Int. J. Cancer: 51,818-821 (1992) 0 1992 Wiley-Liss, Inc.
Publicationof the International Union Against Cancer Publication de I'Union lnternationale Contre le Cancer
INSULIN-LIKE GROWTH-FACTOR-BINDING PROTEIN GENE EXPRESSION AND PROTEIN PRODUCTION BY HUMAN TUMOUR CELL LINES Julie G. REEVE',^, Lemuel B. KIRBY',Ad BRINK MAN^, Stephanie A. HUGHES',Jurg sCHWANDER3 and Norman M. BLEEHEN' Medical Research Council, Clinical OncoloRy and Radiotherapeutics Unit, MRC Centre, Hills Road, Cambridge CB2 2Qfl U K 2PediatricEndocrinology, Erasmus University, Rotterdam, The Netherlands; and 3DepartmentInnere Medizin, Kantonsspital, Basel, Switzerland. The secretion of insulin-like growth-factor-bindingproteins (IGFBPs) and expression of the genes encoding IGFBP-I, IGFBP-2 and IGFBP-3 have been studied in a panel of cell lines derived from breast carcinomas, Wilms' tumour, neuroblastoma, retinoblastoma, colon carcinoma, liver adenocarcinoma, Burkitt's lymphoma and a non-small-cell lung carcinoma. All cell lines, with the exception of the Burkitt's lymphoma cell line, secreted IGFBPs, as detected by affinity labelling. A 34-kDa BP was present in the conditioned media of all IGFBP-secretingcell lines, whereas BPs ranging from I 8 kDa to 53 kDa were variably secreted. All IGFBP-secreting cell lines expressed the IGFBP-2 gene as determined by Northern blot analysis. The Wilms' tumour, the neuroblastoma and the retinoblastoma cell line expressed the IGFBP-2 gene only. All other cell lines, with the exception of the Burkitt's lymphoma, expressed the IGFBP-2 gene and, in addition, either the IGFBP-I gene and/or the IGFBP-3 gene. IGFBP- I gene expression could be detected by reverse transcriptase polymerase chain reaction only. IGFBP-3 gene expression was detected by Northern blot analysis, but transcripts were less abundant than IGFBP-2 mRNAs. These findings indicate that the expression of multiple BP genes and the secretion of BPs may be a common property of tumour cells.
o 1992 Wiley-Liss,Inc. Insulin-like growth factors (IGFs) in the plasma and tissue fluids occur bound to specific IGF-binding proteins (Baxter and Martin, 1989). In the circulation, IGF-binding proteins (BPs) prolong the biological half-life of the IGFs and protect against the acute insulin-like actions of these peptides. BPs in the interstitial fluids are thought to regulate the bio-availability of locally secreted IGFs to their target cells, and to modulate the biological effects of the IGFs by altering their interaction with the IGF receptor. To date, 6 classes of binding protein have been characterized by protein and cDNA sequencing studies. These are IGFBP-1, isolated from human amniotic fluid (Baxter and Martin, 1989); IGFBP-2 (Binkert et al., 1989), the human homologue of rat BRL-BP, isolated from human serum (Kiefer et al., 1991); IGFBP-3, also known as BP-53, the glycosylated IGF binding subunit of the 150-kDa complex that carries most of the IGFs in adult human plasma; IGFBP-4 which is secreted by human osteosarcoma and prostatic carcinoma cell lines in vitro (Mohan et al., 1989; Perkel et al., 1990), and is also present in human serum (Kiefer et al., 1991); IGFBP-5, isolated from cerebrospinal fluid and from human serum (Kiefer et al., 1991), and finally IGFBP-6 isolated recently from pig ovarian follicular fluid (Shimasaki et al., 1991). A variety of human cell types have been shown to produce or contain IGFBPs (Baxter and Martin, 1989). These include foetal and adult liver, endometrium, brain, pituitary, and human fibroblast cell lines. In addition, cell lines derived from human hepatomas (Povoa et al., 1985), carcinomas of the colon (Culouscou and Shoyab, 1991), breast (De Leon et al., 1989), and lung (Reeve et al., 1990), and tumours of the central nervous system (Unterman et al., 1991) have been shown to secrete BPs in vitro. Increased IGFBP-1 levels have been detected in patients with ovarian carcinoma (Iino et al., 1986) and our recent studies indicate increased BP serum levels in patients with lung cancer (Reeve et al., 1990).
The production of BPs by human tumour cells is of particular interest, given the acknowledged role of the IGFs as growth factors for certain tumour types and the ability of BPs to either stimulate (Baxter and Martin, 1989; Clemmons et al., 1990) or inhibit (Baxter and Martin, 1989; Lui et al., 1991) IGF-induced DNA synthesis. These observations raise the possibility that IGFBPs may represent a new class of tumour-growthregulatory factors which may promote or inhibit cell division, depending on the BP produced and the tumour type. The present study examines the distribution of IGFBP production in a panel of tumour cell lines and, as a first step toward studying the role(s) played by BPs in different tumours, characterizes BP production by examining the expression of genes encoding IGFBPs. MATERIAL AND METHODS
Cell lines The following cell lines were obtained from the ATCC (Rockville, MD): ZR-75-1, SK-Br3, T47D (breast carcinomas); WiDr (colon carcinoma); SK-HEP-1 (liver adenocarcinoma); SK-NEP-1 (Wilms' tumour); Y-79 (retinoblastoma); SK-NMC (neuroblastoma); EB-1 (Burkitt's lymphoma). LUDLU-1 (squamous-cell lung carcinoma) was kindly donated by Dr. P.H. Rabbitts of this Unit. Detection of IGF-binding proteins by afinity labelling For the production of conditioned media, cells were grown in serum-free RPMI 1640 medium (Gibco, Paisley, UK) for up to 72 hr at a concentration of approximately lo6 cells/ml. Conditioned media were harvested, clarified by centrifugation at 10,OOOgand stored at -70°C in aliquots until use. Conditioned media (10 PI) diluted 1 : l O in 0.5 M sodium phosphate buffer (pH 7.4) were pre-incubated on ice for 30 min in the presence of approximately 250,000 counts per minute of lz5I-IGF-I (Amersham, Aylesbury, UK). Crosslinking of radiolabelled IGF-I to proteins was accomplished by the addition of 5 ~1 of 20 mM disuccinimidyl suberate (DSS) to give a final concentration of 1 mM, followed by incubation at room temperature for 10 min. To confirm the specificity of the cross-linking, the reaction was carried out in the presence or absence of 500 ng of cold IGF-I (Amersham). Samples were prepared for electrophoresis by the addition of 0.0005% bromophenol blue in 0.015 M Tris-HC1 (pH 8.8). Electrophoresis was performed under non-reducing conditions on 12.5% SDS-polyacrylamide gels overnight at room temperature with a constant current of 8 mA. Gels were fixed in 3.5% acetic acid/ 10.5% methanol and autoradiographed. RNA preparation Cells in the logarithmic phase of growth were pelleted by centrifugation at 300 g for 10 min and suspended in their own
4Towhom requests for reprints should be addressed.
Received: January 20,1992 and in revised form March 7,1992.
819
TUMOUR-DERIVED IGF-BINDING PROTEINS
volume. A solution containing 6.0 M guanidine hydrochloride and 0.2 M sodium acetate (pH 5.5) was added to the cells (20 ml per 5 x lo7 cells) and the DNA was sheared by vigorous homogenization in a homogenizer (Virtis, New York, NY). RNA was precipitated by the addition of a half volume of 95% ethanol followed by incubation at -20°C overnight. The pelleted precipitate was dissolved in a solution containing 7.0 M urea, 0.35 M NaCI, 50 mM Tris, pH 7.5, 1 mM EDTA and 0.2% SDS and was extracted once with phenol-chloroform. RNA was precipitated from the aqueous phase using 2 volumes of ethanol, washed with 70% ethanol, air-dried and dissolved in sterile, double-distilled water. Poly(A)+ RNA was prepared from total RNA using a mRNA purification kit (Pharmacia, Milton Keynes, UK). Northern blot analysis Five micrograms of poly(A)+ RNA in 10 mM sodium phosphate buffer (pH 7.0) was denatured in 1.0 M glyoxal for 1 hr at 50°C. The RNA was electrophoresed in a 1.4% agarose gel in 10 mM sodium phosphate buffer and was transferred by Northern blotting to nylon filters. After treatment for 2 min with ultraviolet light, the nylon filters were baked at 80°C for 2 hr before hybridization. Radiolabelled IGFBP-1 (Brinkman et al., 1988), IGFBP-2 (Binkert et al., 1989), IGFBP-3 (Wood et al., 1988) cDNA probes were prepared using an oligolabelling kit (Pharmacia). The IGFBP-3 cDNA probe was generously supplied by Genentech, South San Francisco, CA. The radiolabelled probes were separated from unincorporated nucleotide triphosphates using Sephadex G50 (Pharmacia) and boiled for 3 rnin before use. A mouse p-actin probe (PRT3) (kindly donated by Dr J. Rogers, Laboratory of Molecular Biology, Cambridge, UK) was similarly labelled to confirm equal loading of RNA. The labelled probe, at a concentration of lo6 counts min-’ ml-l, was hybridized to the filter in 1 M NaCl, 0.1 M trisodium citrate (6 x SSC), 5% dextran sulphate, 0.02% Ficoll, 0.02% BSA, 0.02% polyvinyl pyrrolidone, 0.1% SDS and 150 pg ml-1 sonicated salmon sperm DNA at 65°C for 18 hr. The filter was washed with 0.1 X SSC, 0.1% SDS at 65°C to remove unhybridized probe prior to autoradiography. Reverse transcriptasepolymerase chain reaction (RT-PCR) Synthetic oligonucleotides, designed on the basis of the nucleotide sequence of the mRNA encoding IGFBP-1 (Brinkman et al., 1988), were synthesized using an Applied Biosystems 380 DNA synthesizer. The sequences of the primers were:
ing was for 2 min at 5 5 T , polymerization was at 72°C for 3 rnin and denaturation at 95°C for 1 min. Forty microlitres of the reaction mixture were then electrophoresed on 1.4% agarose gels in the presence of ethidium bromide. Amplified products were detected by ultraviolet light transillumination and by autoradiography following Southern blotting and hybridization with IGFBP-1 cDNA. RESULTS
Detection of IGF-BPs in cell-conditioned media The secretion of IGF-BPs by the cell lines studied is shown in Figure 1. It can be seen that, with the exception of the Burkitt’s lymphoma cell line, all cell lines produced one or more BPs which specifically bound IGF-I. IGFBP complexes ranged in molecular size from 25 kDa to 60 kDa, indicating that cell lines secreted BPs with Mrs of 18 kDa to 53 kDa. A 34-kDa BP was present in the conditioned media of all IGFBP-secreting cell lines, whereas BPs ranging from 18 kDa to 53 kDa were variably secreted. IGFBP-I gene expression Although a 1.5-kb IGFBP-1 transcript was detected by Northern blot analysis in normal liver, no evidence of IGFBP-1 gene expression was observed using this method in any of the cell lines studied (data not shown). However, enzymatic amplification from IGFBP-1 mRNA by RT-PCR (Fig. 2) demonstrated the presence of a 437-bp amplification product which hybridized to the IGFBP-1 cDNA probe in 4 of the 10 cell lines studied, including the breast carcinoma lines ZR-75-1
(Bl) 5’..GCTCCCCATGCTGCAGAGGCAGGG..3’ corresponding to nucleotides 386-409 (B2) 5‘..TACATTAAAATACATCTGGCAGTT..3’ complementary to nucleotides 823-800. The 437 bp amplification product is unique to IGFBP-1. Ten micrograms of total RNA were reversed transcribed into first-strand cDNA by addition of 5 pIO.1 M dithiothreitol, 2.5 p15 mM deoxyribonucleoside triphosphate (dNTP) (Pharmacia), 20 pmoles of oligonucleotide B2 and 5 p15 x reverse transcriptase buffer (500 mM Tris, pH 8.3, 60 mM MgClz and 400 mM KCI). After heating for 10 min at 70T, the reaction mixture was cooled to 25°C and 2 units of avian myeloblastosis virus (AMV) reverse transcriptase (Anglian Biotec, Colchester, UK) were added. Following incubation at 42°C for 1 hr, a 5-1.1.1 aliquot of first-strand cDNA was added to 20 pmoles of oligonucleotide primers B1 and B2,5 ~ 1 mM 5 dNTP, and 5 p1 10 x Thermus aquaticus (Taq) polymerase buffer (0.67 mM Tris, pH 8.8, 9.17 M (NH4)z SO4, 0.1 M MgCl2, 0.1 M bromomercaptoethanol, 2 mg ml-l gelatine) in a total volume of 50 pl. Two units of Taq polymerase (Cetus, Emeryville, CA) were added and amplification (35 cycles) was performed using a PHC-1 automated cycler (Techne, Cambridge, UK). Anneal-
FIGURE1 -Detection of IGF-binding proteins in tumour cell conditioned media by affinity-labelling in the absence (-) and presence (+) of IGF-I. Arrows indicate the position of molecular weight markers. A 41-kDa IGFBP complex is present in the conditioned media of all cell lines with the exception of EB-1, indicating that cell lines secreted a 34-kDA BP.
820
REEVE E T A L .
FIGURE2 - Southern blot analysis of IGFBP-1 gene expression by tumour cell lines following enzymatic amplification from IGFBP-1 mRNA by polymerase chain reaction and hybridization to radiolabelled IGFBP-1 cDNA probe.
FIGURE4 - Northern blot analysis of IGFBP-3 gene expression in tumour cell lines. DISCUSSION
FIGURE3 - Northern blot analysis of IGFBP-2 gene expression in tumour cell lines.
and T47D, the colon carcinoma cell line WiDr and the hepatoma cell line SK-HEP-1.
IGFBP-2 gene wpression Figure 3 shows that IGFBP-2 gene expression was detected by Northern blot analysis in all cell lines studied, with the exception of the EB-1 Burkitt’s lymphoma cell line. It can be seen that the IGFBP-2 probe hybridized to multiple RNA species in several of the cell lines, with the previously reported 1.4-kb gene transcript being most frequently detected. IGFBP-3 gene expression Northern blot analysis of IGFBP-3 gene expression revealed a single 2.5-kb IGFBP-3 transcript in 4 of the 10 cell lines studied, including the breast carcinoma cell lines SK-Br-3 and T47D, the lung carcinoma cell line LUDLU-1 and the hepatoma cell line SK-HEP-1 (Fig. 4).
Recent research has shown that osteosarcoma (Mohan et al., 1989), prostatic carcinoma (Perkel et al., 1990) and colon carcinoma (Culouscou and Shoyab, 1991) cell lines secrete IGFBP-4, and that proteins immunologically related to IGFBP-1 and IGFBP-3 are secreted by the MDA-231 breast carcinoma cell line (Camacho-Hubner et al., 1991) and by certain central-nervous-system tumour cells (Unterman et al., 1991). The findings of the present study extend these observations and demonstrate that the secretion of IGFBPs is a property common to many human tumour cell lines growing in vitro and that in several cases multiple IGFBP genes are expressed. A major finding of the present study is that expression of the IGFBP-2 gene occurred in all IGFBP-secreting cell lines studied. This observation is of particular interest given the restricted normal tissue distribution of IGFBP-2 gene expression. It is likely that this gene encodes the 34-kDa protein that was detected in the conditioned media of all secreting cell lines. A single 2.5-kb IGFBP-3 transcript was detected by Northern blot analysis in 5 of the 10 cell lines examined and, as previously reported, in adult human liver (Wood et al., 1988). IGFBP-3 gene transcripts were much less abundant than IGFBP-2 mRNA, as evidenced by the much longer exposure times required for detection of the expression of this gene by autoradiography . In contrast to IGFBP-2 and IGFBP-3 gene expression, IGFBP-1 mRNA could not be detected by Northern blot analysis in any of the cell lines studied. However, the presence of an amplification product of the expected size, following RT-PCR, which hybridized to the IGFBP-1 cDNA probe demonstrates that this gene is expressed at relatively low levels in a proportion of cell lines. The apparent differences in the relative levels of IGFBP-1, IGFBP-2 and IGFBP-3 gene expression observed in the various cell lines may indicate differences in gene activation and/or mRNA stability, or expression of the IGFBP-1 and IGFBP-3 genes in a proportion of the cells only. At present, little is known about the regulation of IGFBP genes, although a number of observations suggest that the regulation of IGFBP-3 is different from that of IGFBP-1 and IGFBP-2. Whereas serum levels of IGFBP-1 and IGFBP-2 are highest during foetal life, IGFBP-3 levels are higher post-natally, and peak during puberty (Drop et al., 1984). In contrast to IGFBP-1 and IGFBP-2 levels, which decrease in the presence of IGF-I and insulin (BoniSchnetzler et al., 1990), IGFBP-3 levels increase in the presence of these and other growth factors such as epidermal growth factor, phorbol esters, bombesin, vasopressin, and platelet-derived growth factor (Corps and Brown, 1991). Although recent studies indicate that the hepatic nuclear factor LF-B1 may play a role in the tissue-specific and developmental expression of IGFBP-1 (Suwanichkul et al., 1990), other transcription factors in placenta, endometrium,
TUMOUR-DERIVED IGF-BINDING ovary and additional tissues which express IGFBP-1 have yet to be identified. The IGFBP-3 promoter has been shown to contain binding sites for Sp-1 and AP-2 (Cubbage et al., 1990), which may account in part for the expression of IGFBP-3 in multiple tissues throughout development and for the expression of this gene in a number of the tumour cell lines reported here. Interestingly, a number of potential regulatory sites have been identified in the IGFBP-2 gene nucleotide sequence, including sites for Spl, ETF, AP-I, AP-2, LF-B1, Pan and a TC box (Brown and Rechlert, 1990). The relationship between these observations and the ubiquitous expression of the IGFBP-2 gene in the tumour cell lines reported here remains to be determined. The finding in the present study that all but one of the tumour cell lines growing in vitro secreted BPs is of particular interest, since BPs have been shown to modulate the mitogenic actions of the IGFs (Baxter and Martin, 1989; Lui et al., 1991). The ubiquitous expression of the IGFBP-2 gene in the panel of cell lines studied here suggests that IGFBP-2 is intimately associated with the malignant phenotype, and may indicate that secretion of this protein confers some growth advantage on the tumour cell. Studies are now in progress to evaluate the growth-modifying actions of this protein in IGF-responsivc tumour cell lines. Interestingly, both IGFBP-1 and IGFBP-2 possess amino acid sequences typical of the integrin family of cellular adhesion proteins, suggesting that they may serve dual
PROTEINS
821
functions related both to the regulation of proliferation and to cellular anchoring. The in vitro production of IGFBPs by lung tumour cell lines and the presence of elevated serum levels of these proteins in lung-cancer patients has been reported recently (Reeve et al., 1990). In addition, circulating levels of IGFBP-1 have been detected in patients with ovarian cancer (Iino et al., 1986). Given that all but one of the tumour cell lines in the present study secreted BPs in vitro, it seems likely that BPs may serve as markers for a wide variety of tumours. Indeed, we have recently detected elevated BP levels in patients with rhabdomyosarcoma, melanoma, carcinomas of the colon, rectum, endometrium and breast, and in patients with thymomas. These observations support the contention that IGFBP production is a common feature of tumour cells both in vitro and in vivo where they may contribute to the regulation of tumour growth and metastatic spread. Although the expression of the recently cloned IGFBP-4, IGFBP-5 (Kiefer et al., 1991) and IGFBP-6 (Shimasaki et al., 1991) genes has not been investigated here, a growthinhibitory IGFBP which appears identical to IGFBP-4 has been shown to be secreted by the HT29 colon carcinoma cell line (Culouscou and Shoyab, 1991). This, together with the findings of the present study, suggests that a complex and possibly multi-functional array of BPs are typically produced by tumour cells.
REFERENCES
BAXTER, R.C. and MARTIN,J.L., Binding proteins for the insulin-like growth factors: structure, regulation and function. Prog. Growth Factor Res., 1 , 4 9 4 8 (1989). BINKERT, C., LANDWEHR, J., MARY,J.L., SCHWANDER, J. and HEINRICH,G., Cloning, sequence analysis and expression of a cDNA encoding a novel insulin-like growth-factor-binding protein (IGFBP2). EMBOJ., 8,2497-2502 (1989). BONI-SCHNETZLER, M., BINZ, K., MARY,J-L., SCHWANDER, J. and FROESCH,E.R., Insulin regulates the expression of the insulin-like growth-factor-binding protein 2 mRNA in rat hepatocytes. Mol. Endocrinol., 4,1320-1326 (1990). BRINKMAN. A,. GROFFEN. C.. KORTLEVE. D.J.. GUERTSVAN KESSEL.A. and DROP,’S.L.S.,Isolation and characterization of a cDNA encoding the low-molecular-weight insulin-like growth-factor-binding protein (IBP-1). EMBOJ., 7,2417-2425 (1988). BROWN,A.L. and RECHLERT, M.M., Cloning of the rat insulin-like growth-factor-binding protein-2 gene and identification of a functional promoter lacking a TATA box. Mol. Endocrinol., 12,2039-2051 (1990). CAMACHO-HUBNER, C., MCCUSKER, R.H. and CLEMMONS, D.R., Secretion and biological actions of insulin-like growth-factor-binding proteins in two human tumor-derived cell lines in vitro. J. cell. PhysioL, 148, 281-289 (1991). CLEMMONS, D., CASCIERI, M.A., CAMACHO-HUBNER, C., MCCUSKER, R.H. and BAYNE,M.L., Discrete alterations of the insulin-like growth factor I molecule which alter its affinity for insulin-like growth-factorbinding proteins result in changes in bioactivity. J. biol. Chem., 265, 12210-12216 (1990). CORPS,A.N. and BROWN,K.D., Mitogens regulate the production of insulin-like growth-factor-binding protein by Swiss 3T3 cells. Endocrinology, 128,1057-1064 (1991). CUBBAGE, M.L., SUWANICHKUL, A. and POWELL,D.R., Insulin-like growth-factor-binding protein-3. Organisation of the human chromosomal gene and demonstration of promoter activity.J. biol. Chem., 265, 12642-12649 (1990). CULOUSCOU, J-M. and SHOYAB, M., Purification of a colon cancer cell growth inhibitor and its identification as an insulin-like growth-factorbinding protein. Cancer Res., 51,2813-2819 (1991). DE LEON,D.D., WILSON,D.M., BAKKER, B., LAMSON, G., HINIz, R.L. and ROSENFELD, R.G., Characterisation of insulin-like growth-factorbinding proteins from human breast cancer cells. Mol. Endocrinol., 3, 567-574 (1989). DROP,S.L.S., KORTLEVE, D.J., GUYDA, H.J. and POSNER,B.I., Immu-
noassay of a somatomedin-binding protein from human amniotic fluid: levels in fetal, neonatal and adult sera. J. din. endocrinol. Metab., 59, 908-9 15 ( 1984). IINO,K., SE.PPALA, M., HEINONEN, P.K., SIPPONEN, P. and RUTANEN, E-M., Elevated levels of a somatomedin-binding protein PP12 in patients with ovarian cancer. Cancer, 58,2294-2297 (1986). KIEFER,M.C., MASIARZ, F.R., BAUER,D.M. and ZAPF,J., Identification and molecular cloning of two new 30-kDa insulin-like erowth-factorbinding proteins isohTed from adult human serum. J. big[.Chem., 266, 9043-9049 (1991). LUI, W.-Y., BRINKMAN, A,, BLAT,C. and HAREL,L., IGFBP-1, an insulin-like growth-factor-binding protein, is a cell-growth inhibitor. Biochem. Biophys. Res. Commun., 174,673-679 (1991). MOHAN,S., BAUTISTA,C.M., WERGEDAL,J. and BAYLINK,D.J., Isolation of an inhibitory insulin-like growth-factor (1GF)-binding protein from bone cell conditioned medium; a potential local regulator of IGF action. Proc. nut. Acad. Sci (Wash.), 86,8338-8342 (1989). PERKEL,V.S., MOHAN,S., BAYLINK, D.J. and LINKART,T.A., An inhibitory insulin-like growth-factor-binding protein (In-IGFBP) from human prostatic cell conditioned medium reveals N-terminal sequence identity with bone-derived In-IGFBP. J. clin. endocrinol. Metab., 71,533-535 (1990). POVOA,G., ISAKSSON, M., JORNVALL, H. and HALL,K., The somatomedin-binding protein isolated from a human hepatoma cell line is identical to the human amniotic fluid somatomedin-binding protein. Biochem. Biophys. Res. Commun., 128,1071-1078 (1985). REEVE,J.G., PAYNE,J.A. and BLEEHEN, N.M., Production of immunoreactive insulin-like growth factor I (IGF-I) and IGF-I-binding proteins by human lung tumours. Brit. J. Cuncer, 61,727-731 (1990). SHIMASAKI, S., GAO,L., SHIMONAKA, M. and LING,N., Isolation and molecular cloning of insulin-like growth-factor-binding protein-6. Mol. Endocrinol., 5,938-948 (1991). SUWANICHKUL, A,, CUBBAGE, M.L. and POWELL, D.R., The promoter of the human gene for insulin-like growth-factor-binding protein-1.1. biol. Chem., 265,21185-21193 (1990). UNTEKMAN, T.G., GLICK,R.P., WAITES,G.T. and BELL,S.C., Production of insulin-like growth-factor-binding proteins by human centralnervous-system tumors. Cancer Res., 51,3030-3036 (1991). WOOD,W.I., CACHIANES, G., HENZEL,W.J., WINSLOW, G.A., SPENCER, S.A., HELLMISS, R., MARTIN,J.L. and BAXTER,R.C., Cloning and expression of the growth-hormone-dependent insulin-like growthfactor-binding protein. Mol. Endocrinol., 2,1176-1185 (1988).
Publicationof the International Union Against Cancer Publication de I'Union lnternationale Contre le Cancer
INSULIN-LIKE GROWTH-FACTOR-BINDING PROTEIN GENE EXPRESSION AND PROTEIN PRODUCTION BY HUMAN TUMOUR CELL LINES Julie G. REEVE',^, Lemuel B. KIRBY',Ad BRINK MAN^, Stephanie A. HUGHES',Jurg sCHWANDER3 and Norman M. BLEEHEN' Medical Research Council, Clinical OncoloRy and Radiotherapeutics Unit, MRC Centre, Hills Road, Cambridge CB2 2Qfl U K 2PediatricEndocrinology, Erasmus University, Rotterdam, The Netherlands; and 3DepartmentInnere Medizin, Kantonsspital, Basel, Switzerland. The secretion of insulin-like growth-factor-bindingproteins (IGFBPs) and expression of the genes encoding IGFBP-I, IGFBP-2 and IGFBP-3 have been studied in a panel of cell lines derived from breast carcinomas, Wilms' tumour, neuroblastoma, retinoblastoma, colon carcinoma, liver adenocarcinoma, Burkitt's lymphoma and a non-small-cell lung carcinoma. All cell lines, with the exception of the Burkitt's lymphoma cell line, secreted IGFBPs, as detected by affinity labelling. A 34-kDa BP was present in the conditioned media of all IGFBP-secretingcell lines, whereas BPs ranging from I 8 kDa to 53 kDa were variably secreted. All IGFBP-secreting cell lines expressed the IGFBP-2 gene as determined by Northern blot analysis. The Wilms' tumour, the neuroblastoma and the retinoblastoma cell line expressed the IGFBP-2 gene only. All other cell lines, with the exception of the Burkitt's lymphoma, expressed the IGFBP-2 gene and, in addition, either the IGFBP-I gene and/or the IGFBP-3 gene. IGFBP- I gene expression could be detected by reverse transcriptase polymerase chain reaction only. IGFBP-3 gene expression was detected by Northern blot analysis, but transcripts were less abundant than IGFBP-2 mRNAs. These findings indicate that the expression of multiple BP genes and the secretion of BPs may be a common property of tumour cells.
o 1992 Wiley-Liss,Inc. Insulin-like growth factors (IGFs) in the plasma and tissue fluids occur bound to specific IGF-binding proteins (Baxter and Martin, 1989). In the circulation, IGF-binding proteins (BPs) prolong the biological half-life of the IGFs and protect against the acute insulin-like actions of these peptides. BPs in the interstitial fluids are thought to regulate the bio-availability of locally secreted IGFs to their target cells, and to modulate the biological effects of the IGFs by altering their interaction with the IGF receptor. To date, 6 classes of binding protein have been characterized by protein and cDNA sequencing studies. These are IGFBP-1, isolated from human amniotic fluid (Baxter and Martin, 1989); IGFBP-2 (Binkert et al., 1989), the human homologue of rat BRL-BP, isolated from human serum (Kiefer et al., 1991); IGFBP-3, also known as BP-53, the glycosylated IGF binding subunit of the 150-kDa complex that carries most of the IGFs in adult human plasma; IGFBP-4 which is secreted by human osteosarcoma and prostatic carcinoma cell lines in vitro (Mohan et al., 1989; Perkel et al., 1990), and is also present in human serum (Kiefer et al., 1991); IGFBP-5, isolated from cerebrospinal fluid and from human serum (Kiefer et al., 1991), and finally IGFBP-6 isolated recently from pig ovarian follicular fluid (Shimasaki et al., 1991). A variety of human cell types have been shown to produce or contain IGFBPs (Baxter and Martin, 1989). These include foetal and adult liver, endometrium, brain, pituitary, and human fibroblast cell lines. In addition, cell lines derived from human hepatomas (Povoa et al., 1985), carcinomas of the colon (Culouscou and Shoyab, 1991), breast (De Leon et al., 1989), and lung (Reeve et al., 1990), and tumours of the central nervous system (Unterman et al., 1991) have been shown to secrete BPs in vitro. Increased IGFBP-1 levels have been detected in patients with ovarian carcinoma (Iino et al., 1986) and our recent studies indicate increased BP serum levels in patients with lung cancer (Reeve et al., 1990).
The production of BPs by human tumour cells is of particular interest, given the acknowledged role of the IGFs as growth factors for certain tumour types and the ability of BPs to either stimulate (Baxter and Martin, 1989; Clemmons et al., 1990) or inhibit (Baxter and Martin, 1989; Lui et al., 1991) IGF-induced DNA synthesis. These observations raise the possibility that IGFBPs may represent a new class of tumour-growthregulatory factors which may promote or inhibit cell division, depending on the BP produced and the tumour type. The present study examines the distribution of IGFBP production in a panel of tumour cell lines and, as a first step toward studying the role(s) played by BPs in different tumours, characterizes BP production by examining the expression of genes encoding IGFBPs. MATERIAL AND METHODS
Cell lines The following cell lines were obtained from the ATCC (Rockville, MD): ZR-75-1, SK-Br3, T47D (breast carcinomas); WiDr (colon carcinoma); SK-HEP-1 (liver adenocarcinoma); SK-NEP-1 (Wilms' tumour); Y-79 (retinoblastoma); SK-NMC (neuroblastoma); EB-1 (Burkitt's lymphoma). LUDLU-1 (squamous-cell lung carcinoma) was kindly donated by Dr. P.H. Rabbitts of this Unit. Detection of IGF-binding proteins by afinity labelling For the production of conditioned media, cells were grown in serum-free RPMI 1640 medium (Gibco, Paisley, UK) for up to 72 hr at a concentration of approximately lo6 cells/ml. Conditioned media were harvested, clarified by centrifugation at 10,OOOgand stored at -70°C in aliquots until use. Conditioned media (10 PI) diluted 1 : l O in 0.5 M sodium phosphate buffer (pH 7.4) were pre-incubated on ice for 30 min in the presence of approximately 250,000 counts per minute of lz5I-IGF-I (Amersham, Aylesbury, UK). Crosslinking of radiolabelled IGF-I to proteins was accomplished by the addition of 5 ~1 of 20 mM disuccinimidyl suberate (DSS) to give a final concentration of 1 mM, followed by incubation at room temperature for 10 min. To confirm the specificity of the cross-linking, the reaction was carried out in the presence or absence of 500 ng of cold IGF-I (Amersham). Samples were prepared for electrophoresis by the addition of 0.0005% bromophenol blue in 0.015 M Tris-HC1 (pH 8.8). Electrophoresis was performed under non-reducing conditions on 12.5% SDS-polyacrylamide gels overnight at room temperature with a constant current of 8 mA. Gels were fixed in 3.5% acetic acid/ 10.5% methanol and autoradiographed. RNA preparation Cells in the logarithmic phase of growth were pelleted by centrifugation at 300 g for 10 min and suspended in their own
4Towhom requests for reprints should be addressed.
Received: January 20,1992 and in revised form March 7,1992.
819
TUMOUR-DERIVED IGF-BINDING PROTEINS
volume. A solution containing 6.0 M guanidine hydrochloride and 0.2 M sodium acetate (pH 5.5) was added to the cells (20 ml per 5 x lo7 cells) and the DNA was sheared by vigorous homogenization in a homogenizer (Virtis, New York, NY). RNA was precipitated by the addition of a half volume of 95% ethanol followed by incubation at -20°C overnight. The pelleted precipitate was dissolved in a solution containing 7.0 M urea, 0.35 M NaCI, 50 mM Tris, pH 7.5, 1 mM EDTA and 0.2% SDS and was extracted once with phenol-chloroform. RNA was precipitated from the aqueous phase using 2 volumes of ethanol, washed with 70% ethanol, air-dried and dissolved in sterile, double-distilled water. Poly(A)+ RNA was prepared from total RNA using a mRNA purification kit (Pharmacia, Milton Keynes, UK). Northern blot analysis Five micrograms of poly(A)+ RNA in 10 mM sodium phosphate buffer (pH 7.0) was denatured in 1.0 M glyoxal for 1 hr at 50°C. The RNA was electrophoresed in a 1.4% agarose gel in 10 mM sodium phosphate buffer and was transferred by Northern blotting to nylon filters. After treatment for 2 min with ultraviolet light, the nylon filters were baked at 80°C for 2 hr before hybridization. Radiolabelled IGFBP-1 (Brinkman et al., 1988), IGFBP-2 (Binkert et al., 1989), IGFBP-3 (Wood et al., 1988) cDNA probes were prepared using an oligolabelling kit (Pharmacia). The IGFBP-3 cDNA probe was generously supplied by Genentech, South San Francisco, CA. The radiolabelled probes were separated from unincorporated nucleotide triphosphates using Sephadex G50 (Pharmacia) and boiled for 3 rnin before use. A mouse p-actin probe (PRT3) (kindly donated by Dr J. Rogers, Laboratory of Molecular Biology, Cambridge, UK) was similarly labelled to confirm equal loading of RNA. The labelled probe, at a concentration of lo6 counts min-’ ml-l, was hybridized to the filter in 1 M NaCl, 0.1 M trisodium citrate (6 x SSC), 5% dextran sulphate, 0.02% Ficoll, 0.02% BSA, 0.02% polyvinyl pyrrolidone, 0.1% SDS and 150 pg ml-1 sonicated salmon sperm DNA at 65°C for 18 hr. The filter was washed with 0.1 X SSC, 0.1% SDS at 65°C to remove unhybridized probe prior to autoradiography. Reverse transcriptasepolymerase chain reaction (RT-PCR) Synthetic oligonucleotides, designed on the basis of the nucleotide sequence of the mRNA encoding IGFBP-1 (Brinkman et al., 1988), were synthesized using an Applied Biosystems 380 DNA synthesizer. The sequences of the primers were:
ing was for 2 min at 5 5 T , polymerization was at 72°C for 3 rnin and denaturation at 95°C for 1 min. Forty microlitres of the reaction mixture were then electrophoresed on 1.4% agarose gels in the presence of ethidium bromide. Amplified products were detected by ultraviolet light transillumination and by autoradiography following Southern blotting and hybridization with IGFBP-1 cDNA. RESULTS
Detection of IGF-BPs in cell-conditioned media The secretion of IGF-BPs by the cell lines studied is shown in Figure 1. It can be seen that, with the exception of the Burkitt’s lymphoma cell line, all cell lines produced one or more BPs which specifically bound IGF-I. IGFBP complexes ranged in molecular size from 25 kDa to 60 kDa, indicating that cell lines secreted BPs with Mrs of 18 kDa to 53 kDa. A 34-kDa BP was present in the conditioned media of all IGFBP-secreting cell lines, whereas BPs ranging from 18 kDa to 53 kDa were variably secreted. IGFBP-I gene expression Although a 1.5-kb IGFBP-1 transcript was detected by Northern blot analysis in normal liver, no evidence of IGFBP-1 gene expression was observed using this method in any of the cell lines studied (data not shown). However, enzymatic amplification from IGFBP-1 mRNA by RT-PCR (Fig. 2) demonstrated the presence of a 437-bp amplification product which hybridized to the IGFBP-1 cDNA probe in 4 of the 10 cell lines studied, including the breast carcinoma lines ZR-75-1
(Bl) 5’..GCTCCCCATGCTGCAGAGGCAGGG..3’ corresponding to nucleotides 386-409 (B2) 5‘..TACATTAAAATACATCTGGCAGTT..3’ complementary to nucleotides 823-800. The 437 bp amplification product is unique to IGFBP-1. Ten micrograms of total RNA were reversed transcribed into first-strand cDNA by addition of 5 pIO.1 M dithiothreitol, 2.5 p15 mM deoxyribonucleoside triphosphate (dNTP) (Pharmacia), 20 pmoles of oligonucleotide B2 and 5 p15 x reverse transcriptase buffer (500 mM Tris, pH 8.3, 60 mM MgClz and 400 mM KCI). After heating for 10 min at 70T, the reaction mixture was cooled to 25°C and 2 units of avian myeloblastosis virus (AMV) reverse transcriptase (Anglian Biotec, Colchester, UK) were added. Following incubation at 42°C for 1 hr, a 5-1.1.1 aliquot of first-strand cDNA was added to 20 pmoles of oligonucleotide primers B1 and B2,5 ~ 1 mM 5 dNTP, and 5 p1 10 x Thermus aquaticus (Taq) polymerase buffer (0.67 mM Tris, pH 8.8, 9.17 M (NH4)z SO4, 0.1 M MgCl2, 0.1 M bromomercaptoethanol, 2 mg ml-l gelatine) in a total volume of 50 pl. Two units of Taq polymerase (Cetus, Emeryville, CA) were added and amplification (35 cycles) was performed using a PHC-1 automated cycler (Techne, Cambridge, UK). Anneal-
FIGURE1 -Detection of IGF-binding proteins in tumour cell conditioned media by affinity-labelling in the absence (-) and presence (+) of IGF-I. Arrows indicate the position of molecular weight markers. A 41-kDa IGFBP complex is present in the conditioned media of all cell lines with the exception of EB-1, indicating that cell lines secreted a 34-kDA BP.
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REEVE E T A L .
FIGURE2 - Southern blot analysis of IGFBP-1 gene expression by tumour cell lines following enzymatic amplification from IGFBP-1 mRNA by polymerase chain reaction and hybridization to radiolabelled IGFBP-1 cDNA probe.
FIGURE4 - Northern blot analysis of IGFBP-3 gene expression in tumour cell lines. DISCUSSION
FIGURE3 - Northern blot analysis of IGFBP-2 gene expression in tumour cell lines.
and T47D, the colon carcinoma cell line WiDr and the hepatoma cell line SK-HEP-1.
IGFBP-2 gene wpression Figure 3 shows that IGFBP-2 gene expression was detected by Northern blot analysis in all cell lines studied, with the exception of the EB-1 Burkitt’s lymphoma cell line. It can be seen that the IGFBP-2 probe hybridized to multiple RNA species in several of the cell lines, with the previously reported 1.4-kb gene transcript being most frequently detected. IGFBP-3 gene expression Northern blot analysis of IGFBP-3 gene expression revealed a single 2.5-kb IGFBP-3 transcript in 4 of the 10 cell lines studied, including the breast carcinoma cell lines SK-Br-3 and T47D, the lung carcinoma cell line LUDLU-1 and the hepatoma cell line SK-HEP-1 (Fig. 4).
Recent research has shown that osteosarcoma (Mohan et al., 1989), prostatic carcinoma (Perkel et al., 1990) and colon carcinoma (Culouscou and Shoyab, 1991) cell lines secrete IGFBP-4, and that proteins immunologically related to IGFBP-1 and IGFBP-3 are secreted by the MDA-231 breast carcinoma cell line (Camacho-Hubner et al., 1991) and by certain central-nervous-system tumour cells (Unterman et al., 1991). The findings of the present study extend these observations and demonstrate that the secretion of IGFBPs is a property common to many human tumour cell lines growing in vitro and that in several cases multiple IGFBP genes are expressed. A major finding of the present study is that expression of the IGFBP-2 gene occurred in all IGFBP-secreting cell lines studied. This observation is of particular interest given the restricted normal tissue distribution of IGFBP-2 gene expression. It is likely that this gene encodes the 34-kDa protein that was detected in the conditioned media of all secreting cell lines. A single 2.5-kb IGFBP-3 transcript was detected by Northern blot analysis in 5 of the 10 cell lines examined and, as previously reported, in adult human liver (Wood et al., 1988). IGFBP-3 gene transcripts were much less abundant than IGFBP-2 mRNA, as evidenced by the much longer exposure times required for detection of the expression of this gene by autoradiography . In contrast to IGFBP-2 and IGFBP-3 gene expression, IGFBP-1 mRNA could not be detected by Northern blot analysis in any of the cell lines studied. However, the presence of an amplification product of the expected size, following RT-PCR, which hybridized to the IGFBP-1 cDNA probe demonstrates that this gene is expressed at relatively low levels in a proportion of cell lines. The apparent differences in the relative levels of IGFBP-1, IGFBP-2 and IGFBP-3 gene expression observed in the various cell lines may indicate differences in gene activation and/or mRNA stability, or expression of the IGFBP-1 and IGFBP-3 genes in a proportion of the cells only. At present, little is known about the regulation of IGFBP genes, although a number of observations suggest that the regulation of IGFBP-3 is different from that of IGFBP-1 and IGFBP-2. Whereas serum levels of IGFBP-1 and IGFBP-2 are highest during foetal life, IGFBP-3 levels are higher post-natally, and peak during puberty (Drop et al., 1984). In contrast to IGFBP-1 and IGFBP-2 levels, which decrease in the presence of IGF-I and insulin (BoniSchnetzler et al., 1990), IGFBP-3 levels increase in the presence of these and other growth factors such as epidermal growth factor, phorbol esters, bombesin, vasopressin, and platelet-derived growth factor (Corps and Brown, 1991). Although recent studies indicate that the hepatic nuclear factor LF-B1 may play a role in the tissue-specific and developmental expression of IGFBP-1 (Suwanichkul et al., 1990), other transcription factors in placenta, endometrium,
TUMOUR-DERIVED IGF-BINDING ovary and additional tissues which express IGFBP-1 have yet to be identified. The IGFBP-3 promoter has been shown to contain binding sites for Sp-1 and AP-2 (Cubbage et al., 1990), which may account in part for the expression of IGFBP-3 in multiple tissues throughout development and for the expression of this gene in a number of the tumour cell lines reported here. Interestingly, a number of potential regulatory sites have been identified in the IGFBP-2 gene nucleotide sequence, including sites for Spl, ETF, AP-I, AP-2, LF-B1, Pan and a TC box (Brown and Rechlert, 1990). The relationship between these observations and the ubiquitous expression of the IGFBP-2 gene in the tumour cell lines reported here remains to be determined. The finding in the present study that all but one of the tumour cell lines growing in vitro secreted BPs is of particular interest, since BPs have been shown to modulate the mitogenic actions of the IGFs (Baxter and Martin, 1989; Lui et al., 1991). The ubiquitous expression of the IGFBP-2 gene in the panel of cell lines studied here suggests that IGFBP-2 is intimately associated with the malignant phenotype, and may indicate that secretion of this protein confers some growth advantage on the tumour cell. Studies are now in progress to evaluate the growth-modifying actions of this protein in IGF-responsivc tumour cell lines. Interestingly, both IGFBP-1 and IGFBP-2 possess amino acid sequences typical of the integrin family of cellular adhesion proteins, suggesting that they may serve dual
PROTEINS
821
functions related both to the regulation of proliferation and to cellular anchoring. The in vitro production of IGFBPs by lung tumour cell lines and the presence of elevated serum levels of these proteins in lung-cancer patients has been reported recently (Reeve et al., 1990). In addition, circulating levels of IGFBP-1 have been detected in patients with ovarian cancer (Iino et al., 1986). Given that all but one of the tumour cell lines in the present study secreted BPs in vitro, it seems likely that BPs may serve as markers for a wide variety of tumours. Indeed, we have recently detected elevated BP levels in patients with rhabdomyosarcoma, melanoma, carcinomas of the colon, rectum, endometrium and breast, and in patients with thymomas. These observations support the contention that IGFBP production is a common feature of tumour cells both in vitro and in vivo where they may contribute to the regulation of tumour growth and metastatic spread. Although the expression of the recently cloned IGFBP-4, IGFBP-5 (Kiefer et al., 1991) and IGFBP-6 (Shimasaki et al., 1991) genes has not been investigated here, a growthinhibitory IGFBP which appears identical to IGFBP-4 has been shown to be secreted by the HT29 colon carcinoma cell line (Culouscou and Shoyab, 1991). This, together with the findings of the present study, suggests that a complex and possibly multi-functional array of BPs are typically produced by tumour cells.
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