Respiratory Medicine (1991) 8 5 , 479-485
Production of platelet-derived growth factor by human lung cancer M. BRAVO, R. V,~SQUEZ, H. RUBIO, M. SALAZAR, A. PARDO* AND M. SELMANt Instituto Nacional de Enfermedades Respiratorias, SSA, and *Facultad de Ciencias, UNAM, Mexico
The production of platelet-derived growth factor (PDGF) was studied in small cell lung carcinoma, lung squamous carcinoma and lung adenocarcinoma cell lines, and in seven human lung tissues obtained from each type of lung cancer. By indirect immunofluorescence, P D G F was detected in all the cell lines. Likewise, five out of seven biopsies derived from patients with adenocarcinoma and squamous carcinoma, and four out of seven specimens with small cell carcinoma displayed a positive pattern for PDGF. In addition, lung carcinoma cell lines expressed both P D G F - A and PDGF-B/sis genes, as judged by Northern blot analysis. Biologically active, serumfree conditioned media obtained from all three cell lines stimulated the incorporation of [3H]thymidine into quiescent BALB/c-3T3 cells. This effect was abolished when I g G - P D G F antiserum was used. These findings suggest that an abnormal expression of P D G F occurs in the three more frequent types of lung cancer, which can play a potential role in neoplastic transformation and uncontrolled cell growth. Introduction
In recent years, important advances in the knowledge of the possible molecular mechanisms involved in carcinogenesis have been made. However, current information is still fragmentary and inconclusive and the sequence of events responsible for neoplastic transformation remains unclear. One area of progress is that of the role of cellular oncogenes. The characterization of the molecular genomes of some retroviruses which induce cancer in vertebrate species has allowed the identification of oncogenic sequences and, furthermore, an homology matching with the D N A of other vertebrates including humans (1,2). Since some of these oncogenes code for growth factors or their related specific receptors (3,4), it is considered that oncogenes are able to participate in normal cellular differentiation and/or proliferation. Platelet-derived growth factor (PDGF) is a molecule normally expressed by several cell types. It has a variety of biological actions including direct and indirect effects on cell proliferation (5), and is the protein product of the cellular oncogene c-sis, which has 96% homology with its counterpart the v-sis oncogene of the simian sarcoma virus (6). Received 2 October 1990and acceptedin revised form 26 February 1991. "l'Towhom correspondenceand reprint request should be addressed at: Instituto Nacionalde EnfermedadesRespiratorias, Tlalpan4502; CP 14080,MrxicoD.F., Mexico. This study was partially supported by CONACYT, grant P219CCOL891537. 0954-6111/91/060479 + 07 $03.00/0
P D G F is composed of two polypeptide chains, P D G F - A and PDGF-B with approximate molecular weights of 14 000 and 17 000 Da, respectively, linked together by disulfide bonds (5). The PDGF-B polypeptide chain is encoded by the c-sis oncogene (7). In physiological conditions, P D G F is a potent mitogen for cells of mesenchymal origin acting in the cell cycle by preparing these cells through the induction of a competence state, allowing them to enter in an early G0/G 1 phase (8). Under pathological conditions, it is known that a similar, if not identical growth factor is produced by osteosarcoma (9), glioblastoma and fibrosarcoma (10), human breast cancer cell lines (11) and prostate adenocarcinoma cells (12). Becaus~ of the growth-promoting properties of PDGF, it is possible to postulate that, at least partially, the uncontrolled replication of the neoplastic cells can be stimulated in an autocrine fashion by the production of this growth factor by the tumour cells themselves (13). In addition, it is known that P D G F can in turn induce the expression of some oncogenes, such as c-myc and c-fos (14-16), and in this way might participate in neoplastic transformation. Studies concerning oncogenes and lung cancer have demonstrated the amplification of at least three myc genes, c-myc, N-myc, and L-myc, in cell lines from small cell lung carcinoma (17), and the expression, by means of in situ hybridization, of c-myc oncogene in tissue sections from six patients with squamous cell carcinoma and one with small cell carcinoma of primary lung origin (18). In addition, studies made in bronchial adenocarcinoma revealed activation of © 1991 Baillirre Tindall
480
M. Bravo et al.
K-ras oncogene in five out of ten patients studied suggesting that in this malignant type the amplification of this oncogene could be an event in the neoplastic induction (19). In this paper, we report the synthesis of biologically active PDGF-like proteins by three different lung cancer cell lines. Additionally, we demonstrate, in several lung cancer tissues, the presence of the same protein as judged by indirect immunofluorescence. Materials and Methods CELL CULTURES
All cell lines used were obtained from The American Type Culture Collection (Rockville, MD, U.S.A.). The three human lung cancer cell-lines were NCI-H69 from small cell carcinoma, A-427 from bronchial adenocarcinoma, and SK MES-1 derived from lung squamous carcinoma. F o r metabolic activity, mouse cells (BALB/c 3T3; clone A31) were used. All cells were grown in T75 culture flasks (Corning, New York, NY) and were maintained in Earle's modified Eagle's and H A M ' s F-12 K medium containing 10% fetal calf serum supplemented with penicillin (50 units ml -~) and streptomycin (50/tg ml-1). Cells were incubated in humidified 5% co 2 atmosphere at 37°C. TISSUE SAMPLES
Through bronchofiberoscopic or open thoracotomy biopsies, primary lung cancer tissue samples were obtained from 21 patients for conventional diagnosis. One part of the tissue specimen was fixed in buffered formaldehyde for morphologic study and the other was immediately frozen in liquid nitrogen before being stored at - 80°C. Pathologically, primary lung cancers were classified as squamous cell carcinoma (seven cases), bronchial adenocarcinoma (seven cases) and small cell carcinoma (seven cases). Three control samples without histologic evidence of cancer or interstitial fibrosis were selected from among a group of individuals who had lobectomy for removal bronchectasis.
reaction with the P D G F antisera was tested by control studies based on the pre-incubation of the antisera with excess (50/xg) of purified human P D G F . The antisera were diluted 1:500 in !% human serum albumin (HSA). Control studies included incubation with normal rabbit serum or excess purified P D G F . After incubation, the tissues were rinsed twice with PBS, treated with fluorescein isothiocyanate (FITC) IgG (Cappel, Malvern, PA) diluted 1:500 in 1% HSA, and incubated in darkness at room temperature for 30 min. A final wash with PBS was made in order to remove background and/or non-specific binding proteins. Samples were coated with mounting solution (90% glycerol, 10% PBS) and covered with glass coverslips. Immunofluorescence was examined under epifluorescence illumination on a Zeiss standard 16 microscope. For both fluorescent and phase-contrast microscopy, the following settings were used: Plan-neofluar t"25/0.8 and neofluar f40/0.75; micrographs were taken with an HBO 100 Hg illumination and MC 63 camera system. F o r cell immunofluorescence staining, cells were grown into eight-chamber slides, then in serum-free medium for 24 h. Cells were then fixed in 4% paraformaldehyde in PBS at room temperature for 15 min; slides were treated in the same way as tissues. NORTHERN BLOT ANALYSIS
Total cellular R N A was purified from the three cell li0es by the guanidine thiocyanate cesium chloride method (21). R N A (20/Lg) was ataalysed by electrophoresis on 1% agarose formaldehyde gels followed by Northern blot transfer to nitrocellulose filters. The filters were prehybridized at 42°C for 18 h in buffer consisting of 5 x SSC, 0.01% SDS, 5 x Denhardt's solution, and 200/zg ml -j salmon sperm DNA. The R N A blots were hybridized to specific P D G F - A and P D G F - B human complementary D N A probes (20) for 16-24h at 42°C with 1-2 x 106cpm nick-translated probe ml -~ hybridization buffer. After this step, the blots were washed twice in 2 x SSC-0.01% SDS at room temperature for 60min. The blots were then dried and exposed to X-ray films at - 70°C.
INDIRECT IMMUNOFLUORESCENCE
Frozen samples were sectioned between 4 and 6/zm in a microtome 'cryostat' model CTI at - 2 5 ° C , washed with phosphate-buffered saline solution (PBS) followed by a 10-min pre-incubation with 1% bovine serum albumin (Fraction V, Sigma, St Louis, MO). The preparations were incubated for 30 min at room temperature with rabbit polyclonal P D G F antisera raised against pure human P D G F . These antisera recognize the P D G F heterodimer and both P D G F - A and P D G F - B homodimers (20). The specificity of the
ASSAY FOR MITOGENIC ACTIVITY
Serum-free media conditioned for 24 h by confluent lung cancer cell lines approx. 4 × l06 were collected, cleared of particulate material by centrifugation, heated at 100°C for l0 rain and lyophilized. The dried powder was dissolved in 1.0 ml distilled water and dialysed against 0.15 M NaCI at 4°C. The concentrated media were tested for their ability to stimulate D N A synthesis by [3H]thymidine incorporation into quiescent BALB/c-3T3 cells, as previously described
P D G F in lung cancer
(22). As a positive control, we used the mitogenic response of the 3T3 cells to purified P D G F (2.5 ng ml- ~; Collaborative Research, Waltham, MA). Finally, in order to correlate DNA synthesis induced by serumfree media obtained from neoplastic cells with a specific effect of PDGF, IgG fractions of P D G F antiserum were added to identical wells. All the experiments were performed in triplicate.
Results
Intracellular PDGF-like proteins were detected in all the three lung cancer cell lines by indirect immunofluorescence using antiserum to P D G F (Plate !). Immunofluorescence staining showed a homogeneous cytoplasmic distribution and occasionally some fluorescence was observed in the perinuclear membrane. Indirect immunofluorescence in tissue sections correlated with the results obtained with the cell lines (Plate 2). Five out of seven biopsies derived from patients with bronchial adenocarcinoma and lung squamous carcinoma (71%), and four out of seven specimens with small cell carcinoma (59%) displayed a positive pattern for PDGF-Iike molecules. Three normal lungs used as controls were negative as previously demonstrated (20). By Northern blot analysis, PDGF-A and PDGF-B mRNAs were detected in the three lung cancer cell lines. Plate 3 illustrates the results of this assay. Both 4.2-kb and 2"3-kb hybridization bands were detectable in NCI-H69, A-427 and SK MES-I carcinoma cell lines. We further examined whether the PDGF-like proteins produced were released into the culture medium and if they were biologically active. Conditioned serum-free media from the three lung cancer cell lines stimulated [3H]thymidine incorporation in BALB/c 3T3 cells (Fig. I). The strongest stimulation was obtained with the small cell lung carcinoma cell line (P < 0-001 for both concentrations). On the other hand, DNA synthesis assayed with 75/11 conditioned media from the three neoplastic cell lines was abolished in the presence of the IgG fraction of the PDGF-antiserum.
Discussion
In the present study we demonstrate the synthesis and release of biologically active PDGF-Iike proteins by human lung small cell carcinoma, adenocarcinoma, and squamous carcinoma cell lines. In addition, 60-70% of the lung cancer tissue samples examined, representing these three major histological types,
481
exhibited a positive immunofluorescence staining for PDGF. The production of PDGF-like proteins corresponded to the presence of detectable levels of m R N A transcripts coded for by both P D G F - A and PDGF-B/sis genes in the cell lines, which agrees with previous findings supporting that both genes can be expressed by human carcinoma cell lines (12,23). Conversely, these findings contrast with those of Soderdahl et al. (24) who did not find P D G F m R N A in four small-cell human lung carcinoma cell lines. Although we only examined one small carcinoma cell line in our study, the expression of the genes for P D G F in this cell line, together with the positive immunofluorescence staining for the protein in half of the tissue specimens suggest that in some patients small cell lung carcinoma is able to produce PDGF. The variable pattern may be related to the phenotypic heterogeneity of the lung cancer. The discovery of the close homology between the PDGF-B amino acid sequence and the peptide coded by the simian sarcoma virus oncogene (v-sis), and the effects of P D G F on cellular growth have allowed speculation on the mechanisms involved in carcinogenesis. Thus, the constitutive expression of this growth factor, which acts as a competence factor in the cell cycle, could induce the unregulated, sustained cell proliferation characteristic of neoplastic cells. Furthermore, it is known that P D G F induces the expression of other cellular oncogenes, such as c-myc and c-fos, and strong evidence exists supporting that c-myc expression can block cell differentiation (25). Regarding lung cancer, overexpression of the myc family genes has been reported in small cell carcinoma cell lines and, moreover, the exaggerated expression of c-myc seems to induce a different phenotype even more malignant (26). More recently, amplification Of c-myc has been demonstrated in lung tissue of patients with squamous carcinoma and small cell carcinoma using in situ hybridization (18). Some authors have proposed that carcinogenesis comprises at least two steps: immortalization and transformation (27,28). This hypothesis can be applied to lung cancer. Theoretically, both steps could be the consequence of an alteration in the regulatory mechanisms controlling the cell cycle. Based on this interpretation, immortalization might be provoked by a modification of regulatory reactions involved at G0/early G1 phase, where P D G F exerts its action. In addition, since P D G F induces the expression of at least two other oncogenes, it can be suggested that this molecule may have some influence during the transformation step. This statement is suggested by experiments showing that transfection with c-sis oncogene permits transformation in normal ceils (29).
Ii
ill
Plate I Immunofluorescence staining for PDGF-like proteins (a, d, and g) of the three lung carcinoma cell lines. A bright fluorescence is present in ~mall cell carcinoma (a), epidermoid carcinoma (d) and adenocarcinoma (g). Panels (b), (e), and (h) represent phase contrast photomicrographs of the same sections stained in panels (a), (d), and (g). Negative controls incubated with normal rabbit serum are shown in panels (c), (f), and (i).
13
P D G F in lung cancer
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Plate 2 Immunofluorescent pictures of lung tissues with adenocarcinoma (a), small cell carcinoma (c), and epidermoid carcinoma (e). Numerous PDGF-positive malignant cells can be observed. Negative controls incubated with normal rabbit serum show non-specific staining (b,d,f).
Accordingly, it has been established that the cooperation o f at least two oncogenes is required in order to obtain full transformation. F o r example, in primary embryo fibroblasts cooperation is necessary from c-myc, which provides the immortalization function,
and an activated ras gene, providing the transformation of the pbenotype (30). Interestingly, both oncogenes have been found in lung carcinoma. Our understanding of the complex series of sequential mechanisms involved in the development of lung
M . Bravo et al.
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Plate 3 Northern blot analysis for PDGF-A and c-sis/PDGF-B genes in the lung carcinoma cell lines (lane b: SK-M ES- 1; lane c: NCI-H69 and lane d: A-427). Positive control (lane a) includes RNA from a human glioblastoma cell line.
cal~cer is far from complete, and, probably, some light will emerge when we get to know, with precision, the interactions between oncogenes, growth factors and the cell cycle control. In this context, our findings support the notion that the autocrine and paracrine production of platelet-derived growth factor can play a role in lung carcinogenesis and that this p h e n o m e n o n seems to be independent o f histological type.
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References
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Fig. 1 Mitogenic activity induced by conditioned media on 3T3 cells. FCS, Fetal calf serum; P, 2.5 ng of purified PDGF; A-427, SK-MES-I and NCI-H69, lung carcinoma cell lines. (IS]), and ([]), represent the response to 50 and 75pl of conditioned media, respectively; ( • ) indicate the response to 75pl of conditioned media after neutralization with the anti-PDGF IgG.
I. Bishop J. Cellular oncogenes and retrovirus. Ann Rev Biochem 1983; 52:301-354. 2. Antoniades HN, Hunkapiller M. Human plateletderived growth factor (PDGF): amino terminal amino acid sequence. Science 1983; 220: 963-966. 3. Chiu I, Reddy E, Givol D, Robbins KC, Tronick SR, Aaronson SA. Nucleotide sequence analysis identifies the human c-sis proto-oncogen as a structural gene for platelet-derived growth factor. Cell 1984; 37:123-129. 4. Yamamoto T, Kamata N, Kawano H et al. High incidence of amplification of the epidermal growth factor receptor gene in human squamous carcinoma cell lines. Cancer Res 1986; 46: 414-416. 5. Ross R, Raines E, Bowen-Pope D. The biology of platelet-derived growth factor. Cell 1986; 46:155-169. 6. Stiles C. The molecular biology of platelet-derived growth factor. Cell 1983; 33: 653-655.
P D G F in lung cancer
7. Doolittle R, Hunkapiller M, Hood L et al. Simian sarcoma virus oncogene, v-sis, is derived from the gene (or genes) encoding a platelet-derived growth factor. Science 1983; 221: 275-277. 8. Pledger W, Stiles C, Antoniades H, Scher C. Induction of DNA synthesis in BALB/c 3T3 cells by serum components; re-evaluation of the commitment process. Proc Natl Acad Sci USA 1977; 74: 4481-4485. 9. Graves DT, Owen A J, Barth RK et al. Detection of c-sis transcript and synthesis of PDGF-Iike proteins by human osteosarcoma cells. Science 1984; 226: 972-974. 10. Pantazis P, Pellicci P, Dalla-Favera R, Antoniades HN. Synthesis and secretion of proteins resembling plateletderived growth factor by human glioblastoma and fibrosarcoma cells in culture. Proe Natl Acad Sci USA 1985; 82: 2404-2408. 11. Rozengurt E, Sinnett-Smith J, Taylor-Papadimitriou J. Production of PDGF-like growth factor by breast cancer cell-lines, lnt J Cancer 1985; 36: 247-252. 12. Nikolaos M, Sariban E, Bravo M, Pantazis P, Antoniades HN. Constitutive production of plateletderived growth factor-like proteins by human prostate carcinoma cell lines. Cancer Res 1988; 48: 1930-1935. 13. Sporn MB, Todaro GJ. Autocrine secretion and malignant transformation of cells. N Engl J Med 1980; 303: 878-880. 14. Kelly K, Cochran BH, Stiles CD, Leder P. Cell-specific regulation of the c-myc gene by lymphocytes mitogens and platelet-derived growth factor. Cell 1983; 35: 603-610. 15. Greenberg ME, Ziff EB. Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene. Nature 1984; 311: 433-436. 16. Cochran BH, Zullo J, Verma IM, Stiles CD. Expression of the c-fos gene and of an fos-related gene is stimulated by platelet-derived growth factor. Science 1984; 226: 1080-1082. 17. Wong A, Ruppert J, Eggleston J, Hamilton S, Baylin S, Vogelstein B. Gene amplification ofc-myc and N-myc in small cell lung carcinoma of the lung. Science 1986; 233: 461-463. 18. Lee JH, Lee DH, Park SS, Seok SE, Lee JD. Oncogene expression detected by in situ hybridization in human primary lung cancer. Chest 1988; 94: 1046-1049.
485
19. Rodenhuis S, van der Wetering M, Moot W, Evers S, van Zandwijk N, Bos J. Mutational activation of K-ras oncogene. A possible pathogenic factor in adenocarcinoma of the lung. N EnglJ Med 1987; 317: 929-935. 20. Antoniades HN, Bravo M, Galanopoulus T, NevilleGolden J, Maxwell M, Selman M. Platelet-derived growth factor in idiopathic pulmonary fibrosis. J Clin Invest 1990; 86: 1055-1064. 21. Chirgwin JM, Przybyla AE, MacDonald R J, Rutter WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 1979; 18: 5294-5299. 22. Antoniades HN, Pantazis P. Structural and functional identification of platelet-derived growth factor-like proteins produced by mammalian cells. Methods Enzytool 1987; 146: 22-40. 23. Johnsson A, Heldin CH, Wasteson A et al. The c-sis gene encodes a precursor of the beta-chain of platelet-derived growth factor. EMBO J 1984; 3- 921-928. 24. Soderdahl G, Betsholtz C, Johansson A, Nilsson K, Bergh J. Differential expression of platelet-derived growth factor and transforming growth factor genes in small and non-small-cell human lung carcinoma cell lines, lnt J Cancer 1988; 41: 636-641. 25. Prochownik EV, Kukowska J. Deregulated expression of c-myc by murine erythroleukaemia cells prevents differentiation. Nature 1986; 322: 848-850. 26. Funa K, Steinholtz L, Nou E, Bergh J. Increased expression of N-myc in human small cell lung cancer biopsies predicts lack of response to chemotherapy and poor prognosis. Am J Clin Patho11987; 88: 216-220. 27. Parada LF, Land H, Weinberg RA, Wolf D, Rotter V. Cooperation between gene encoding p53 tumour antigen and ras in cellular transformation. Nature 1984; 312: 649-65 I. 28. Jenkins JR, Rudge K, Currie GA. Cellular immortalization by a cDNA clone encoding the transformationassociated phosphoprotein p53. Nature 1984; 312: 651-654. 29. Josephs SF, Ratner L, Clarke MF, Westin EH, Reitz MS, Wong-Staal F. Transforming potential of human c-sis nucleotide sequences encoding platelet-derived growth factor. Science 1984; 225: 636-639. 30. Land H, Parada L, Weiberg R. Tumorigenic conversion of primary embryo fibroblast requires at least two cooperating oncogenes. Nature 1983; 304: 596-602.
Production of platelet-derived growth factor by human lung cancer M. BRAVO, R. V,~SQUEZ, H. RUBIO, M. SALAZAR, A. PARDO* AND M. SELMANt Instituto Nacional de Enfermedades Respiratorias, SSA, and *Facultad de Ciencias, UNAM, Mexico
The production of platelet-derived growth factor (PDGF) was studied in small cell lung carcinoma, lung squamous carcinoma and lung adenocarcinoma cell lines, and in seven human lung tissues obtained from each type of lung cancer. By indirect immunofluorescence, P D G F was detected in all the cell lines. Likewise, five out of seven biopsies derived from patients with adenocarcinoma and squamous carcinoma, and four out of seven specimens with small cell carcinoma displayed a positive pattern for PDGF. In addition, lung carcinoma cell lines expressed both P D G F - A and PDGF-B/sis genes, as judged by Northern blot analysis. Biologically active, serumfree conditioned media obtained from all three cell lines stimulated the incorporation of [3H]thymidine into quiescent BALB/c-3T3 cells. This effect was abolished when I g G - P D G F antiserum was used. These findings suggest that an abnormal expression of P D G F occurs in the three more frequent types of lung cancer, which can play a potential role in neoplastic transformation and uncontrolled cell growth. Introduction
In recent years, important advances in the knowledge of the possible molecular mechanisms involved in carcinogenesis have been made. However, current information is still fragmentary and inconclusive and the sequence of events responsible for neoplastic transformation remains unclear. One area of progress is that of the role of cellular oncogenes. The characterization of the molecular genomes of some retroviruses which induce cancer in vertebrate species has allowed the identification of oncogenic sequences and, furthermore, an homology matching with the D N A of other vertebrates including humans (1,2). Since some of these oncogenes code for growth factors or their related specific receptors (3,4), it is considered that oncogenes are able to participate in normal cellular differentiation and/or proliferation. Platelet-derived growth factor (PDGF) is a molecule normally expressed by several cell types. It has a variety of biological actions including direct and indirect effects on cell proliferation (5), and is the protein product of the cellular oncogene c-sis, which has 96% homology with its counterpart the v-sis oncogene of the simian sarcoma virus (6). Received 2 October 1990and acceptedin revised form 26 February 1991. "l'Towhom correspondenceand reprint request should be addressed at: Instituto Nacionalde EnfermedadesRespiratorias, Tlalpan4502; CP 14080,MrxicoD.F., Mexico. This study was partially supported by CONACYT, grant P219CCOL891537. 0954-6111/91/060479 + 07 $03.00/0
P D G F is composed of two polypeptide chains, P D G F - A and PDGF-B with approximate molecular weights of 14 000 and 17 000 Da, respectively, linked together by disulfide bonds (5). The PDGF-B polypeptide chain is encoded by the c-sis oncogene (7). In physiological conditions, P D G F is a potent mitogen for cells of mesenchymal origin acting in the cell cycle by preparing these cells through the induction of a competence state, allowing them to enter in an early G0/G 1 phase (8). Under pathological conditions, it is known that a similar, if not identical growth factor is produced by osteosarcoma (9), glioblastoma and fibrosarcoma (10), human breast cancer cell lines (11) and prostate adenocarcinoma cells (12). Becaus~ of the growth-promoting properties of PDGF, it is possible to postulate that, at least partially, the uncontrolled replication of the neoplastic cells can be stimulated in an autocrine fashion by the production of this growth factor by the tumour cells themselves (13). In addition, it is known that P D G F can in turn induce the expression of some oncogenes, such as c-myc and c-fos (14-16), and in this way might participate in neoplastic transformation. Studies concerning oncogenes and lung cancer have demonstrated the amplification of at least three myc genes, c-myc, N-myc, and L-myc, in cell lines from small cell lung carcinoma (17), and the expression, by means of in situ hybridization, of c-myc oncogene in tissue sections from six patients with squamous cell carcinoma and one with small cell carcinoma of primary lung origin (18). In addition, studies made in bronchial adenocarcinoma revealed activation of © 1991 Baillirre Tindall
480
M. Bravo et al.
K-ras oncogene in five out of ten patients studied suggesting that in this malignant type the amplification of this oncogene could be an event in the neoplastic induction (19). In this paper, we report the synthesis of biologically active PDGF-like proteins by three different lung cancer cell lines. Additionally, we demonstrate, in several lung cancer tissues, the presence of the same protein as judged by indirect immunofluorescence. Materials and Methods CELL CULTURES
All cell lines used were obtained from The American Type Culture Collection (Rockville, MD, U.S.A.). The three human lung cancer cell-lines were NCI-H69 from small cell carcinoma, A-427 from bronchial adenocarcinoma, and SK MES-1 derived from lung squamous carcinoma. F o r metabolic activity, mouse cells (BALB/c 3T3; clone A31) were used. All cells were grown in T75 culture flasks (Corning, New York, NY) and were maintained in Earle's modified Eagle's and H A M ' s F-12 K medium containing 10% fetal calf serum supplemented with penicillin (50 units ml -~) and streptomycin (50/tg ml-1). Cells were incubated in humidified 5% co 2 atmosphere at 37°C. TISSUE SAMPLES
Through bronchofiberoscopic or open thoracotomy biopsies, primary lung cancer tissue samples were obtained from 21 patients for conventional diagnosis. One part of the tissue specimen was fixed in buffered formaldehyde for morphologic study and the other was immediately frozen in liquid nitrogen before being stored at - 80°C. Pathologically, primary lung cancers were classified as squamous cell carcinoma (seven cases), bronchial adenocarcinoma (seven cases) and small cell carcinoma (seven cases). Three control samples without histologic evidence of cancer or interstitial fibrosis were selected from among a group of individuals who had lobectomy for removal bronchectasis.
reaction with the P D G F antisera was tested by control studies based on the pre-incubation of the antisera with excess (50/xg) of purified human P D G F . The antisera were diluted 1:500 in !% human serum albumin (HSA). Control studies included incubation with normal rabbit serum or excess purified P D G F . After incubation, the tissues were rinsed twice with PBS, treated with fluorescein isothiocyanate (FITC) IgG (Cappel, Malvern, PA) diluted 1:500 in 1% HSA, and incubated in darkness at room temperature for 30 min. A final wash with PBS was made in order to remove background and/or non-specific binding proteins. Samples were coated with mounting solution (90% glycerol, 10% PBS) and covered with glass coverslips. Immunofluorescence was examined under epifluorescence illumination on a Zeiss standard 16 microscope. For both fluorescent and phase-contrast microscopy, the following settings were used: Plan-neofluar t"25/0.8 and neofluar f40/0.75; micrographs were taken with an HBO 100 Hg illumination and MC 63 camera system. F o r cell immunofluorescence staining, cells were grown into eight-chamber slides, then in serum-free medium for 24 h. Cells were then fixed in 4% paraformaldehyde in PBS at room temperature for 15 min; slides were treated in the same way as tissues. NORTHERN BLOT ANALYSIS
Total cellular R N A was purified from the three cell li0es by the guanidine thiocyanate cesium chloride method (21). R N A (20/Lg) was ataalysed by electrophoresis on 1% agarose formaldehyde gels followed by Northern blot transfer to nitrocellulose filters. The filters were prehybridized at 42°C for 18 h in buffer consisting of 5 x SSC, 0.01% SDS, 5 x Denhardt's solution, and 200/zg ml -j salmon sperm DNA. The R N A blots were hybridized to specific P D G F - A and P D G F - B human complementary D N A probes (20) for 16-24h at 42°C with 1-2 x 106cpm nick-translated probe ml -~ hybridization buffer. After this step, the blots were washed twice in 2 x SSC-0.01% SDS at room temperature for 60min. The blots were then dried and exposed to X-ray films at - 70°C.
INDIRECT IMMUNOFLUORESCENCE
Frozen samples were sectioned between 4 and 6/zm in a microtome 'cryostat' model CTI at - 2 5 ° C , washed with phosphate-buffered saline solution (PBS) followed by a 10-min pre-incubation with 1% bovine serum albumin (Fraction V, Sigma, St Louis, MO). The preparations were incubated for 30 min at room temperature with rabbit polyclonal P D G F antisera raised against pure human P D G F . These antisera recognize the P D G F heterodimer and both P D G F - A and P D G F - B homodimers (20). The specificity of the
ASSAY FOR MITOGENIC ACTIVITY
Serum-free media conditioned for 24 h by confluent lung cancer cell lines approx. 4 × l06 were collected, cleared of particulate material by centrifugation, heated at 100°C for l0 rain and lyophilized. The dried powder was dissolved in 1.0 ml distilled water and dialysed against 0.15 M NaCI at 4°C. The concentrated media were tested for their ability to stimulate D N A synthesis by [3H]thymidine incorporation into quiescent BALB/c-3T3 cells, as previously described
P D G F in lung cancer
(22). As a positive control, we used the mitogenic response of the 3T3 cells to purified P D G F (2.5 ng ml- ~; Collaborative Research, Waltham, MA). Finally, in order to correlate DNA synthesis induced by serumfree media obtained from neoplastic cells with a specific effect of PDGF, IgG fractions of P D G F antiserum were added to identical wells. All the experiments were performed in triplicate.
Results
Intracellular PDGF-like proteins were detected in all the three lung cancer cell lines by indirect immunofluorescence using antiserum to P D G F (Plate !). Immunofluorescence staining showed a homogeneous cytoplasmic distribution and occasionally some fluorescence was observed in the perinuclear membrane. Indirect immunofluorescence in tissue sections correlated with the results obtained with the cell lines (Plate 2). Five out of seven biopsies derived from patients with bronchial adenocarcinoma and lung squamous carcinoma (71%), and four out of seven specimens with small cell carcinoma (59%) displayed a positive pattern for PDGF-Iike molecules. Three normal lungs used as controls were negative as previously demonstrated (20). By Northern blot analysis, PDGF-A and PDGF-B mRNAs were detected in the three lung cancer cell lines. Plate 3 illustrates the results of this assay. Both 4.2-kb and 2"3-kb hybridization bands were detectable in NCI-H69, A-427 and SK MES-I carcinoma cell lines. We further examined whether the PDGF-like proteins produced were released into the culture medium and if they were biologically active. Conditioned serum-free media from the three lung cancer cell lines stimulated [3H]thymidine incorporation in BALB/c 3T3 cells (Fig. I). The strongest stimulation was obtained with the small cell lung carcinoma cell line (P < 0-001 for both concentrations). On the other hand, DNA synthesis assayed with 75/11 conditioned media from the three neoplastic cell lines was abolished in the presence of the IgG fraction of the PDGF-antiserum.
Discussion
In the present study we demonstrate the synthesis and release of biologically active PDGF-Iike proteins by human lung small cell carcinoma, adenocarcinoma, and squamous carcinoma cell lines. In addition, 60-70% of the lung cancer tissue samples examined, representing these three major histological types,
481
exhibited a positive immunofluorescence staining for PDGF. The production of PDGF-like proteins corresponded to the presence of detectable levels of m R N A transcripts coded for by both P D G F - A and PDGF-B/sis genes in the cell lines, which agrees with previous findings supporting that both genes can be expressed by human carcinoma cell lines (12,23). Conversely, these findings contrast with those of Soderdahl et al. (24) who did not find P D G F m R N A in four small-cell human lung carcinoma cell lines. Although we only examined one small carcinoma cell line in our study, the expression of the genes for P D G F in this cell line, together with the positive immunofluorescence staining for the protein in half of the tissue specimens suggest that in some patients small cell lung carcinoma is able to produce PDGF. The variable pattern may be related to the phenotypic heterogeneity of the lung cancer. The discovery of the close homology between the PDGF-B amino acid sequence and the peptide coded by the simian sarcoma virus oncogene (v-sis), and the effects of P D G F on cellular growth have allowed speculation on the mechanisms involved in carcinogenesis. Thus, the constitutive expression of this growth factor, which acts as a competence factor in the cell cycle, could induce the unregulated, sustained cell proliferation characteristic of neoplastic cells. Furthermore, it is known that P D G F induces the expression of other cellular oncogenes, such as c-myc and c-fos, and strong evidence exists supporting that c-myc expression can block cell differentiation (25). Regarding lung cancer, overexpression of the myc family genes has been reported in small cell carcinoma cell lines and, moreover, the exaggerated expression of c-myc seems to induce a different phenotype even more malignant (26). More recently, amplification Of c-myc has been demonstrated in lung tissue of patients with squamous carcinoma and small cell carcinoma using in situ hybridization (18). Some authors have proposed that carcinogenesis comprises at least two steps: immortalization and transformation (27,28). This hypothesis can be applied to lung cancer. Theoretically, both steps could be the consequence of an alteration in the regulatory mechanisms controlling the cell cycle. Based on this interpretation, immortalization might be provoked by a modification of regulatory reactions involved at G0/early G1 phase, where P D G F exerts its action. In addition, since P D G F induces the expression of at least two other oncogenes, it can be suggested that this molecule may have some influence during the transformation step. This statement is suggested by experiments showing that transfection with c-sis oncogene permits transformation in normal ceils (29).
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Plate I Immunofluorescence staining for PDGF-like proteins (a, d, and g) of the three lung carcinoma cell lines. A bright fluorescence is present in ~mall cell carcinoma (a), epidermoid carcinoma (d) and adenocarcinoma (g). Panels (b), (e), and (h) represent phase contrast photomicrographs of the same sections stained in panels (a), (d), and (g). Negative controls incubated with normal rabbit serum are shown in panels (c), (f), and (i).
13
P D G F in lung cancer
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Plate 2 Immunofluorescent pictures of lung tissues with adenocarcinoma (a), small cell carcinoma (c), and epidermoid carcinoma (e). Numerous PDGF-positive malignant cells can be observed. Negative controls incubated with normal rabbit serum show non-specific staining (b,d,f).
Accordingly, it has been established that the cooperation o f at least two oncogenes is required in order to obtain full transformation. F o r example, in primary embryo fibroblasts cooperation is necessary from c-myc, which provides the immortalization function,
and an activated ras gene, providing the transformation of the pbenotype (30). Interestingly, both oncogenes have been found in lung carcinoma. Our understanding of the complex series of sequential mechanisms involved in the development of lung
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Plate 3 Northern blot analysis for PDGF-A and c-sis/PDGF-B genes in the lung carcinoma cell lines (lane b: SK-M ES- 1; lane c: NCI-H69 and lane d: A-427). Positive control (lane a) includes RNA from a human glioblastoma cell line.
cal~cer is far from complete, and, probably, some light will emerge when we get to know, with precision, the interactions between oncogenes, growth factors and the cell cycle control. In this context, our findings support the notion that the autocrine and paracrine production of platelet-derived growth factor can play a role in lung carcinogenesis and that this p h e n o m e n o n seems to be independent o f histological type.
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References
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Fig. 1 Mitogenic activity induced by conditioned media on 3T3 cells. FCS, Fetal calf serum; P, 2.5 ng of purified PDGF; A-427, SK-MES-I and NCI-H69, lung carcinoma cell lines. (IS]), and ([]), represent the response to 50 and 75pl of conditioned media, respectively; ( • ) indicate the response to 75pl of conditioned media after neutralization with the anti-PDGF IgG.
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P D G F in lung cancer
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