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Experimental Therapeutics |
Ludwig Institute for Cancer Research, S-751 24 Uppsala, Sweden [T. S., A. S., K. P. O., A. Ö., C-H. H.]; Department of Molecular Medicine, Clinical Genetics Unit, Karolinska Hospital, S-171 76 Stockholm, Sweden [K. P. O., J. P. D.]; Centre for Human Genetics, University of Leuven, B-3000 Leuven, Belgium [P. D. C.]; and Novartis Pharma AG, Oncology Research, CH-4002 Basel, Switzerland [E. B.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1 (COLIA1) gene to the platelet-derived growth factor B-chain (PDGFB) gene. It has been shown that the resulting COL1A1/PDGF-B fusion protein is processed to mature PDGF-BB. Autocrine PDGF receptor stimulation has therefore been predicted to contribute to DFSP and GCF tumor development and growth. Here we demonstrate presence of activated PDGF receptors in primary cultures derived from six different DFSP and GCF tumors. Three of the primary cultures were further characterized; their in vitro growth displayed an increased sensitivity to treatment with the PDGF receptor tyrosine kinase inhibitor STI571, as compared with normal fibroblasts. Transplantable tumors, displaying a DFSP-like histology, were established from one of the DFSP primary cultures. Treatment of tumor-bearing severe combined immunodeficient mice with STI571 reduced tumor growth. The growth-inhibitory effects in vitro and in vivo occurred predominantly through induction of tumor cell apoptosis. Our study demonstrates growth-inhibitory effects of PDGF receptor antagonists on human DFSP- and GCF-derived tumor cells and demonstrates that autocrine PDGF receptor stimulation provides antiapoptotic signals contributing to the growth of these cells. These findings suggest targeting of PDGF receptors as a novel treatment strategy for DFSP and GCF. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Various types of PDGF and PDGF receptor antagonists have been developed over recent years, and these candidate drugs have allowed investigations of the role of PDGF receptor activation in various pathological situations (7) . Encouraging results have been observed in animal models of restenosis, glomerulonephritis, and lung fibrosis. PDGF antagonists have also been used in cellular and animal models of malignancies associated with autocrine PDGF receptor activation. PDGF receptor-dependent growth has been demonstrated in s.c. and intracranially grown glioblastomas (8 , 9) . In mouse models of chronic myelomonocytic leukemia as well, PDGF antagonists have been used to establish a causal link between activated PDGF receptors and disease development (10) .
STI571 is a selective low-molecular-weight inhibitor of the PDGF receptor tyrosine kinase that displays in vivo activity after oral administration (11) . At the concentrations required for inhibition of the PDGF receptor, STI571 also blocks the cytosolic c-Abl and v-Abl tyrosine kinases, as well as the leukemia-associated Tel-Abl, Tel-PDGFßR, and Bcr-Abl tyrosine kinases (12 , 13) . The efficient blocking of Bcr-Abl by STI571 has led to clinical trials using this compound for treatment of chronic myeloid leukemia (14 , 15) .
A dependency on PDGF receptor signaling in fibroblasts transformed by transfection of DFSP-derived fusion genes has been demonstrated previously (4, 5, 6) . To what extent this requirement for PDGF receptor signaling also occurs in the case of tumor-derived DFSP or GCF cells has not been investigated. The purpose of this study was to investigate whether autocrine PDGF receptor activation contributes to DFSP and GCF growth by assessing the STI571 sensitivity of primary tumor cells derived from DFSP and GCF tumors.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immunoprecipitation and Immunoblotting.
The rabbit antiserum PDGFR-3, recognizing the PDGF ß-receptor, has been described previously (18)
. The goat PDGF ß-receptor antiserum 958 (sc-432) and the monoclonal anti-phosphotyrosine antibody PY99 (sc-7020) were acquired from Santa Cruz Biotechnology, Santa Cruz, CA, and used as recommended by the supplier. Prior to harvest, cells were incubated overnight in the presence or absence of 1 µM STI571 in serum-free DMEM containing 1 mg/ml BSA. Immunoprecipitations of the PDGF ß-receptor and phosphotyrosine and PDGF ß-receptor immunoblotting were performed as described (4)
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In Vitro Growth Characterization.
To study the in vitro growth rate of the three low-passage DFSP cultures, cells were plated in six-well plates (5 x 104 cells/well) in DMEM supplemented with 10 or 1% FCS in the presence or absence of 1 µM STI571. Medium was changed three times/week. After trypsinization, cells were counted using a Coulter particle counter.
TUNEL staining was performed on 105 149333 cells derived from transplanted 149333 tumors and NF control cells, respectively, grown for 48 h on glass coverslips in DMEM with 1 or 10% FCS in the absence or presence of 1 µM STI571. After fixation in 2% buffered PFA, apoptotic cells were labeled with FITC using TUNEL Label (Roche) according to the protocol of the manufacturer. Omission of TdT enzyme served as negative control. After mounting in Fluoromount-G, images were captured at 200-fold magnification in a fluorescence microscope (Zeiss Axioplan 2).
Establishment of Tumor Xenografts from 149333 Cells and Treatment Studies with STI571.
The animal work in this study was approved by the local board of animal experimentation and carried out in accordance with the United Kingdom Coordinating Committee on Cancer Research (UKCCCR) guidelines (19)
. All manipulations were performed in isoflurane gas anesthesia (Forene; Abbott). Upon arrival from the supplier (M&B, Ry, Denmark), the animals were acclimated, caged in groups of five or fewer, and had their backs shaved. To establish tumors from primary DFSP, 3 x 106 low-passage 149333 cells in 50 µl of DMEM were injected s. c. in four 6–8-week-old, athymic BALB/c mice. One of the established tumors was kept as a serial transplant in Fox-Chase SCID mice. The expression of the COL1A1/PDGFB fusion gene was verified by RT-PCR at passages 4, 9, and 17.
STI571 treatment studies were performed in 6–8-week-old, female Fox-Chase SCID mice inoculated s. c. with 5 x 105 149333 cells in 50 µl of DMEM. When tumors reached a volume of 100 mm3, the animals were randomly assigned to receive 200 mg/kg per day STI571 in PBS (n = 7) or vehicle alone (n = 7). STI571 was administered by gavage in doses of 100 mg/kg at 12-h intervals in 200 µl of PBS. Tumor dimensions were measured with calipers, and tumor volume was calculated according to the formula 
, where a represents the shorter and b the longer dimensions of the tumor, respectively. Three h before sacrifice, 100 mg/kg BrdUrd in 0.9% NaCl was administered by i.p. injection. Animals were sacrificed by means of a lethal dose of pentobarbitone and perfused through the left cardiac ventricle with PBS, followed by 4% PFA in PBS (pH 7.4). After excision, tumors were weighed, fixed overnight in 4% PFA, and embedded in paraffin. Sections were cut at 4 µm on Superfrost Plus slides (Histolab, Göteburg, Sweden).
Effects of STI571 on PDGF ß-receptor phosphorylation in vivo were studied in SCID mice bearing s.c. 149333 tumors of 1.5 cm3. Four h before sacrifice, two animals received 100 mg/kg STI571 in PBS by oral gavage, and two animals received PBS alone. Treated and control tumors were excised, weighed, and homogenized in RIPA lysis buffer containing 100 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% deoxycholic acid, 1% Triton X-100, 0.1% SDS, Complete protease inhibitor mixture (Roche), and 100 µM orthovanadate. Lysates were clarified by centrifugation, and an equivalent of 100 mg of tumor tissue was subjected to immunoprecipitation with preimmune serum, followed by PDGF ß-receptor immunoprecipitation and phosphotyrosine and PDGF ß-receptor immunoblotting (4) .
Immunohistochemistry.
For the detection of capillary blood vessels, sections were deparaffinized and pretreated by boiling in 10 mM citrate buffer (pH 6.0) for 2 x 7 min at 750 W in a microwave oven. Tissue peroxidase activity was quenched by incubation in 3% H2O2 in PBS for 10 min, followed by blocking in 1% BSA. Immunohistochemistry was performed with a goat antimouse CD31/PECAM-1 antibody (sc-1506, 1:100; Santa Cruz Biotechnology). Positive reactions were developed using DAB (Vector) as a peroxidase substrate. Immunohistochemical detection of BrdUrd was performed on deparaffinized, citrate-pretreated sections preincubated in 55% formamide/SSC/0.1% Tween 20 for 30 min at 72°C. Staining with a monoclonal mouse anti-BrdUrd antibody (1:50; Becton Dickinson) was done on a NexES immunostainer equipped with DAB substrate kit (Ventana Medical Systems, Tucson, AZ). Omission of primary antibody, in the case of CD31/PECAM-1 staining, or replacement of the primary antibody with an irrelevant mouse IgG, in the case of BrdUrd staining, was used as a negative control. TUNEL staining for detection of apoptotic cells in tumor sections was performed as described (20)
with omission of Proteinase K digestion. Nick-end labeling was carried out using TdT enzyme and digoxigenin-11-dUTP (Roche) as recommended by the manufacturer. Positive nuclei were stained with peroxidase-coupled F(ab) fragments raised against dUTP-digoxigenin (1:500; Roche) and DAB peroxidase substrate.
Sections were counterstained in Mayer’s hematoxylin, dehydrated, and coverslipped in Mountex resin (Histolab). Nuclear stainings (BrdUrd; TUNEL) were quantitated and presented as the percentage of positive nuclei of 2000 counted at x400 magnification under a microscope (VANOX-T; Olympus). Tumor angiogenesis was assessed by stereological quantification (21
, 22)
of CD31+ vascular structures counted at x400 with an eyepiece grid of 10 x 10 squares (0.30 x 0.30 mm). The grid was placed at random in the upper left-hand corner of the section and systematically advanced in steps of 2 mm in both directions. Morphological parameters of 25–50 vision fields were quantified from each tumor. The presence of viable tissue in the uppermost square to the far right of the grid (nvc, number of grids with viable corner) was noted and used as an estimator of the fraction of viable tumor tissue and in the calculation of vascular parameters (defined in Table 2
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. To determine whether expression of COLIA1/PDGF-B fusion transcripts was accompanied by the presence of activated PDGF ß-receptor, we investigated the steady-state level of PDGF ß-receptor tyrosine phosphorylation in the six primary cultures. DFSP, GCF, and normal human fibroblast cells were seeded at equal density; after overnight serum starvation, 3 x 106 cells were lysed, and PDGF ß-receptors were isolated by immunoprecipitation and subjected to phosphotyrosine immunoblotting. All DFSP and GCF cultures displayed tyrosine phosphorylated receptors in contrast to normal human fibroblast cells (Fig. 1A)
. We thus conclude that autocrine PDGF ß-receptor stimulation occurs in all six analyzed primary cultures derived from DFSP and GCF.
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. Three of the DFSP primary cultures and normal human fibroblast cells were, therefore, grown in the absence or presence of 1 µM STI571 in DMEM containing 10% FCS. STI571 reduced the growth rate of DFSP cells and led to a 60–75% reduction in cell number after 20 days of treatment, as compared with untreated cells (Fig. 2B)
. Similar results were also obtained when STI571 was used at a concentration of 0.5 µM (data not shown). STI571 had a smaller effect on the growth of normal fibroblasts, leading to <20% reduction in cell number (Fig. 2B)
. We next investigated the effects of STI571 on the growth of DFSP cells and normal human fibroblast cells in DMEM containing 1% FCS. Treatment under these conditions with 1 µm (Fig. 2C)
or 0.3 µM (data not shown) of STI571 reduced the growth of DFSP cells; in contrast, the growth of normal human fibroblast cells was not affected by STI571. These results indicate that autocrine PDGF ß-receptor stimulation contributes to the in vitro growth of DFSP and GCF cells.
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To investigate the dependence of autocrine PDGF receptor stimulation for growth of the 149333 tumors, a treatment study on serially transplanted 149333 tumors using the PDGF receptor kinase inhibitor STI571 was performed. Peroral treatment with 200 mg/kg per day STI571 reduced 149333 tumor volume to one-third the size of control tumors (Fig. 3A)
. This difference was also reflected in postmortem tumor weight (0.7 ± 0.1 g versus 2.0 ± 0.3 g, mean and SD; P < 0.001). In a similar experiment, peroral administration of 100 mg/kg per day STI571 once daily resulted in a 2-fold reduction of tumor volume at the end of treatment (data not shown). To confirm that STI571 treatment indeed reduced PDGF receptor activation, the tyrosine phosphorylation status of tumor-derived PDGF receptors was examined. PDGF ß-receptors were extracted from tumors 4 h after administration of 100 mg/kg STI571. As shown in Fig. 3B
, treatment with STI571 decreased tyrosine phosphorylation of PDGF ß-receptors in 149333 tumors, as compared with PBS controls, without affecting expression levels or the ratio of mature and precursor form of the PDGF ß-receptor.
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). Tumor cells in S-phase of the cell cycle were labeled by i.p. injection of BrdUrd and visualized by immunostaining with anti-BrdUrd antibodies. Microscopic quantification showed similar levels of tumor cell proliferation in STI571-treated tumors as compared with vehicle control (Fig. 4B)
. Control and STI571-treated tumors were also subjected to TUNEL labeling for detection of tumor cell apoptosis. As shown in Fig. 4B
, treatment with STI571 resulted in a 3-fold increase in tumor cell apoptosis as compared with vehicle control.
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, addition of STI571 to 149333 cells in low serum concentration induced apoptosis. In contrast, apoptosis was not induced in normal fibroblasts by STI571.
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STI571 Treatment Alters the Vascular Morphology of 149333 Tumors and Decreases Tissue Necrosis.
Potential antiangiogenic effects of STI571 were assessed by staining of capillary blood vessels in control and treated 149333 tumors by CD31/PECAM-1 immunohistochemistry and subsequent stereological quantification. The length density, volumetric density, and surface density of blood vessels were not significantly reduced in tumors treated with STI571, as compared with control-treated tumors (Table 2)
. On the contrary, mean vessel area, boundary length, and diameter were decreased by administration of STI571. This decrease is attributable to the relative absence in STI571-treated tumors of dilated, presumably venous, vascular structures present in vehicle-treated tumors (Fig. 4A)
. Notably, necrosis was virtually absent in the STI571-treated tumors. This is reflected by an increase in the fraction of viable tumor tissue by 42% as compared with PBS control. From the characterization of the vascular morphology of treated and control tumors, we conclude that antiangiogenic effects do not contribute to the antitumor effect of STI571 on 149333 tumors.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DFSP is not the only type of soft tissue sarcoma where autocrine PDGF receptor stimulation has been suggested to contribute to disease development. Coexpression of PDGF ligand and receptor has been documented in clinical samples of a variety of fibroblast-derived tumors (23 , 24) . The effects of PDGF antagonists on cell lines derived from these types of tumors have not been characterized.
Our characterization of the effects of STI571 on proliferation and apoptosis in vivo and in vitro indicated that the growth-inhibitory effects in vivo are predominately achieved through induction of apoptosis. Although PDGF stimulation of normal cells is traditionally considered to induce a proliferative response, antiapoptotic effects by PDGF stimulation have also been demonstrated in normal cells (25) . In this context, it is noteworthy that the recently reported growth-inhibitory effect of STI571 on human glioblastoma cells does not appear to occur through induction of apoptosis (9) .
Our characterization of the vascular morphology failed to provide any evidence for primary antiangiogenic effects of STI571 in this tumor model. STI571 treatment of 149333 tumors decreased the mean vessel section area, boundary length, and diameter while not significantly affecting blood vessel density. This difference reflects the relative absence in STI571-treated tumors of large-diameter vessels found in the untreated group. This vascular phenotype is clearly different from what has been observed using angiogenesis inhibitors, where the length density, volume density, and surface density of vessels are decreased (20) . Relieved stasis of tumor blood vessels because of a high rate of tumor cell apoptosis and decreased cell density might explain the effect of STI571. We conclude that the altered vascular morphology of 149333 tumors upon STI571 treatment is attributable to effects on the tumor cells rather than being direct effects on the endothelial compartment itself. Furthermore, the overall tissue viability is increased upon STI571 treatment. To what extent this change in tumor microenvironment by STI571 treatment will lead to a sensitization to the therapeutic effect of chemotherapy administered at the same time constitutes a valid purpose for future studies.
The PDGF receptor antagonist used in this study, STI571, belongs to a growing class of low-molecular-weight compounds that block PDGF receptor signaling by interfering with ATP binding to the receptor tyrosine kinase (7)
. Similar to other tyrosine kinase inhibitors, STI571 does not display absolute selectivity for the PDGF receptors. A recent study characterized in some detail the activity of STI571 against PDGF receptor-related tyrosine kinases (11)
. STI571 was found to block PDGF - and ß-receptor and the structurally related stem cell factor receptor, c-Kit, with similar efficiency. However, the closely related receptor for CSF-1, c-Fms, or the VEGF receptors VEGF-R1 and VEGF-R2 showed much lower sensitivity to STI571. The fact that no major side effects have been observed in the early clinical trials using STI571 in treatment of chronic myeloid leukemia makes STI571 well suited for treatment of DFSP (15)
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In conclusion, our study has provided experimental support for the notion that PDGF receptors are potential drug targets for treatment of DFSP. Obvious ways to build on these observations include investigations of the effects of STI571 on other fibroblast-derived tumors, as well as to investigate whether STI571 will show synergistic effects when coadministered with conventional chemotherapeutic drugs.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 These authors contributed equally to this work. 
2 Present address: Department of Dermatology, Yamanashi Medical University, 1110 Shimokato, Tamaho, Nakakoma, Yamanashi 409-38, Japan.
3 Present address: Department of Dermatology, Research Center M3:02, Karolinska Hospital, S-171 76 Stockholm, Sweden.
4 Present address: Department of Pathology, Brigham and Women’s Hospital, Boston, MA 02115.
5 Present address: Department of Genetics and Pathology, Uppsala University, S-751 85 Uppsala, Sweden.
6 To whom requests for reprints should be addressed, at Ludwig Institute for Cancer Research, Box 595, S-751 24 Uppsala, Sweden. Phone: 46-18-160401; Fax: 46-18-160420; E-mail: c-h.heldin{at}licr.uu.se
7 The abbreviations used are: DFSP, dermatofibrosarcoma protuberans; GCF, giant cell fibroblastoma; COLIA1, collagen type I1; PDGFB, platelet-derived growth factor B-chain; BrdUrd, bromodeoxyuridine; DAB, 3, 3'-diaminobenzidine; PECAM, platelet/endothelial cell adhesion molecule; PFA, paraformaldehyde; RT-PCR, reverse transcription polymerase chain reaction; SCID, severe combined immunodeficient; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; VEGF, vascular endothelial growth factor.
Received 1/19/01. Accepted 6/ 9/01.
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R. F. Hwang, K. Yokoi, C. D. Bucana, R. Tsan, J. J. Killion, D. B. Evans, and I. J. Fidler Inhibition of Platelet-Derived Growth Factor Receptor Phosphorylation by STI571 (Gleevec) Reduces Growth and Metastasis of Human Pancreatic Carcinoma in an Orthotopic Nude Mouse Model Clin. Cancer Res., December 15, 2003; 9(17): 6534 - 6544. [Abstract] [Full Text] [PDF]
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K. Pietras, M. Stumm, M. Hubert, E. Buchdunger, K. Rubin, C.-H. Heldin, P. McSheehy, M. Wartmann, and A. Ostman STI571 Enhances the Therapeutic Index of Epothilone B by a Tumor-selective Increase of Drug Uptake Clin. Cancer Res., September 1, 2003; 9(10): 3779 - 3787. [Abstract] [Full Text] [PDF]
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P. Lindblom, H. Gerhardt, S. Liebner, A. Abramsson, M. Enge, M. Hellstrom, G. Backstrom, S. Fredriksson, U. Landegren, H. C. Nystrom, et al. Endothelial PDGF-B retention is required for proper investment of pericytes in the microvessel wall Genes & Dev., August 1, 2003; 17(15): 1835 - 1840. [Abstract] [Full Text] [PDF]
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C. A. London, A. L. Hannah, R. Zadovoskaya, M. B. Chien, C. Kollias-Baker, M. Rosenberg, S. Downing, G. Post, J. Boucher, N. Shenoy, et al. Phase I Dose-Escalating Study of SU11654, a Small Molecule Receptor Tyrosine Kinase Inhibitor, in Dogs with Spontaneous Malignancies Clin. Cancer Res., July 1, 2003; 9(7): 2755 - 2768. [Abstract] [Full Text] [PDF]
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T. J. Abrams, L. B. Lee, L. J. Murray, N. K. Pryer, and J. M. Cherrington SU11248 Inhibits KIT and Platelet-derived Growth Factor Receptor {beta} in Preclinical Models of Human Small Cell Lung Cancer Mol. Cancer Ther., May 1, 2003; 2(5): 471 - 478. [Abstract] [Full Text] [PDF]
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M. S. Merchant, C.-W. Woo, C. L. Mackall, and C. J. Thiele Potential Use of Imatinib in Ewing's Sarcoma: Evidence for In Vitro and In Vivo Activity J Natl Cancer Inst, November 20, 2002; 94(22): 1673 - 1679. [Abstract] [Full Text] [PDF]
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E. C. McGary, K. Weber, L. Mills, M. Doucet, V. Lewis, D. C. Lev, I. J. Fidler, and M. Bar-Eli Inhibition of Platelet-derived Growth Factor-mediated Proliferation of Osteosarcoma Cells by the Novel Tyrosine Kinase Inhibitor STI571 Clin. Cancer Res., November 1, 2002; 8(11): 3584 - 3591. [Abstract] [Full Text] [PDF]
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K. Pietras, K. Rubin, T. Sjoblom, E. Buchdunger, M. Sjoquist, C.-H. Heldin, and A. Ostman Inhibition of PDGF Receptor Signaling in Tumor Stroma Enhances Antitumor Effect of Chemotherapy Cancer Res., October 1, 2002; 62(19): 5476 - 5484. [Abstract] [Full Text] [PDF]
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S. Attoub, C. Rivat, S. Rodrigues, S. Van Bocxlaer, M. Bedin, E. Bruyneel, C. Louvet, M. Kornprobst, T. Andre, M. Mareel, et al. The c-kit Tyrosine Kinase Inhibitor STI571 for Colorectal Cancer Therapy Cancer Res., September 1, 2002; 62(17): 4879 - 4883. [Abstract] [Full Text] [PDF]
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B. P. Rubin, S. M. Schuetze, J. F. Eary, T. H. Norwood, S. Mirza, E. U. Conrad, and J. D. Bruckner Molecular Targeting of Platelet-Derived Growth Factor B by Imatinib Mesylate in a Patient With Metastatic Dermatofibrosarcoma Protuberans J. Clin. Oncol., September 1, 2002; 20(17): 3586 - 3591. [Abstract] [Full Text] [PDF]
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J. L. Arbiser, B. Govindarajan, X. Bai, H. Onda, A. Kazlauskas, S. D. Lim, M. B. Amin, and L. Claesson-Welsh Functional Tyrosine Kinase Inhibitor Profiling : A Generally Applicable Method Points to a Novel Role of Platelet-Derived Growth Factor Receptor-{beta} in Tuberous Sclerosis Am. J. Pathol., September 1, 2002; 161(3): 781 - 786. [Abstract] [Full Text] [PDF]
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) or presence (
) of 1 µM STI571. At the indicated times, cells were trypsinized, and cell numbers were determined with a Coulter counter. C, normal fibroblasts (NF) and DFSP cells were plated in six-well plates at a concentration of 5 x 104 cells/well in DMEM containing 1% FCS and grown in the absence or presence of 1 µM STI571 for 12 days. Cells were trypsinized, and cell numbers were determined with a Coulter counter. Each experiment was performed four times with similar results.
; n = 7) or PBS (
; n = 7) was started when tumors reached a volume of 100 mm3. After 9 days of treatment, tumors of STI571-treated animals were only one-third the size of control tumors. Data are means; bars, SD. ***, P < 0.001 (Student’s t test). B, lysates from two control (PBS) and two STI571-treated tumors were subjected to consecutive immunoprecipitations (IP) with preimmune serum, followed by PDGF ß-receptor antiserum. After separation in 7% SDS-PAGE and transfer to blotting membranes, the PDGF ß-receptor immunoprecipitations were analyzed by immunoblotting using antibodies against phosphotyrosine (upper panel) and antisera against the PDGF ß-receptor (lower panel). The two bands seen in the lower panel represent the Mr 180,000 mature receptor and Mr 160,000 precursor, respectively. IB, immunoblot; P-Tyr, phosphotyrosine.
