Pancreas Vol. 6 , No. 2 , pp. 142-149 0 1991 Raven Press, Ltd., New York
Regulation of Transforming Growth Factor-a mRNA Expression in T,M, Human Pancreatic Carcinoma Cells Betty J. Glinsmann-Gibson and "Murray Korc Department of Microbiology and Immunology, University of Arizona, Tucson, Arizona, and *Departments of Medicine and Biochemistry, University of California, Irvine, California, U.S.A.
Summary: Cultured human pancreatic cancer cells produce transforming growth factor-a (TGF-a), a potent mitogenic polypeptide. In the present study, we investigated the regulation of TGF-a mRNA expression in T,M, human pancreatic carcinoma cells. TGF-a mRNA levels were quantitated by densitometric analysis of autoradiographs obtained following hybridization of sizefractionated cytoplasmic RNA with 3zP-labeled cRNA coding for human TGF-a. There was a twofold increase in TGF-a mRNA levels at 2 h following addition of either epidermal growth factor (EGF) or TGF-a. However, TGF-a mRNA levels declined to near basal levels by 10 h. At 2 h, one-half maximal stimulation of TGF-a mRNA levels occurred at 1 nM and maximal stimulation at 4 nM of either EGF or TGF-a. The transcriptional inhibitor actinomycin D (Act D) and the phorbol ester, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), mimicked the actions of EGF and TGF-a. These findings indicate that the regulation of TGF-a mRNA expression in T,M, cells is complex, and is mediated, in part, via the EGF receptor. Key Words: Transforming growth factor-a-Epidermal growth factor receptor-Autocrine regulation-pancreatic cancer.
Transforming growth factor-a (TGF-a) is a 50 amino acid polypeptide that was originally isolated from the culture medium of retrovirally transformed fibroblasts (1,2). It binds to the epidermal growth factor (EGF) receptor, and induces a variety of biological responses, including EGF receptor phosphorylation (3,4). To date, a distinct receptor for TGF-a has not been identified. Therefore, all of the biological actions of TGF-a are thought to be mediated via the EGF receptor. The ability of TGF-a to bind and activate the EGF receptor is believed to be due to the amino
acid sequence homology between TGF-a and EGF (5,6), and to the similar spacing of the six cysteine residues in both polypeptides (7). However, the TGF-a gene (8) is distinct from the EGF gene (9), and is transcribed into a 4.5-4.8 kbase messenger RNA (5). The latter is translated into a large transmembrane precursor protein (10,ll). The extracellular portion of the glycosylated precursor is cleaved to release mature TGF-a, but is also biologically active prior to release from the cell membrane (12,13). TGF-a mRNA and protein are synthesized by many tumor cell lines and solid tumors (14). Furthermore, overexpression of TGF-a in transfected cells confers onto these cells the transformed phenotype (15). Therefore, it has been suggested that TGF-a may play a role in the initiation or mainte-
Manuscript received December 19, 1989; revised manuscript accepted February 26, 1990. Address correspondence and reprint requests to Dr. M. Korc at Division of Endocrinology and Metabolism, Med Sci I, C240, University of California, Irvine, CA 92717, U.S.A.
TGF-a EXPRESSION IN PANCREATIC CANCER CELLS
nance of malignant transformation. However, TGF-a has also been isolated from the developing rat embryo (16) and normal adult mammalian tissues (13,17), indicating that it has a role in the regulation of fetal and normal cell growth. We have previously reported that cultured human pancreatic carcinoma cells exhibit a high number of EGF receptors (18) and produce TGF-a mRNA and protein (19). The aims of this study were to investigate the regulation of TGF-a mRNA expression in T3M4human pancreatic carcinoma cells. We now report that both TGF-a and EGF increase TGF-a mRNA levels in this cell line. MATERIALS AND METHODS Reagents The following were purchased: TGF-a from Bachem Inc. (Torrance, CA, U.S.A.); EGF from Collaborative Research Inc. (Bedford, MA, U.S.A.); Act D from Sigma Chemical Company (St. Louis, MO, U.S.A.); Nytran blotting membranes from Schleicher & Schuell (Keene, NH, U.S.A.); [ w ~ ~ P ] C Tand P dCTP (3,000 Ci/mmol) from New England Nuclear (Boston, MA, U.S.A.); transcription buffers and nonradioactive nucleotides from Promega (Madison, WI, U.S.A.); restriction enzymes from New England Bio-Labs (Beverley, MA, U.S.A.); random labeling kit from Boehringer Mannheim (Indianapolis, IN, U.S.A.); and recombinant human TGF-a from Bachem, Inc. (Torrance, CA, U.S.A.). T3M4 cells (20) were obtained from R. S. Metzgar at Duke University. Human TGF-a cDNA (7) was obtained from R. Derynck at Genentech, Inc. (South San Francisco, CA, U.S.A.) and mouse ribosomal cDNA from T. Bowden at the University of Arizona (21). Recombinant human EGF was obtained from Dr. George-Nascimento at Chiron Co. (Emeryville, CA, U.S.A.). Cell culture T3M4cells were grown in monolayer culture in 75-cm2tissue culture flasks, in RPMI 1640 medium supplemented with antibiotics (100 unitdm1 of penicillin; 100 p,g/ml of streptomycin), and 5% bovine and 5% fetal bovine serum (FBS). Cells were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO,. For the individual experiments, rapidly growing cultures were harvested by rinsing the cells once with calcium- and magnesium-free Dulbecco's phosphate-buffered saline (PBS), and once
with 0.25% trypsin and 0.02% EDTA. Following incubation for 5 min at 37"C, the cells were incubated in RPMI 1640 medium supplemented with 10% FBS to stop the action of trypsin. Cells were then plated in RPMI 1640 medium supplemented with 10% FBS at a density of 5 X lo6 per 100 mm dish, and allowed to settle for 20 to 24 h. Medium was then changed to RPMI 1640 that was supplemented with the above antibiotics and 0.5% FBS. The indicated additions were made 30 min later. Incubations were terminated by placing the cell plates at 4"C, aspirating the medium, and washing the cells twice with PBS at 4°C. The recombinant human EGF and TGF-a that were used in the present study were tested in competition-inhibition binding experiments, in order to determine whether they were equipotent with respect to EGF receptor occupancy. To this end, both growth factors were iodinated by a modification of the chloramine-T method to an approximate specific activity of 120 pCilpg for EGF and 150 pCi/p.g for TGF-a (22). One-half maximal inhibition of binding for '251-EGF and '251-EGF-a was observed at 4 ng/ml (0.7 nM) and 5.5 ng/ml (1.0 nM), respectively. Furthermore, the binding of both ligands was inhibited to the same extent at concentrations of TGF-a greater than 1 nM. Therefore, concentrations above and below 1 nM were used in experiments comparing the dose-dependent effects of EGF and TGF-a on TGF-a mRNA levels. RNA extraction and hybridization studies To prepare cytoplasmic RNA, T,M4 cells were scraped with a cell scraper in PBS at 4°C. Cells were then lysed at 4°C in a hypotonic solution containing the nonionic detergent Nonidet P-40 (0.5%), 20 mM NaCl, 20 mM Tris-HC1 (pH 7 . 3 , 2 mM MgCl,, and 200 @g/mlof heparin as an RNase inhibitor (23). Following centrifugation (12,000 X g ) at 23"C, the supernatants were transferred to microfuge tubes containing 8% sodium dodecyl sulfate (SDS) and 0.8 M NaCl and subjected to phenolchloroform extraction. The RNA was precipitated, and stored at -80°C until use. Poly(Af) mRNA was prepared from total RNA by oligo(dT)-cellulose chromatography (24). RNA was size-fractionated by electrophoresis in a 0.8% agarosel2.2 M formaldehyde gel, stained with ethidium bromide in order to verify its integrity and assess equivalent loading, blot-transferred onto Nytran membranes, and cross-linked to the membrane by ultraviolet
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(UV) irradiation in a Stratalinker apparatus (Stratagene, La Jolla, CA, U.S.A.). The blots containing the cross-linked RNA were prehybridized for 2.5 h at 65°C in a prehybridization buffer (buffer A) optimized for use with the TGF-a cRNA probe, based on a previously described hybridization protocol (25). Buffer A contained 50% formamide, 0.5% sodium dodecyl sulfate (SDS), 5x SSC (0.75 M NaCl and 0.75 M sodium citrate), 5x Denhardt’s (0.5% Ficoll, 0.5% polyvinylpyrrolidone, and 0.5% bovine serum albumin), 250 pg/ml of salmon sperm DNA, and 50 mM sodium phosphate (pH 6.5). The blots were then hybridized for 15.5 h at 65°C in modified buffer A containing 50% formamide, 0.2% SDS, 4X SSC, 2.5x Denhardt’s, 100 pg/ml of salmon sperm DNA, 20 mM Na2P04 (pH 6.5), 10% dextran sulfate, and the labeled cRNA (3 x lo6 cpm/l0 ml of solution). The blots were then washed twice at 65°C in a solution containing 1x SSPE (150 mM NaCl, 10 mM NaH2P04, and 1 mM EDTA) and 0.5% SDS, and twice at 68°C in a solution containing 0.1 X SSPE and 0.5% SDS. Following exposure at - 80°C to Kodak XAR-5 film with Kodak intensifying screens, the bound radioactivity was removed by incubating the blots for 20 min at 95°C in 0.1 x SSC. The blots were then prehybridized for 1 h at 42°C in a prehybridization buffer optimized for use with the 7 s cDNA probe. Buffer B contained 50% formamide, 0.1% SDS, 5X SSC, 2x Denhardt’s, 250 kg/ml of salmon sperm DNA, and 50 mM Na,P04 (pH 6.5). Hybridization with the 32P-labeled cDNA corresponding to 7 s mouse ribosomal RNA was next carried out at 42°C for 16 h in fresh buffer B that also contained 10% dextran sulfate and the labeled cDNA (6 x lo5 cpm/ 10 ml of solution). The blots were then washed twice at 23°C in 6X SSPE/O.5% SDS, twice at 37°C in 1 x SSPE/O.5% SDS, and once at 57°C in 0.1 x SSPE/O.5% SDS. Following autoradiography , the intensity of the radiographic bands was quantified by laser densitometry . The plasmid for antisense TGF-a cRNA synthesis contained a 1,270 base pair fragment corresponding to the human TGF-a cDNA sequence (5) in the pSP65 vector. Following linearization with Xbal and HindIII, the plasmid containing the TGF-a cDNA sequence in reverse orientation to the SP65 promoter was purified by electrophoresis through agarose and transcribed with SP6 polymerase in the presence of 32P-labeled CTP (26). The resulting RNA probe was complementary to mRNA
Pancreas, Vul. 6, N u . 2, 1991
for TGF-a, and had a specific activity of 1 x lo9 cpm/pg. cRNA probes were stored at -80°C and used within 5 days of preparation. The cDNA corresponding to the 7 s mouse ribosomal RNA was labeled with [32P]dCTP using random hexanucleotide primers and Klenow enzyme (27), and had a specific activity of 1 x 10’ cpm/pg. Statistical analysis Statistical analysis of multiple comparisons was carried out using analysis of variance (ANOVA) and a two-tailed Student’s t test.
RESULTS Effects of growth factors on TGF-cw mRNA levels Both EGF and TGF-a increased TGF-a mRNA levels in a time- (Figs. 1 and 2) and dose-dependent (Figs. 3 and 4) manner in T,M4 cells. Densitometric analysis of the time-course data revealed that at a concentration of 4 nM, both EGF and TGF-a caused a twofold increase in the level of TGF-a mRNA after a 2 h incubation, by comparison with the levels observed in control cells. The magnitude of the increase remained relatively stable during the subsequent 4 h, and declined to near basal levels after 10 h of incubation (Fig. 2). Densitometric anaL ysis of the dose-response data indicated that onehalf maximal stimulation of TGF-ci mRNA levels occurred at a concentration of 1 nM, and maximal stimulation at 4 nM of either growth factor (Figs. 3 and 4). Although 0.1 nM TGF-a exerted a greater effect than 0.1 nM EGF on TGF-a mRNA levels, the actions of neither factor were statistically significant at this concentration. Furthermore, the differences between the effects of EGF and TGF-a were not statistically significant at any of the concentrations or time points that were examined. Effects of TPA and actinomycin D on TGF-cw mRNA levels To determine whether activation of protein kinase C altered TGF-a mRNA levels in T3M4cells, the effects of TPA were examined next. Preliminary experiments with TPA indicated that maximal induction of TGF-a mRNA accumulation occurred at 6 h (data not shown). Therefore, the effects of varying concentrations of TPA were studied at this time point (Fig. 5). TPA increased TGF-a mRNA levels in a dose-dependent manner, and this effect was
TGF-(YEXPRESSION IN PANCREATIC CANCER CELLS
0 2 4 6 8x) A
FIG. 2. Densitometric analysis of the time course data. T,M4 cells were incubated and RNA processed as described in the legend to Fig. 1. The resulting autoradiographs were analyzed by laser densitometry , and corrected for loading variations by using the corresponding densities obtained with the 7s cDNA. Data were calculated as % increase above control, and are the means -t SEM of three to six separate experiments. *p < 0.05, as compared with the respective control.
FIG. 1. Time-l_-pendent induction a TGF-a mRNA. T3M, cells were seeded in 100 mm plates (5 x O6 cells/plate) in RPMI 1640 medium supplemented with 10% FBS, and allowed to settle for 20 to 24 h. Cells were then incubated for 30 min at 37°C in fresh medium containing 0.5% FBS prior to the addition of 4 nM EGF (A$) or 4 nM TGF-a (B). Incubations were either terminated immediately (zero time), or 2, 4, 6, 8, and 10 h later, as indicated at the top of panel A. Cytoplasmic RNA was isolated, electrophoresed, trans-blotted to a nylon membrane, and probed with either the TGF-a cRNA (A,B) or the 7s cDNA (C) as described in the Materials and Methods section. Data shown are from a representative (of three to six) experiment. RNA size was determined by comparison with ribosomal RNA markers. TGF-a (A,B) and 7s (C) mRNAs migrated as 4.6 and 0.4 kbase species, respectively.
significant at concentrations of 10 and 40 nM. At the latter concentration, TPA caused a threefold increase in TGF-a mRNA levels. The effects of higher concentrations of TPA were not analyzed because of enhanced background in the hybridiza-
tion signal, perhaps as a result of spurious RNA degradation. To determine whether TGF-a mRNA levels in T,M, cells are regulated by inhibitors of transcription, the effects of 8 pg/ml of actinomycin D (Act D) were examined next. This concentration of Act D, which is known to inhibit more than 95% of RNA synthesis, did not alter the viability and growth characteristics of T3M, cells (data not shown). Act D caused a time-dependent increase in TGF-a mRNA levels (Fig. 6). Maximal stimulation occurred at 90 min following addition of Act D, and resulted in a 12-fold increase above control values in TGF-a mRNA levels without a concomitant change in 7s mRNA levels. DISCUSSION
Many different types of cancer cell lines and malignant tumors exhibit increased EGF receptor number (28-32). This receptor is a transmembrane protein with intrinsic tyrosine kinase activity (33). Its overexpression appears to provide cancer cells with a growth advantage and enhanced ability to metastasize (34,35). In addition to the growth advantage inherent in EGF receptor overexpression, some cancer cells produce TGF-a that binds and activates the EGF receptor (14). TGF-a, by acting in an autocrine manner on the EGF receptor, may allow cells to exhibit a reduced dependence on exogenous growth factors. In the present study, we have determined that EGF and TGF-a increased TGF-a mRNA levels in
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B . J . GLINSMANN-GIBSON A N D M . KORC
TEGF 1 r T G F * a i 1 2 3 4 5 6
FIG. 3. Effects of varying concentrations of EGF and TGF-a on TGF-a mRNA levels. T,M, cells were incubated for 2 h at 37°C in the absence (lanes 1, 2 , 8) or presence of varying concentrations of EGF (lanes 3-7) and TGF-a (lanes 9-13). Lanes 3 and 9: 0.01 nM; lanes 4 and 10: 0.04 nM; lanes 5 and 11: 1.0 nM; lanes 6 and 12: 4.0 nM; and lanes 7 and 13: 10.0 nM of the indicated growth factor. Lane 1: poly(A+) mRNA from control T,M, cells, demonstrating the presence of the 4.6 kbase TGF-a mRNA species and the absence of the 0.4 kbase 7 s ribosomal RNA species. Incubation conditions and RNA analysis were camed out as described in the legend to Fig. 1. Data are from a representative (of four) experiment.
T,M, cells, a human pancreatic cancer cell line that produces large quantities of TGF-a protein (19). TGF-a and EGF were equipotent with respect to this effect: half-maximal induction of TGF-a mRNA levels occurring at 1 nM of either growth factor. The kinetics of this induction by the two growth factors were also similar. In contrast, TGF-a is more potent than EGF in enhancing the anchorage-independent growth of T,M, cells (19), whereas EGF is more potent than TGF-a in inducing EGF receptor downregulation in these cells (36). Taken together, these observations indicate that EGF and TGF-a may be equipotent with re-
spect to some actions, but exhibit different potencies with respect to other actions in the same cell type. Our findings also suggest that TGF-a may act as an endogenous stimulator of TGF-a mRNA expression in T,M, cells. Furthermore, human pancreatic cancer cells avidly internalize EGF and release a large percentage of the internalized ligand in the form of intact EGF that rebinds to the cellsurface EGF receptor (36,37). Therefore, it is possible that EGF released by these cells may also act 400
u = E g
22 -n -
Iig EGF TGFa
0 0 1
FIG. 4. Densitometric analysis of the dose-response data. T,M, cells were incubated and RNA processed as described in the legend to Fig. 3. The resulting autoradiographs were analyzed by laser densitometry , and corrected for loading variations by using the corresponding densities obtained with the 7 s cDNA. Data were calculated as % increase above control, and are the means 2 SEM of four separate experiments. *p < 0.05, as compared with the respective control.
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FIG. 5. Effects of TPA on TGF-a mRNA levels. T,M, cells were incubated and RNA processed as described in the legend to Fig. 1, but in the absence or presence of the indicated concentrations of TPA for 6 h. The resulting autoradiographs were analyzed by laser densitometry, and corrected for loading variations by using the corresponding densities obtained with the 7s cDNA. Data were calculated as % increase above control, and are the means 2 SEM of three separate experiments. * p < 0.05, as compared with the respective control.
TGF-a EXPRESSION IN PANCREATIC CANCER CELLS
T1 1 *
FIG. 6. Effects of actinomycin D on TGF-a mRNA levels. T,M4 cells were incubated and RNA processed as described in the legend to Fig. 1 , but in the absence or presence of Act D (8 &ml) for the indicated times. The resulting autoradiographs were analyzed by laser densitometry, and corrected for loading variations by using the correspondingdensities obtained with the 7s cDNA. Data were calculated as % increase above control, and are the means k SEM of three separate experiments. * p < 0.05, as compared with the respective control.
in an autocrine manner to induce TGF-a mRNA expression. The induction of TGF-a mRNA by EGF and TGF-a was originally demonstrated in normal keratinocytes (13). Subsequent studies have described TGF-a mRNA induction by EGF in MDA468 breast cancer cells (38) and in bovine anterior pituitaryderived cell cultures (39). In the latter studies, TGF-a mRNA levels were also increased by 120-tetradecanoyl-phorbol-13-acetate (TPA) (38,39). Recently, TPA has been shown to exert a similar effect in keratinocytes (40). In the present study, TPA also mimicked the actions of EGF and TGF-a. Thus, in T3M4cells, as in other cell types, TGF-a mRNA expression may be regulated by protein kinase C. Therefore, it is conceivable that other growth factors, hormones, and neurotransmitters that activate protein kinase C may also regulate TGF-a mRNA expression in pancreatic cancer cells. The regulation of TGF-a gene transcription is known to occur at the level of a promoter region that is located 5’ to the mRNA start site (41). This region is GC-rich, and is devoid of TATA and CCAAT boxes (41). In contrast to other promoter regions that exhibit these characteristics, the TGF-a promoter directs transcriptional initiation
from a single start site (41). In the present study, Act D markedly increased TGF-a mRNA levels. It is unlikely that this transcriptional inhibitor somehow activates the promoter region to transcribe more TGF-a mRNA molecules. Instead, it is probable that Act D inhibits the synthesis of short-lived proteins that degrade TGF-a mRNA, thereby leading to enhanced accumulation of this mRNA. These observations suggest that in T3M4 cells, there is rapid degradation of TGF-a mRNA in the basal state. Therefore, the induction of TGF-a mRNA levels by EGF and TGF-a may be mediated via several distinct mechanisms that include direct effects on the TGF-a gene promoter, repression of inhibitors of this promoter, activation of protein kinase C, and modulation of TGF-a RNA degradation. Several lines of evidence suggest that routine TGF-a determinations may not provide a clear understanding of the autocrine role of this growth factor. First, TGF-a is extensively degraded by cancer cells, including human pancreatic carcinoma cells (36). Second, in addition to authentic TGF-a, cancer cells produce nonmeasurable variant TGF-a molecules (10). Third, cancerous cells may have large quantities of surface-bound but biologically active TGF-a precursor moieties that are not readily quantitated (8,9). Although we have measured TGF-a in conditioned medium from cultured human pancreatic cancer cells (19), the above observations suggest that these determinations grossly underestimate the amount of TGF mRNA that is translated into protein. Nonetheless, it is likely that TGF-a participates in the autocrine regulation of the growth of human pancreatic cancer cells. Thus, these cells produce and bind TGF-a (19). Furthermore, as little as 0.1 ng/ml of TGF-a enhances their growth in soft agar (19), whereas their basal anchorage-independent growth is significantly inhibited by anti-TGF-a antibodies (42). Our current findings demonstrate that the EGF receptor is an important modulator of TGF-a gene transcription in T3M4 cells. Inasmuch as the EGF receptor is activated by TGF-a in these cells (36), our observations provide further support for the concept of a functional EGF receptor/TGF-a autocrine cycle in human pancreatic cancer cells. Acknowledgment: This investigation was supported by USPHA grant CA-40162 awarded from the National Cancer Institute, Department of Health and Human Services. A portion of the manuscript was submitted in partial ful-
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fillment of the requirements for the degree of Master of Sciences in Microbiology at the University of Arizona by B .J.G .-G .
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