultured GH-3 cells and P3 cells were pulse labelled with [35S]methionine (Materials and Methods) and analyzed by 12.5% SDS-PAGE. Labelled proteins from GH-3 cell extract (lane A) and culture media (lane B) are shown. Two major labelled secretory products can be seen in culture media corresponding to rat prolactin (PRL) and rat growth hormone ((3H). Labelled proteins from P3 cell extract (lane C) and culture media (lane D) are shown. A single labelled secretory protein corresponding to rat G H is observed in the culture media. Lane M contains molecular weight markers.
m M T - I induction The m M T - I T R E has been shown to direct the inducible expression of a number of heterologous genes in a variety of cultured cell lines (Mayo et al., 1982; Selden et al., 1986; Kelder et al., 1988). Induction of the mMT-1 T R E by ZnC12 as measured by C A T activity can be seen in Fig. 5. The mMT-1 T R E responded to ZnC12 in both G H - 3 and P3 cells. In P3 cells, mMT-1 directed gene expression peaked at 100 /~M ZnC12, whereas in G H - 3 cells gene expression continued to increase at 150/~M ZnC12. With 100 # M ZnC12, a 17-fold and 23-fold increase in C A T activity was found in G H - 3 and P3 cells, respectively, as compared to p M T 1 - C A T transfected cells maintained in the absence of ZnCI 2. 150/~M ZnC12 yielded a 26-fold increase in C A T activity in G H - 3 cells and a 23% decrease in C A T activity in P3 cells relative to the 100 /tM level. It must be noted that at 150 #M, the amount of total protein in each cell line decreased apparently due to the cytotoxic effects of high concentrations of ZnC12.
97
Nuclear pDNA analysis
GH-3
O n e possibility for differential expression (or lack of expression) of T R E s in t r a n s i e n t l y transfected cells could be due to differential cellular u p t a k e of the p D N A s . T o test this possibility we isolated p D N A from the t r a n s i e n t l y transfected cells. Fig. 6 represents a n a u t o r a d i o g r a p h of hyb r i d i z a t i o n analysis from one such experiment. T h e h y b r i d i z a t i o n p r o b e used i n p a n e l A was a synthetic oligonucleotide c o r r e s p o n d i n g to a 28S r R N A gene. This was used as a c o n t r o l to n o r m a l ize for the a m o u n t of D N A applied to the m e m brane. Essentially equal signals were o b t a i n e d from either mock transfected cells or those transfected with the different C A T plasmids. Panel B represents results of a n e q u i v a l e n t b l o t in which the C A T gene was used as h y b r i d i z a t i o n probe. All p D N A s were identified i n the nuclei of b o t h G H - 3 a n d P3 cells t h o u g h the absolute a m o u n t s differed slightly. A l t h o u g h p C M V I E - C A T a p p e a r e d to be t a k e n up less efficiently t h a n other p l a s m i d s ( p B L C A T - 3 , p B L C A T - 2 , pSV2-CAT), it was exp r e s s e d m o s t efficiently in each cell line. pBLCAT-3, pBLCAT-2 and pSV-2CAT DNAs also were present i n the nucleus of each cell type
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Fig. 6. Slot blot hybridization of plasmid DNA isolated from transiently transfected pituitary cells. Equal volumes of DNA isolated from transiently transfected cells were applied to Nytran membranes in duplicate as indicated. Panel A was hybridized with a 32p-labelled oligonucleotide corresponding to the 28S rRNA gene and used to normalize the amount of DNA applied to the membrane. Panel B was hybridized with a [32p]CAT specific probe. No CAT specific DNA is seen in mock transfected cells ( - DNA, panel B) while intense signals were obtained with the different plasmids used. Essentially equal amounts of DNA were applied to the filter as judged by equivalent signals from all the samples in panel A. The numbers (0-150) represent the ZnCI 2 concentration (/~M) in culture fluids of cells transfected with pMT-1 CAT DNA.
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yet fail to direct reporter gene expression. Finally, p M T 1 - C A T was present in the n u c l e u s of each cell type at c o m p a r a b l e levels i n d e p e n d e n t of the ZnC12 c o n c e n t r a t i o n .
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Fig. 5. Comparison of mMT1-CAT induction by increasing concentrations of ZnCI 2. CAT activity was determined after transient transfection of 2/xg pMT1-CAT into GH-3 (hatched boxes) or P3 (open boxes) cells and subsequent treatment with increasing concentrations of ZnCI 2 as described. Values expressed as cpm/h/#g of cell protein/~g of transfected pDNA represent the mean+SD of four values from two separate transfections (Materials and Methods). Differences between groups were determined by t-test ( *p < 0.01 vs. GH-3).
Discussion
The a n t e r i o r p i t u i t a r y is c o m p o s e d of several different cell types which are r e s p o n s i b l e for expression a n d secretion of a variety of p e p t i d e hormones. G H - 3 cells are t u m o r cells derived from a class of cells termed s o m a t o m a m m o t r o p h s which express a n d secrete b o t h G H a n d PRL. These cells are t h o u g h t to be p r o g e n i t o r cells that can dif-
98 ferentiate into either somatotrophs ( G H secretors) or m a m m o t r o p h s (PRL secretors) (Hoeffler et al., 1985; Boockfor and Schwarz, 1988). The results presented in the present study demonstrate that transformed (GH-3) and non-transformed (P3) rat anterior pituitary somatomammotroph cells can be distinguished by several parameters. Morphologically, each cell line appears to be epithelial-like; however, a difference in their growth characteristics was noted. The transformed line continues to grow independent of cell density, whereas the P3 line demonstrates density dependent growth inhibition characteristic of a non-transformed cell type (Chomczynski et al., 1988). Under the growth conditions used in this study, P3 cells express and secrete predominantly G H and therefore would be classified as somatotrophs. Previous studies had shown that P3 cells express and secrete both G H and PRL (Chomczynski et al., 1988). The difference between these results and those presented here could be due, in part, to the differences in culture medium used to maintain the cells. T 3, corticosterone and G R H were not included in the culture medium used for the present study. In addition, the medium was supplemented with Nu-serum rather than the 10% horse serum that was previously used. The extent to which these differences were responsible for the loss of PRL gene expression is presently unknown. One goal of this study was to ascertain whether differences existed between the transformed and non-transformed pituitary cells in their ability to utilize various TREs. Because transformation of normal cells often involves activation of genes involved in transcription, alterations in the transcriptional apparatus may be associated with the transformed phenotype which in turn could result in differences in T R E activity. We have shown that the C M V - I E T R E is most efficient in directing reporter gene expression in each cell line. Similar results were observed in a comparison of bovine growth hormone ( b G H ) gene expression directed by the C M V - I E T R E and RSV-LTR in transiently transfected G H - 3 cells (Pasleau et al., 1985). Based on the criteria used in the present study, a comparison of CMV-IE T R E activity in G H - 3 versus P3 cells demonstrated a significantly higher activity in the transformed line which may
suggest a difference in trans-acting transcription factors required for efficient C M V - I E T R E utilization. This observation is supported by the fact that the RSV-LTR directed equivalent levels of gene expression in each cell line. In addition, the RSV-LTR was shown to direct C A T gene expression at 28% that of C M V - I E in G H - 3 cells, a value virtually identical to the ratio observed when b G H was used as a reported gene (Pasleau et al., 1985). Assuming the enhanced activity of the C M V - I E T R E found in transformed pituitary cells is a result of alterations in trans-acting transcription factors, this enhancer element may be utilized to identify such factor(s). We also have demonstrated that the mMT-1 T R E is inducible in both pituitary cell lines. The mMT-1 T R E directs basal levels of reporter gene expression in the absence of ZnCI 2. U p o n addition of increasing concentrations of ZnC12 to the culture medium, a concomitant increase in C A T enzyme activity was found. In G H - 3 cells, a 26-fold increase in C A T activity was observed by addition of 150 ~ M ZnCI 2 to the culture medium. P3 cells demonstrated a maximal 23-fold increase in C A T activity upon addition of 100 /~M ZnC12. The mMT-1 T R E can therefore be used to direct inducible expression of other heterologous genes in these pituitary lines (Brar et al., 1990). We have observed the apparent inability of the H S V - T K and SV-40E T R E s to direct reporter gene expression in either pituitary cell line. These plasmids, when transiently transfected into H e L a cells or mouse L cells are capable of directing CAT gene expression (data not shown). Others have also observed a similar low activity of the SV-40E T R E in G H - 3 cells (Schirm et al., 1987). This viral T R E is normally a potent transcriptional enhancer. Binding sites for several transactivators of the SV-40E T R E have been identified including Spl (Gidoni et al., 1985), AP-1 through AP-5 (Lee et al., 1987; Mermod et al., 1988; Mercurio and Karin, 1989), and octamer binding factor (Rosales et al., 1987; Sturm et al., 1987). The H S V - T K T R E also has been well characterized and shown to contain binding sites for Spl and C T F (Jones et al., 1985). The inability of these two T R E s to direct reporter gene expression in the present study is not due to an inability of the plasmids to be taken up by pituitary cells. We
99
have demonstrated that both H S V - T K and SV-40E plasmids can be reisolated from nuclei of transiently transfected cells. Graessmann et al. (1989) have also reported that both these plasmids can be taken up by rat-2 cells, and that the SV-40E T R E can be taken up more efficiently due to a 'helper function' localized near the AP-1 binding site of the 72 bp repeat. Because each plasmid is present in the nucleus, the question remains why neither can direct reporter gene expression in the transient assay. The possibilities include lack of a trans-activator required for efficient transcription or presence of a trans-acting repressor. Although the two cell lines were derived from rat anterior pituitaries, they originated from rats of different strains and sex. In particular, G H - 3 cells were derived from a female W i s t a r / F u r t h rat while the P3 cells were derived from male Sprague-Dawley rats. The contribution of these differences to this study has not been examined and we cannot exclude the possibility that either sex or strain differences between the two cell lines might contribute to the difference in T R E utilization. In summary, both transformed and non-transformed cells derived from rat anterior pituitary are capable of directing reporter gene expression from heterologous TREs. N o t all T R E s are equally active suggesting the involvement of tissue-specific transcription factors in modulating T R E activity. We have found that the C M V - I E T R E is most efficient, the R S V - L T R intermediate, and the mMT-1 weak though inducible while both the SV-40E and H S V - T K are apparently inactive in directing reporter gene expression in the cell lines used in this study. These results may be important to those interested in directing constitutive or inducible levels of gene expression in cultured rat anterior pituitary cells. Finally, the enhanced effects of the C M V - I E T R E in directing CAT gene expression in transformed, as compared to nontransformed cells, raise the possibility that transformation results in an altered regulation of one or more trans-acting factors.
Acknowledgements J.J.K. is supported in part by the State of Ohio's Eminent Scholar Program which includes a
grant from Milton and Lawrence Goll. L.A.F. is supported in part by N I H G r a n t DK-30667 and P.C. is supported in part by N I H G r a n t DK-41326.
References Bancroft, C.F. (1981) in Functionally Differentiated Cell Lines (Sato, G., ed.), pp. 47-59, A.R. Liss, New York. Barbu, V. and Dautry, F. (1989) Nucleic Acids Res. 17, 7115. Boockfor, F.R. and Schwarz, L.K. (1988) Endocrinology 122, 762-764. Brar, A.K., Coleman, T.A., Kopchick, J.J. and Frohman, L.A. (1990) Mol. Cell. Endocrinol. 71,105-115. Brinster, R.L., Chen, H.Y., Trumbauer, M., Senear, A.W., Warren, R. and Palmiter, R.D. (1982) Cell 27, 223-231. Chomczynski, P., Brat, A. and Frohman, L.A. (1988) Endocrinology 123, 2276-2283. Dada, M.O., Campbell, G.T. and Blake, C.A. (1984) J. Endocrinol. 101, 87-94. Farquhar, M.G., Skutelsky, E.H. and Hopkins, C.R. (1975) in The Anterior Pituitary (Tivier-Vidal, A. and Farquhar, M.G., eds.), pp. 83-135, Academic Press, New York. Flug, F., Copp, R.P., Casanova, J., Horowitz, Z.D., Janocko, L.J., Plotnick, M. and Samuels, H.H. (1987) J. Biol. Chem. 262, 6373-6382. Gidoni, D., Kadonaga, J.T., Barrera-Saldana, H., Takahashi, K., Chambon, P. and Tijan, R. (1985) Science 230, 511-517. Gorman, C.M., Moffat, L.F. and Howard, B.H. (1982a) Mol. Cell. Biol. 2, 1044-1051. Gorman, C.M., Merlino, G.T., Willingham, M.C., Pastan, I. and Howard, B.H. (1982b) Proc. Natl. Acad. Sci. U.S.A. 79, 6777-6781. Graessmann, M., Menne, J., Liebler, M., Graeber, I. and Graessmann, A. (1989) Nucleic Acids Res. 17, 6603-6612. Hirt, B. (1967) J. Mol. Biol. 26, 365-369. Hocffler, J.P., Boockfor, F.R. and Frawley, L.S. (1985) Endocrinology 117, 187-185. Jones, K.A., Yamamoto, K.R. and Tjian, R. (1985) Cell 42, 559-572. Kelder, B., Chert, H. and Kopchick, J.J. (1989) Gene 76, 75-80. Laemmli, U.K. (1970) Nature 227, 680-685. Lee, W., Mitchell, P. and Tjian, R. (1987) Cell 49, 741-752. Lopata, M.A., Cleveland, D.W. and Sollner-Webb, B. (1984) Nucleic Acids Res. 12, 5707-5717. Luckow, B. and Schutz, G. (1987) Nucleic Acids Res. 15, 5490. Maniatas, T., Fritsch, E.F. and Sambrook, J. (1982) in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Martial, J.A., Seeburg, P.H., Guenzi, D., Goodman, H.M. and Baxter, J.D. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 42934295. Mayo, K.E., Warren, R. and Palmiter, R.D. (1982) Cell 29, 99-108. McCormick, A., Wu, D., Castrillo, J.L., Dana, S., Strobl, J., Thompson, E.B. and Karin, M. (1988) Cell 55, 379-389. Mercurio, F. and Karin, M. (1989) EMBO J. 8, 1455-1460.
100 Mermod, N., Williams, T.J. and Tjian, R. (1988) Nature 332, 557-561. Miller, W.L. and Eberhardt, N.L. (1983) Endocr. Rev. 4, 97-129. Nachtigal, M.W., Nickel, B.E., Klassen, M.E., Zhang, W., Eberhardt, N.L. and Cattini, P.A. (1989) Nucleic Acids Res. 17, 4327-4337. Neumann, J.R., Morency, C.A. and Russian, K.O. (1987) BioTech 5, 444-447. Pasleau, F., Tocci, M.J., Leung, F.C. and Kopchick, J.J. (1985) Gene 38, 227-232.
Peterson, D.O. (1985) Mol. Cell. Biol. 5, 1104-1110. Rosales, R., Vigneron, M., Macchi, M., Davidson, I., Xiao, J.H. and Chambon, P. (1987) EMBO J. 6, 3015-3025. Schirm, S., Jiricny, J. and Schaffner, W. (1987) Genes Dev. 1, 65-74. Seldon, R.F., Howie, K.B., Rowe, M.E., Goodman, H.M. and Moore, D.D. (1986) Mol. Cell. Biol. 6, 3173-3179. Sturm, R., Baumruker, B.R. and Herr, W. (1987) Genes Dev. 1, 1147-1160. Tashjian, A.H., Yasumura, Y., Levine, L., Sato, G.H. and Parker, M.L. (1968) Endocrinology 82, 342-352.
91
MOLCEL 02423
A comparison of transcriptional regulatory element activities in transformed and non-transformed rat anterior pituitary cells Timothy A. Coleman 1, Piotr Chomczynski 2, Lawrence A. Frohman 2 and John J. Kopchick 1 1 Department of Zoological and Biomedical Sciences, Molecular and Cellular Biology Program and Edison Animal Biotechnology Center, Ohio University, Athens, OH, U.S.A., and e Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH, U.S.A. (Received 14 September 1990; accepted 17 October 1990)
Key words: GH-3 and P3 (rat pituitary) cells; Transient transfection; Transcriptional regulatory elements; Metallothionein promoter induction
Summary Transformed (GH-3) and non-transformed (P3) rat anterior pituitary cells were compared in their ability to direct expression of plasmids containing a variety of eukaryotic transcriptional regulatory elements (TREs). These include the herpes simplex virus thymidine kinase (HSV-TK), Rous sarcoma virus long terminal repeat (RSV-LTR), simian virus 40 early (SV-40E), human cytomegalovirus immediate-early (CMV-IE) and mouse metallothionein 1 (mMT-1) TREs. Chloramphenicol acetyl transferase (CAT) gene expression served as a reporter in this study. Following transient transfection, the cell lines exhibited similar profiles of TRE utilization. In each cell fine, CMV-IE was most efficient in directing reporter gene expression, although 2-fold greater activity was observed in GH-3 versus P3 cells. RSV-LTR directed gene expression was lower than that of CMV-IE while both HSV-TK and SV-40E were inactive in each cell line. Also, the mMT-1 promoter was inducible by addition of ZnC12 to the culture media, though the level required for maximal activation differed between the two cell lines. Transfected GH-3 and P3 cells, therefore, displayed similar TRE utilization profiles yet significant differences were observed in the ability of these cell lines to respond to specific regulatory elements.
Introduction
The mammalian anterior pituitary gland contains five phenotypically distinct cell types which are defined by the hormones which they produce (Farquhar et al., 1975; Dada et al., 1984). Two
Address for correspondence: Dr. John J. Kopchick, Edison Animal Biotechnology Center, 201 Wilson Hall, Ohio University, Athens, OH 45701, U.S.A.
closely related pituitary cell types are somatotrophs and lactotrophs which secrete predominantly growth hormone (GH) and prolactin (PRL), respectively. Tashjian et al. (1968) described isolation of three clonal strains of epithelial cells from two, 7-month-old female W i s t a r / F u r t h rats bearing a pituitary tumor. One clonal line, GH-3 (ATCC CCL 82.1), was shown to secrete high levels of rat GH while maintaining the ability to respond to the hormonal effectors, thyroid hormone (T3) and
92 glucocorticoids (Martial et al., 1977). GH-3 cells have been used extensively to study hormone ( G H and PRL) gene expression (Bancroft, 1981). Recently, a non-transformed rat anterior pituitary somatomammotroph cell line (PO) has been isolated without the use of transforming agents and successfully propagated in culture for more than 2 years (Chomczynski et al., 1988). The original cell line contained epithelial-like cells that were heterogenous in their secretory properties yet maintained their ability to respond to hormonal stimulation by T3, cortisol and growth hormone releasing hormone (GRH). Clonal derivatives of PO cells exhibited markedly different ratios of G H and PRL secretion, e.g. one line (P3) secreted barely detectable amounts of PRL. In addition, PO cells unlike GH-3 cells, do not form tumors when injected into syngeneic animals (Chomczynski et al., 1988). Pituitary tumor cell lines have been used extensively as host cells for both transient and stable transfection studies (Pasleau et al., 1985; Flug et al., 1987; McCormick et al., 1988). The properties of these cell lines that make them suitable for such studies are unknown, but may relate to the fact that they are transformed. Although transformation of normal cells may lead to activation of genes involved in transcription, there have been few studies designed specifically to address this issue. The availability of the non-transformed PO cell line has now permitted an examination of whether utilization of specific eukaryotic transcriptional regulatory elements (TREs) is altered in transformed as compared to non-transformed cells from the same tissue and species. Plasmids containing the herpes simplex virus thymidine kinase (HSV-TK), Rous sarcoma virus long terminal repeat (RSV-LTR), simian virus 40 early (SV-40E), cytomegalovirus immediate-early (CMV-IE), and the mouse metallothionein 1 (mMT-1) TREs attached to the reporter gene, chloramphenicol acetyl transferase (CAT), were introduced into GH-3 and P3 cells and their ability to direct C A T gene expression evaluated. The goals of these studies were: (1) Elucidation of the relative ability of five commonly utilized TREs in directing reporter gene expression in GH-3 and P3 cells. (2) Examination of differences in TRE utilization in GH-3 and P3 cells which may be indica-
tive of differences in trans-acting transcription factors involved in rat pituitary cell transformation. Materials and methods
Cell culture and transient transfection GH-3 and P3 cell lines were maintained in culture medium consisting of Dulbecco's modified Eagle's medium (DMEM) containing 10% Nuserum (Collaborative Research, Bedford, MA, U.S.A.), 2 mM L-glutamine and 50 # g / m l gentamicin (Gibco, Grand Island, NY, U.S.A.). A modification of the DEAE-dextran mediated transfection system (Lopata et al., 1984; Pasleau et al., 1985) was used to introduce plasmid DNAs into both P3 and GH-3 cells. Between 1.0 and 1.5 )< 10 6 cells were plated onto 35-mm tissue culture plates. After 24 h incubation in culture medium, the cells were rinsed twice with serumfree DMEM. Cells were transfected with 2 /~g of p D N A diluted in 1.0 ml of serum-free D M E M containing 0.2 mg of DEAE-dextran. After incubation for 30 min at 37 ° C, the D N A / D E A E dextran solution was removed and the cells washed sequentially with phosphate buffered saline (PBS), serum-free DMEM, and finally cultured in 2.0 ml of culture medium. Medium was changed daily until day 5 post-transfection. For pulse labelling studies, culture medium was removed and cells incubated in 2 ml of methionine-free DMEM. After 1 h incubation, medium was removed and 1 ml of methionine-free D M E M containing 20/~Ci L-[35S]methionine (1163 Ci/mmol, NEG-009T, DuPont NEN, Wilmington, DE, U.S.A.) added. Following a 6 h incubation period, culture media and cell extracts were individually analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE; Laemmli, 1970). Plasmid construction Bacterial strains transformed with plasmids pRSV-CAT (RSV-LTR; ATCC-37152) and pSV2CAT (SV-40E; ATCC-37155) were purchased from American Type Culture Collection (Rockville, MD, U.S.A.) (Gorman et al., 1982a, b). Plasmids pBLCAT-3 (promoterless) and pBLCAT-2 (HSVTK) were gifts from B. Luckow and G. Schutz
93 Cla I
I
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o~ Fig. 1. Construction of pCMVIE-CAT and pMT1-CAT. A 1200 bp ClaI to BgllI linear fragment containing the CMV-IE TRE (top left, open box) and a 1700 bp EcoRI to BgllI linear fragment containing the mMT-1 TRE (top fight, open box) were ligated into the unique BgllI site of the multiple cloning region located 5' of the CAT reporter gene. The plasmid pBLCAT-3 contains the bacterial CAT gene (black box) linked to the SV-40 small t intron and polyadenylation signal (stippled box) in a pUC 18 vector. Open arrows denote the plasmid ampicillin (Amp) resistance gene and origin of replication (ORI). Closed arrows represent the primary CAT transcript. The translational start (A TG) and stop (TAA) codons are indicated.
94 (Institut ftir Zell- und Tumorbiologie, Heidelberg, F.R.G.). pBLCAT-3 contains the entire CAT gene as well as the SV-40 small t intron and polyadenylation signals inserted into the polylinker region of pUC18 (Luckow and Schutz, 1987). pBLCAT-2 includes a 156 bp BamHI/BgllI fragment spanning the HSV-TK promoter that was inserted into the corresponding sites of pBLCAT-3. pBLCAT-3 was used for the construction of pCMVIE-CAT and pMT1-CAT. Briefly, the vector DNA was linearized at a unique BgllI site located 5' of the CAT gene (Fig. 1). The termini of the linear DNA fragments were made flush by incubation with Klenow polymerase and dNTPs. Other DNA fragments containing TREs were derived from plasmids pCMV-BGHb and pMKMT-1. Plasmid pCMV-BGHb (Pasleau et al., 1985) was digested with ClaI and BgllI which generated three linear DNA fragments, the termini of which were made flush as described above. A 1200 bp fragment corresponding to the CMV-IE T R E was isolated by 1% agarose gel electrophoresis, electroeluted, and further purified using an Elutip-D column (Schleicher & Schuell, Keene, N H , U.S.A.). Plasmid pMK-MT-1 (Brinster et al., 1981) was digested with EcoRI and BgllI which generated three linear D N A fragments and the entire reaction treated with Klenow polymerase and dNTPs as above. A 1700 bp fragment corresponding to the mMT-1 T R E was isolated by 1% agarose gel electrophoresis and purified as described. The T R E containing fragments were mixed with the linear pBLCAT-3 vector at 30:1 molar ratios in the presence of T4 DNA ligase (New England BioLabs, Beverly, MA, U.S.A.) and the resulting ligation mixture used to transform competent Escherichia coli (MC 1061). Bacterial colonies were grown on LB containing 50 # g / m l ampicillin and resistent colonies were screened by restriction enzymatic digestion analyses. Plasmid DNAs were prepared by lysozyme-Triton X-100 lysis and cesium chloride-ethidium bromide equilibrium gradient centrifugation (Maniatis et al., 1982).
CA T assays CAT assays were performed as described (Neumann et al., 1987) with the following modifications. Five days after transfection, cells were rinsed with PBS and harvested using 1.0 ml of PBS
containing 1 mM EDTA. After centrifugation at 1300 x g, the cells were resuspended in 100/~1 of Tris-HC1 (0.1 M Tris pH 8.0) aznd lysed by the addition of 10 #1 2% Triton X-100 (Nachtigal et al., 1989). The suspension was incubated on ice for 10 min, frozen at - 7 0 ° C and thawed at 37 ° C. Insoluble material was removed by centrifugation (16,000 x g, 10 min), and the extracts inactivated by incubation at 6 8 ° C for 10 min. Denatured material was removed by centrifugation as described above. Cell extracts (100/~1) were added to 7 ml scintillation vials. A reaction mixture (150/~1) containing 2 ~tl [3H]acetyl-CoA (200.0 m C i / m m o l ; 1 #Ci per assay; NET-290L, DuPont NEN, Wilmington, DE, U.S.A.), 50 t~l of 5 m M chloramphenicol (in water), 15/~1 of 1.0 M Tris-HC1, pH 8.0 and 83/~1 of water was added to each vial. The reactions then were overlayed with 5 ml of scintillation fluid (Econo-fluor, DuPont NEN, Wilmington, DE, U.S.A.) and radioactivity determined after 1 and 2 h incubation at room temperature. CAT activity was expressed as counts per m i n u t e / p e r hour of incubation/per/xg of cell p r o t e i n / p e r #g of transfected p D N A ( c p m / h / / ~ g protein//xg pDNA).
mMT-1 induction In order to measure the inducibility of the mMT-1 T R E by heavy metals, GH-3 and P3 cells were transiently transfected with 2 /~g of pMT1CAT as described. After overnight incubation, the culture medium was removed and the cells were incubated with culture medium containing 0, 25 ~M, 50 ~tM, 75 #M, 100 # M and 150 t~M ZnC12. Media containing the various ZnC12 concentrations were replaced daily and the cells assayed for CAT activity on day 5 post-transfection. Nuclear DNA uptake and hybridization Nuclei from transfected cells were released by a hypotonic lysis procedure (Peterson, 1985) and plasmid D N A isolated as described (Hirt, 1967). Pelleted nuclei were resuspended in 250 /xl T r i s / E D T A (10 mM Tris-HCl 8.0; 10 mM EDTA), and lysed by the addition of 250/~1 lysis buffer (10 mM Tris-HC1 pH 8.0; 10 mM EDTA; 1.2% w / v SDS). NaCI was added to a final concentration of 1 M and chromosomal D N A precipitated on ice for approximately 2 h. Chromosomal D N A was
95 pelleted by centrifugation at 16,000 x g for 15 min and the aqueous phase transferred to a clean 1.5 ml Eppendorf tube. Protein was extracted with an equal volume of p h e n o l / c h l o r o f o r m / iso-amyl alcohol ( 2 5 : 2 4 : 1 ) and nuclear plasmid D N A precipitated with 2 volumes of - 2 0 ° C EtOH. The D N A was pelleted, washed 1 x with 70% EtOH ( - 20 ° C), dried briefly, and resuspended in 30/xl TE (10 mM Tris-HC1 pH 8.0; 1 mM EDTA) containing 2 0 / ~ g / m l RNAse A. D N A samples were diluted to 270/tl with TE, denatured by addition of 30 /L1 3 M N a O H and incubation at 65 ° C for 30 min, and neutralized by addition of an equal volume of 2 M NH4OAc, pH 7.4. Equal volumes (300 #l) of each sample were immediately vacuum blotted in duplicate to Nytran membranes (0.45/~m; Schleicher & Schuell, Keene, NH, U.S.A.). The membranes were hybridized to either a rRNA specific or CAT specific D N A probe and processed according to the manufacturer's directions. A 551 bp HindIII to NcoI fragment encoding 29 bp of the 5' non-translated region, translational start signal and 174 amino acids of protein coding sequence was isolated from pSV-2 CAT. This DNA fragment, common to all the CAT containing plasmids, was radiolabelled and hybridized with D N A isolated from transiently transfected cells. A synthetic oligonucleotide corresponding to nucleotides 4011-4036 of the human 28S rRNA (Barbu and Dautry, 1989) was prepared and used as a control to normalize for the amount of D N A applied to the membrane.
Results Cellular morphology A comparison of the morphology of transformed (GH-3) and non-transformed (P3) rat anterior pituitary cells is shown in Fig. 2. Both cell lines consist of epithelial-like cells. Neither cell line exhibited growth to confluency though GH-3 cells continued to grow in batches (or foci) while P3 cells exhibited density-dependent growth inhibition as described previously (Chomczynski et al., 1988). Hormone secretion To compare the secretory products of each cultured line, the cells were incubated for 6 h with methionine-free D M E M containing 20 /~Ci [3SS]methionine and the resulting culture media and cell extracts analyzed by SDS-PAGE. Two radioactive bands were found in culture media derived from GH-3 cells (Fig. 3, lane B). Their apparent molecular masses of 24 kDa and 22 kDa correspond to PRL and GH, respectively (Miller and Eberhardt, 1983; Chomczynski et al., 1988). P3 cells secreted predominantly G H (Fig. 3, lane D). T R E utilization The two cell lines were compared in their ability to direct CAT reporter gene expression after transient transfection of pDNAs. A summary of the results is shown in Fig. 4. The promoterless plasmid, pBL-CAT3, served as a negative control
© Fig. 2. Photomicrograph(200x) of cultured rat anterior pituitary tumor GH-3 cells (panel a) and non-transformed somatomammotroph P3 cells (panel b).
96
because no significant difference in C A T activity was observed between mock transfected and transfected cells (data not shown). The values represent the mean + the standard deviation of four different assay values from two separate transfection experiments. Differences between group means were compared by t-test. The C M V - I E T R E was most efficient in directing reporter gene expression in both G H - 3 and P3 cells (Fig. 4). The C M V - I E T R E was approximately 2-fold more active in G H - 3 cells than in P3 cells ( p < 0.01). The RSV-LTR directed reporter gene expression at 28% and 54% that of C M V - I E in G H - 3 and P3 cells, respectively. mMT-1 directed expression at a low (2.4% and 2.9% that of C M V - I E in G H - 3 and P3, respectively) but reproducible level in the absence of ZnC12. The H S V - T K and SV-40E T R E s did not
M
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B
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Fig. 4. Comparison of T R E utilization in GH-3 and P3 cells. C A T activity was determined after transient transfection of reporter plasmids containing different T R E s into GH-3 (hatched boxes) or P3 (open boxes) cells. Values expressed as c p m / h / # g of cell protein//~g of transfected p D N A represent the m e a n ± SD of four values from two separate transfections (Materials and Methods). Differences between groups were determined by t-test ( *p < 0.01 vs. GH-3).
direct detectable levels of reporter gene expression in either G H - 3 or P3 cell lines.
PRLI~ GH I~
Fig. 3. Autoradiograph of pulse labelled cell extracts and culture media from rat anterior pituitary cells. Cultured GH-3 cells and P3 cells were pulse labelled with [35S]methionine (Materials and Methods) and analyzed by 12.5% SDS-PAGE. Labelled proteins from GH-3 cell extract (lane A) and culture media (lane B) are shown. Two major labelled secretory products can be seen in culture media corresponding to rat prolactin (PRL) and rat growth hormone ((3H). Labelled proteins from P3 cell extract (lane C) and culture media (lane D) are shown. A single labelled secretory protein corresponding to rat G H is observed in the culture media. Lane M contains molecular weight markers.
m M T - I induction The m M T - I T R E has been shown to direct the inducible expression of a number of heterologous genes in a variety of cultured cell lines (Mayo et al., 1982; Selden et al., 1986; Kelder et al., 1988). Induction of the mMT-1 T R E by ZnC12 as measured by C A T activity can be seen in Fig. 5. The mMT-1 T R E responded to ZnC12 in both G H - 3 and P3 cells. In P3 cells, mMT-1 directed gene expression peaked at 100 /~M ZnC12, whereas in G H - 3 cells gene expression continued to increase at 150/~M ZnC12. With 100 # M ZnC12, a 17-fold and 23-fold increase in C A T activity was found in G H - 3 and P3 cells, respectively, as compared to p M T 1 - C A T transfected cells maintained in the absence of ZnCI 2. 150/~M ZnC12 yielded a 26-fold increase in C A T activity in G H - 3 cells and a 23% decrease in C A T activity in P3 cells relative to the 100 /tM level. It must be noted that at 150 #M, the amount of total protein in each cell line decreased apparently due to the cytotoxic effects of high concentrations of ZnC12.
97
Nuclear pDNA analysis
GH-3
O n e possibility for differential expression (or lack of expression) of T R E s in t r a n s i e n t l y transfected cells could be due to differential cellular u p t a k e of the p D N A s . T o test this possibility we isolated p D N A from the t r a n s i e n t l y transfected cells. Fig. 6 represents a n a u t o r a d i o g r a p h of hyb r i d i z a t i o n analysis from one such experiment. T h e h y b r i d i z a t i o n p r o b e used i n p a n e l A was a synthetic oligonucleotide c o r r e s p o n d i n g to a 28S r R N A gene. This was used as a c o n t r o l to n o r m a l ize for the a m o u n t of D N A applied to the m e m brane. Essentially equal signals were o b t a i n e d from either mock transfected cells or those transfected with the different C A T plasmids. Panel B represents results of a n e q u i v a l e n t b l o t in which the C A T gene was used as h y b r i d i z a t i o n probe. All p D N A s were identified i n the nuclei of b o t h G H - 3 a n d P3 cells t h o u g h the absolute a m o u n t s differed slightly. A l t h o u g h p C M V I E - C A T a p p e a r e d to be t a k e n up less efficiently t h a n other p l a s m i d s ( p B L C A T - 3 , p B L C A T - 2 , pSV2-CAT), it was exp r e s s e d m o s t efficiently in each cell line. pBLCAT-3, pBLCAT-2 and pSV-2CAT DNAs also were present i n the nucleus of each cell type
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Fig. 6. Slot blot hybridization of plasmid DNA isolated from transiently transfected pituitary cells. Equal volumes of DNA isolated from transiently transfected cells were applied to Nytran membranes in duplicate as indicated. Panel A was hybridized with a 32p-labelled oligonucleotide corresponding to the 28S rRNA gene and used to normalize the amount of DNA applied to the membrane. Panel B was hybridized with a [32p]CAT specific probe. No CAT specific DNA is seen in mock transfected cells ( - DNA, panel B) while intense signals were obtained with the different plasmids used. Essentially equal amounts of DNA were applied to the filter as judged by equivalent signals from all the samples in panel A. The numbers (0-150) represent the ZnCI 2 concentration (/~M) in culture fluids of cells transfected with pMT-1 CAT DNA.
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yet fail to direct reporter gene expression. Finally, p M T 1 - C A T was present in the n u c l e u s of each cell type at c o m p a r a b l e levels i n d e p e n d e n t of the ZnC12 c o n c e n t r a t i o n .
s
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50
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Fig. 5. Comparison of mMT1-CAT induction by increasing concentrations of ZnCI 2. CAT activity was determined after transient transfection of 2/xg pMT1-CAT into GH-3 (hatched boxes) or P3 (open boxes) cells and subsequent treatment with increasing concentrations of ZnCI 2 as described. Values expressed as cpm/h/#g of cell protein/~g of transfected pDNA represent the mean+SD of four values from two separate transfections (Materials and Methods). Differences between groups were determined by t-test ( *p < 0.01 vs. GH-3).
Discussion
The a n t e r i o r p i t u i t a r y is c o m p o s e d of several different cell types which are r e s p o n s i b l e for expression a n d secretion of a variety of p e p t i d e hormones. G H - 3 cells are t u m o r cells derived from a class of cells termed s o m a t o m a m m o t r o p h s which express a n d secrete b o t h G H a n d PRL. These cells are t h o u g h t to be p r o g e n i t o r cells that can dif-
98 ferentiate into either somatotrophs ( G H secretors) or m a m m o t r o p h s (PRL secretors) (Hoeffler et al., 1985; Boockfor and Schwarz, 1988). The results presented in the present study demonstrate that transformed (GH-3) and non-transformed (P3) rat anterior pituitary somatomammotroph cells can be distinguished by several parameters. Morphologically, each cell line appears to be epithelial-like; however, a difference in their growth characteristics was noted. The transformed line continues to grow independent of cell density, whereas the P3 line demonstrates density dependent growth inhibition characteristic of a non-transformed cell type (Chomczynski et al., 1988). Under the growth conditions used in this study, P3 cells express and secrete predominantly G H and therefore would be classified as somatotrophs. Previous studies had shown that P3 cells express and secrete both G H and PRL (Chomczynski et al., 1988). The difference between these results and those presented here could be due, in part, to the differences in culture medium used to maintain the cells. T 3, corticosterone and G R H were not included in the culture medium used for the present study. In addition, the medium was supplemented with Nu-serum rather than the 10% horse serum that was previously used. The extent to which these differences were responsible for the loss of PRL gene expression is presently unknown. One goal of this study was to ascertain whether differences existed between the transformed and non-transformed pituitary cells in their ability to utilize various TREs. Because transformation of normal cells often involves activation of genes involved in transcription, alterations in the transcriptional apparatus may be associated with the transformed phenotype which in turn could result in differences in T R E activity. We have shown that the C M V - I E T R E is most efficient in directing reporter gene expression in each cell line. Similar results were observed in a comparison of bovine growth hormone ( b G H ) gene expression directed by the C M V - I E T R E and RSV-LTR in transiently transfected G H - 3 cells (Pasleau et al., 1985). Based on the criteria used in the present study, a comparison of CMV-IE T R E activity in G H - 3 versus P3 cells demonstrated a significantly higher activity in the transformed line which may
suggest a difference in trans-acting transcription factors required for efficient C M V - I E T R E utilization. This observation is supported by the fact that the RSV-LTR directed equivalent levels of gene expression in each cell line. In addition, the RSV-LTR was shown to direct C A T gene expression at 28% that of C M V - I E in G H - 3 cells, a value virtually identical to the ratio observed when b G H was used as a reported gene (Pasleau et al., 1985). Assuming the enhanced activity of the C M V - I E T R E found in transformed pituitary cells is a result of alterations in trans-acting transcription factors, this enhancer element may be utilized to identify such factor(s). We also have demonstrated that the mMT-1 T R E is inducible in both pituitary cell lines. The mMT-1 T R E directs basal levels of reporter gene expression in the absence of ZnCI 2. U p o n addition of increasing concentrations of ZnC12 to the culture medium, a concomitant increase in C A T enzyme activity was found. In G H - 3 cells, a 26-fold increase in C A T activity was observed by addition of 150 ~ M ZnCI 2 to the culture medium. P3 cells demonstrated a maximal 23-fold increase in C A T activity upon addition of 100 /~M ZnC12. The mMT-1 T R E can therefore be used to direct inducible expression of other heterologous genes in these pituitary lines (Brar et al., 1990). We have observed the apparent inability of the H S V - T K and SV-40E T R E s to direct reporter gene expression in either pituitary cell line. These plasmids, when transiently transfected into H e L a cells or mouse L cells are capable of directing CAT gene expression (data not shown). Others have also observed a similar low activity of the SV-40E T R E in G H - 3 cells (Schirm et al., 1987). This viral T R E is normally a potent transcriptional enhancer. Binding sites for several transactivators of the SV-40E T R E have been identified including Spl (Gidoni et al., 1985), AP-1 through AP-5 (Lee et al., 1987; Mermod et al., 1988; Mercurio and Karin, 1989), and octamer binding factor (Rosales et al., 1987; Sturm et al., 1987). The H S V - T K T R E also has been well characterized and shown to contain binding sites for Spl and C T F (Jones et al., 1985). The inability of these two T R E s to direct reporter gene expression in the present study is not due to an inability of the plasmids to be taken up by pituitary cells. We
99
have demonstrated that both H S V - T K and SV-40E plasmids can be reisolated from nuclei of transiently transfected cells. Graessmann et al. (1989) have also reported that both these plasmids can be taken up by rat-2 cells, and that the SV-40E T R E can be taken up more efficiently due to a 'helper function' localized near the AP-1 binding site of the 72 bp repeat. Because each plasmid is present in the nucleus, the question remains why neither can direct reporter gene expression in the transient assay. The possibilities include lack of a trans-activator required for efficient transcription or presence of a trans-acting repressor. Although the two cell lines were derived from rat anterior pituitaries, they originated from rats of different strains and sex. In particular, G H - 3 cells were derived from a female W i s t a r / F u r t h rat while the P3 cells were derived from male Sprague-Dawley rats. The contribution of these differences to this study has not been examined and we cannot exclude the possibility that either sex or strain differences between the two cell lines might contribute to the difference in T R E utilization. In summary, both transformed and non-transformed cells derived from rat anterior pituitary are capable of directing reporter gene expression from heterologous TREs. N o t all T R E s are equally active suggesting the involvement of tissue-specific transcription factors in modulating T R E activity. We have found that the C M V - I E T R E is most efficient, the R S V - L T R intermediate, and the mMT-1 weak though inducible while both the SV-40E and H S V - T K are apparently inactive in directing reporter gene expression in the cell lines used in this study. These results may be important to those interested in directing constitutive or inducible levels of gene expression in cultured rat anterior pituitary cells. Finally, the enhanced effects of the C M V - I E T R E in directing CAT gene expression in transformed, as compared to nontransformed cells, raise the possibility that transformation results in an altered regulation of one or more trans-acting factors.
Acknowledgements J.J.K. is supported in part by the State of Ohio's Eminent Scholar Program which includes a
grant from Milton and Lawrence Goll. L.A.F. is supported in part by N I H G r a n t DK-30667 and P.C. is supported in part by N I H G r a n t DK-41326.
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