Affiliation of authors: H. Uehara, S. J. Kim, T. Karashima, D. L. Shepherd, D. Fan, R. Tsan, J. J. Killion, I. J. Fidler (Department of Cancer Biology), C. Logothetis, P. Mathew (Department of Genitourinary Medical Oncology), The University of Texas M. D. Anderson Cancer Center, Houston.
Correspondence to: Isaiah J. Fidler, D.V.M., Ph.D., Dept. of Cancer Biology-173, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030 (e-mail: ifidler{at}mdanderson.org).
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ABSTRACT |
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INTRODUCTION |
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To produce a metastasis, tumor cells must complete a series of sequential and highly selective steps whose outcome is determined by homeostatic host mechanisms (79). Preferential metastasis of tumor cells to certain organs is independent of vascular anatomy, rate of blood flow, and the number of tumor cells delivered to each organ (7). Paget (10) proposed that some tissues may provide a better environment than other tissues for the growth of certain tumors and suggested that metastasis occurs when certain tumor cells (the seed) are compatible with a particular organ tissue (the soil). Angiogenesis is a prime example of how the organ microenvironment can contribute to the growth and metastasis of cancer (6,11,12). The onset of angiogenesis involves a change in the local equilibrium between proangiogenic and antiangiogenic molecules. Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), basic fibroblast growth factor (bFGF), interleukin 8 (IL-8), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) are among the major proangiogenic molecules (1218).
The PDGF receptor (PDGF-R), a member of a family of protein tyrosine kinases that includes many oncogenes and proto-oncogenes (1922), is encoded by two genes (PDGF-R and PDGF-R
) (22). PDGF itself is a potent mitogen in both normal and tumor cells (23). PDGF is a dimer that consists of AA, BB, and AB proteins (22). PDGF and PDGF-R are co-expressed in many human carcinomas, including those of the stomach, pancreas, lung, and prostate (24). The binding of PDGF to PDGF-R can stimulate cell division (2527), cell migration (28), and angiogenesis (29). PDGF binding causes PDGF-R activation, which involves dimerization and autophosphorylation (i.e., activation) of specific tyrosines in the cytoplasmic domain of PDGF-R. The phosphotyrosines serve as targets for cytoplasmic effector proteins involved in signal transduction. Activation of PDGF-R has also been shown to inhibit some apoptotic pathways in normal cells and in tumor cells (30,31). Hence, inhibition of PDGF-R activation may decrease cell proliferation and increase the rate of cell death. PDGF and PDGF-R may play critical roles in the osteotropism of human prostate cancer cells and the development of bone metastases (3537).
STI571, a derivative of 2-phenylaminopyrimidine, was originally developed as a competitor for an ATP-binding site of the Abl protein tyrosine kinase (32,33). STI571 is also a potent inhibitor of the tyrosine kinase activities of c-KIT and PDGF-R (38), and it inhibits cell proliferation and induces apoptosis (32,33). We examined the effects of oral STI571 administration on the PDGF-R signaling pathway and the progressive growth of experimental bone metastases in nude mice bearing intratibial injections of human prostate cancer cells. We used STI571 alone and in combination with injected paclitaxel because of their potential additive therapeutic effects.
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MATERIALS AND METHODS |
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Androgen-independent human prostate cancer PC-3MM2 cells (39) were maintained as monolayer cultures in Dulbeccos modified Eagle medium (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS), sodium pyruvate, nonessential amino acids, L-glutamine, a two-fold concentrated vitamin solution (Life Technologies), and penicillinstreptomycin (Life Technologies). Cell cultures were incubated in 5% CO2/95% air at 37 °C. Cultures were free of Mycoplasma and the following murine viruses: reovirus type 3, pneumonia virus, K virus, Theilers encephalitis virus, Sendai virus, minute virus, mouse adenovirus, mouse hepatitis virus, lymphocytic choriomeningitis virus, ectromelia virus, and lactate dehydrogenase virus (assayed by BioWhittaker, Walkersville, MD).
Mice
Male athymic nude mice (NCI-nu) were purchased from the Animal Production Area of the National Cancer Institute-Frederick Cancer Research Facility (Frederick, MD). The mice were housed and maintained in specific pathogen-free conditions. The animal facilities were approved by the American Association for Accreditation of Laboratory Animal Care and met all current regulations and standards of the U.S. Department of Agriculture, U.S. Department of Health and Human Services, and the National Institutes of Health. The mice were used in accordance with institutional guidelines when they were 812 weeks old.
Intratibial Injections of PC-3MM2 Cells
To produce bone tumors, we first harvested PC-3MM2 cells from subconfluent cultures by briefly exposing them to 0.25% trypsin and 0.02% EDTA. Trypsinization was stopped by the addition of medium containing 10% FBS, and the detached cells were collected, washed once in serum-free medium, and resuspended in Ca2+- and Mg2+-free Hanks balanced salt solution. Cell viability was determined by trypan blue dye exclusion; only single-cell suspensions with greater than 95% cell viability were used to produce tumors in the tibias of nude mice. Before intratibial injection, the mice were anesthetized with Nembutal (0.5 mg/g body weight) (Abbott Laboratories, North Chicago, IL). A percutaneous intraosseal injection was made by drilling a 27-gauge needle into the tibia immediately proximal to the tuberositas tibia. After penetration of the cortical bone, the needle was further inserted into the shaft of the tibia and was used to deposit 20 µL of the tumor cell suspension (2 x 105 cells) in the cortex with the use of a calibrated, push button-controlled dispensing device (Hamilton Syringe Co., Reno, NV). To prevent leakage of cell suspensions, a cotton swab was held against the injection site for 1 minute. The animals tolerated this surgical procedure well, and none died during the procedure.
Treatment of Nude Mice Bearing Tumors Derived From Injected PC-3MM2 Cells
STI571 (imatinib mesylate, Gleevec) was provided by Novartis Pharma (Basel, Switzerland). For each oral administration, STI571 was dissolved in distilled water at 6.25 mg/mL. For each intraperitoneal injection, paclitaxel (Taxol; Bristol-Myers Squibb, Princeton, NJ) was diluted in distilled water at 1 mg/mL.
Three days after the intratibial PC-3MM2 cell injections were performed, five mice were killed and the injected bones were examined histologically to identify actively growing cancer cells. The remaining mice were randomly assigned to receive one of the following four treatments (10 mice in each treatment group): 1) a daily oral dose of vehicle solution (water) and one intraperitoneal injection per week of distilled water (control group); 2) one intraperitoneal injection per week of paclitaxel at 8 mg/kg and no oral medication (Taxol group); 3) a daily oral dose of STI571 at 50 mg/kg and no intraperitoneal injections (STI571 group); and 4) a daily oral dose of STI571 at 50 mg/kg and one intraperitoneal injection per week of paclitaxel at 8 mg/kg (STI571 plus Taxol group). The mice were treated for 5 weeks. Tumor size and osteolysis of the injected bone were evaluated by gross observation and by digital radiography as described below. This experiment was repeated once.
Digital Radiography and Harvesting of Bone Tumors and Lymph Nodes
After 34 weeks of treatment, we randomly selected three mice from each treatment group, anesthetized them with Nembutal (0.5 mg/g body weight), placed them in a prone position, and subjected their hind limbs to digital radiography with the use of a Faxitron digital radiography system (Faxitron X-ray Corp., Wheeling, IL) to monitor progression of disease in the bone. After the mice recovered from anesthesia, they resumed their respective treatments. During week 6 of the study (i.e., after 5 weeks of treatment), all mice were killed by injection with Nembutal (1.0 mg/g body weight) and weighed. Digital radiography was carried out on the hind limbs of each mouse, and tumor incidence and size were recorded. The tumor-bearing injected leg and the tumor-free uninjected contralateral leg of each mouse were resected at the head of the femur and weighed. The net tumor weight was calculated by subtracting the weight of the tumor-free leg from that of the tumor-bearing leg. We also harvested macroscopically enlarged lymph nodes and histologically examined them for the presence of metastasis.
Western Blot Analysis of PDGF-R Autophosphorylation in Cells Cultured From Harvested Bone Tumors
PC-3MM2 cells were injected into the tibia of six nude mice. Three mice were treated with STI571 (as described above), and three mice received water (control). After 3 weeks of daily oral treatments, the mice were killed with Nembutal (0.5 mg/g body weight). The tumor-bearing legs of control and STI571-treated mice were rinsed with alcohol and iodine and resected. Tumor tissue confined to the bone was isolated while it was viewed through a dissecting microscope and minced. The tissues were dissociated by incubation with collagenase I at 200 U/mL and DNAse I at 270 U/mL (Sigma Chemical Co., St. Louis, MO) for 2 hours at 37 °C in a shaking water bath as described previously (40). Single-cell suspensions were filtered through sterile gauze, washed three times in serum-free medium, and incubated for 72 hours in tissue culture flasks that contained Dulbeccos modified Eagle medium with 10% FBS. The cells were then washed and cultured for 24 hours in serum-free medium, then incubated in the presence or absence of 1.6 µM STI571 for 60 minutes, followed by an additional 15-minute incubation in the presence or absence of recombinant human PDGF BB at 10 ng/mL (Life Technologies, Rockville, MD). The adherent cells were washed with phosphate-buffered saline (PBS) containing 5 mM EDTA and 1 mM sodium orthovanadate and then scraped into a lysis buffer (20 mM TrisHCl [pH 8.0], 137 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 20 µM leupeptin, and 0.15 U/mL aprotinin) and incubated for 20 minutes on ice. The lysed cells were centrifuged at 16 000g for 15 minutes at 4 °C, and the supernatant was collected. Proteins in the supernatant were quantified by spectrophotometry, and a constant amount of protein was loaded per lane and resolved on 7.5% sodium dodecyl sulfatepolyacrylamide gels and transferred onto 0.45-µm nitrocellulose membranes. The membranes were incubated with 3% bovine serum albumin in Tris-buffered saline (TBS) (20 mM TrisHCl [pH 7.5], 150 mM NaCl) to block nonspecific binding and then probed with either a sheep polyclonal anti-human PDGF-R antibody (1 : 1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) or a mouse monoclonal anti-phosphotyrosine antibody (monoclonal antibody 4G10; 1 : 2000 dilution) (Upstate Biotechnology, Lake Placid, NY) in TweenTBS (TTBS) (0.1% Tween 20 in TBS) and incubated with horseradish peroxidase (HRP)-conjugated donkey anti-sheep immunoglobulin G (IgG) (1 : 2000 dilution; Sigma Chemical Co.) or sheep anti-mouse IgG (1 : 2000 dilution), respectively, in TTBS. Antibody-reactive protein bands were visualized with the use of an enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ).
Preparation of Tissues
Tumors harvested from the tibia and the surrounding muscles were cut into 2- to 3-mm pieces, fixed in 10% buffered formalin for 24 hours at room temperature, washed with PBS for 30 minutes, decalcified by incubation with 15% EDTA (pH 7.4) for 710 days at 4 °C, and embedded in paraffin. We also prepared frozen sections of the harvested tumors according to the method described by Mori et al. (42), with the following modifications. Tumors cut into 2- to 3-mm pieces were fixed in 4% paraformaldehyde containing 0.075 M lysine and 0.01 M sodium periodate (PLP) for 24 hours and then washed with PBS containing 10% sucrose for 4 hours, with PBS containing 15% sucrose for 4 hours, and with PBS containing 20% sucrose for 16 hours. All procedures were carried out at 4 °C. The tissues were then embedded in OCT compound (Miles, Inc., Elkhart, IN), rapidly frozen in liquid nitrogen, and stored at 70 °C.
Immunohistochemistry and Single-Label Immunofluorescence
Paraffin-embedded tissues were sectioned (4- to 6-µm thick) and used to detect expression of PDGF, PDGF-R, activated PDGF-R, VEGF, bFGF, IL-8, and proliferating cell nuclear antigen (PCNA). We used the following primary antibodies for immunohistochemistry and immunofluorescence: rabbit polyclonal anti-VEGF/VPF, anti-FGF-2 (which recognizes bFGF), anti-PDGF A, anti-PDGF B, anti-PDGF-R, and anti-PDGF-R
antibodies (Santa Cruz Biotechnology); goat polyclonal anti-phospho-PDGF-R
(which recognizes activated PDGF-R
) (Santa Cruz Biotechnology); rabbit polyclonal anti-IL-8 (Biosource International, Camarillo, CA); rat monoclonal anti-mouse CD31/plateletendothelial cell adhesion molecule-1 (PECAM-1), which recognizes PECAM-1 (PharMingen, San Diego, CA); mouse monoclonal anti-PCNA (which recognizes PCNA) clone PC-10 (DAKO A/S, Copenhagen, Denmark). We used the following secondary antibodies: HRP-conjugated goat anti-rabbit IgG, HRP-conjugated goat anti-rat IgG, Texas Red-conjugated goat anti-rat IgG, and fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Jackson Research Laboratories, West Grove, PA); HRP-conjugated rat anti-mouse IgG2a (Serotec, Harlan Bioproducts for Science, Inc., Indianapolis, IN); Alexa Fluor 594-conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR); and Biogenex multilink and Biogenex label used for enhancing detection of antibodies (BioGenex, San Ramon, CA). Stable 3,3'-diaminobenzidine (Research Genetics, Huntsville, AL) was used to visualize antibody reactivity, and all sections were counterstained with Gills hematoxylin (Sigma Chemical Co.).
Tissue sections were mounted on positively charged Superfrost slides (Fisher Scientific Co., Houston, TX) and dried overnight. The sections were deparaffinized in xylene, dehydrated in a graded alcohol series (100%, 95%, and 80% ethanol/water [vol/vol]), and rehydrated in PBS (pH 7.5). The sections used to detect PCNA expression were microwaved at 1000W for 5 minutes to improve antigen retrieval. All other paraffin-embedded tissues were treated with pepsin (Biomeda, Foster City, CA) for 15 minutes at 37 °C and then washed with PBS (40,41). PLP-fixed frozen tissues that were used to detect CD31/PECAM-1 expression were sectioned (8- to 10-µm thick), mounted onto positively charged Plus slides (Fisher Scientific Co.), and air-dried for 30 minutes. Frozen sections were fixed in 4 °C acetone for 5 minutes, in 1 : 1 acetone/chloroform (vol/vol) for 5 minutes, and in acetone for 5 minutes and then washed with PBS. Immunohistochemical procedures were performed as described previously (40,41). Nonspecific binding of the anti-phospho-PDGF-R antibody was blocked by incubating sections with 4% fish gel (Cold Water Fish Skin Gelatin, 40% Aurion; Electron Microscopy Sciences, Fort Washington, PA) in PBS. Positive antibody reactions were visualized by incubating the slides with stable 3,3'-diaminobenzidine for 1020 minutes. The sections were rinsed with distilled water, counterstained with Gills hematoxylin for 1 minute, and mounted onto slides with the use of Universal Mount (Research Genetics). Control samples, which were exposed to secondary antibody alone, showed no specific staining. Dilutions of primary antibodies were as follows: IL-8 (1 : 25); PDGF A, PDGF-R
, phosphorylated PDGF-R
, bFGF, PCNA, VEGF (all at 1 : 100); PDGF B (1 : 200); and CD31/PECAM-1 (1 : 400). HRP-conjugated secondary antibodies were used for immunohistochemical detection of the binding of primary antibodies specific for PDGF, PDGF-R, VEGF, bFGF, IL-8, and PCNA. An Alexa Fluor 594-conjugated secondary antibody, at a 1 : 400 dilution, was used to visualize binding of the anti-phospho-PDGF-R
antibody. The sections treated with Alexa Fluor were rinsed with distilled water and mounted with Vectashield (mounting medium with 4',6-diamidino-2-phenylindole [DAPI]; Vector Laboratories, Inc., Burlingame, CA), which produced blue fluorescence in the cell nuclei.
Immunofluorescence Double Staining for CD31/PECAM-1 (Endothelial Cells) and PDGF-R or TUNEL (Apoptotic Cells)
Frozen sections were incubated with a protein-blocking solution (5% normal horse serum and 1% normal goat serum in PBS) for 20 minutes at room temperature and then incubated with a rat monoclonal anti-mouse CD31/PECAM-1 antibody, which recognizes human and mouse PECAM-1 (1 : 400 dilution; PharMingen) for 18 hours at 4 °C. After the samples were rinsed four times with PBS for 3 minutes each, the slides were incubated in the dark with Texas Red-conjugated goat anti-rat antibody (1 : 200 dilution) for 1 hour at room temperature. Samples were then washed twice with PBS containing 0.1% Brij (Fisher Scientific, Pittsburgh, PA) and once with PBS for 5 minutes and then mounted onto slides with the use of Vectashield.
The terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) assay was performed with the use of a commercial apoptosis detection kit (Promega Corp., Madison, WI) with the following modifications. Tissue samples were fixed with 4% paraformaldehyde (methanol-free) for 10 minutes at room temperature, washed twice with PBS for 5 minutes, and then incubated with 0.2% Triton X-100 for 15 minutes at room temperature. The samples were washed twice with PBS for 5 minutes and then incubated with equilibration buffer for 10 minutes at room temperature. The equilibration buffer was drained, and reaction buffer containing equilibration buffer, nucleotide mix, and terminal deoxynucleotidyltransferase was added to the tissue sections, which were then incubated in a dark, humid environment at 37 °C for 1 hour. The reaction was terminated by immersing the samples in 2x SSC (NaCl at 17.5 g/L, citric acid at 8.8 g/L [pH 7.0]) for 15 minutes. The samples were washed three times for 5 minutes each time to remove unincorporated fluorescein-dUTP and then incubated with 300 µg/mL Hoechst stain for 10 minutes at room temperature. Fluorescent bleaching was minimized by treating the slides with an enhancing reagent (Prolong solution, Prolong Antifade Kit; Molecular Probes). Immunofluorescence microscopy was performed with the use of a Zeiss epifluorescence microscope (Carl Zeiss, Thornwood, NY) equipped with a x40 objective and with narrow band-pass excitation filters mounted on a filter wheel (Ludl Electronic Products, Hawthorne, NY). Images were captured with the use of a Sony three-chip camera (Sony Corporation of America, Montvale, NJ) and Optimas Image Analysis software (Bioscan, Edmond, WA) installed on a Compaq computer with a Pentium chip, a frame grabber, an optical disk storage system, and a Sony Mavigraph UP-D7000 digital color printer (Sony, Tokyo, Japan). Images were further processed with the use of Adobe Photoshop software (Adobe Systems, Mountain View, CA). Endothelial cells were identified by red fluorescence, and DNA fragmentation (i.e., TUNEL-positive apoptotic cells) was detected by localized green fluorescence within cell nuclei. We quantified the total number of TUNEL-positive tumor cells by counting 10 randomly selected 0.159-mm2 microscope fields in tumors adjacent to bone and within muscles at x100 magnification. Quantification of apoptotic endothelial cells (yellow fluorescence) was expressed as the average of the ratio of apoptotic endothelial cells to the total number of endothelial cells in 510 random 0.011-mm2 fields at x400 magnification.
Quantification of Microvessel Density and PCNA-Expressing Cells
For the quantification of microvessel density, we captured the images, at x100 magnification, of 10 randomly chosen 0.159-mm2 microscope fields adjacent to the bone and 10 randomly chosen fields in the muscles for each tumor and used those images to count microvessel-like structures consisting of endothelial cells that were stained with the anti-CD31/PECAM-1 antibody, as described previously (43). We counted the number of cells that stained with the anti-PCNA antibody in the same 10 randomly chosen 0.159-mm2 fields at x100 magnification.
Statistical Analysis
The statistical significance of differences between pairs of treatment groups in tumor incidence, tumor weight, incidence of lymph node metastasis, and number of cells positive for PCNA, TUNEL, CD31/PECAM-1, or both CD31 and TUNEL among the treatment groups was compared by using 2 x 2 factorial analysis. The combined data from the STI571 and STI571 + Taxol groups were compared with those from the Taxol and control groups, and data from the Taxol and STI571 + Taxol groups were compared with those from the STI571 and control groups. The KruskalWallis rank sum test was used to determine the statistical significance of differences between two sets of combined experimental groups. For adjustments of unequal variances among the groups, we used the X1/4 transformation (44) for TUNEL-positive cells in bone lesions, the X1/2 transformation for CD31-positive cells in bone lesions, and the X1/1.5 transformation for the percentages of TUNEL-positive cells in bone lesions and PCNA-positive cells in muscle lesions. The log (x) transformation was used to adjust for unequal variances among measurements of the numbers of TUNEL-positive cells in muscle lesions.
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RESULTS |
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Preliminary results from our laboratory determined that PC-3MM2 cells growing in vitro do not express detectable levels of the PDGF-R, whereas cells growing in bones of nude mice do. To determine whether exposure of PC-3MM2 cells to STI571 affects autophosphorylation of the PDGF-R, we established cultures of PC-3MM2 cells from cells that were harvested from bone lesions of control mice or mice treated with STI571. After 4 days in culture, the cells were incubated for 15 minutes in serum-free medium containing PDGF BB and then analyzed for levels of autophosphorylated PDGF-R by western blotting using an antibody specific for the phosphorylated PDGF-R (Fig. 1). Whereas these cells exhibited high levels of phosphorylated PDGF-R, PC-3MM2 cells that were pretreated with STI571 for 1 hour before incubation in serum-free medium that contained PDGF BB displayed lower PDGF-R autophosphorylation. The intensity of the PDGF-R [labeled "PY (4G10)" in Fig. 1
] band for each sample was divided by the intensity of the
-actin band for each sample to yield comparative values (Fig. 1
). After 4 weeks of continuous culture, PC-3MM2 cells treated with PDFG BB did not show high levels of PDGF-R phosphorylation (data not shown). Under the same experimental conditions (4 days), paclitaxel (10 µM) did not affect the level of phosphorylated PDGF-R in PC-3MM2 cells that were harvested from bone lesions (data not shown).
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We next examined the effects of treatment with STI571 alone or in combination with paclitaxel on the growth and spread of human prostate cancer cells implanted into the bone of nude mice. All mice in the four treatment groups were killed on day 3840 (i.e., during the 5th week of treatment), because by that time the mice in the control group (i.e., the group that received only water) had developed large tumors in the legs that were injected with PC-3MM2 cells. The data collected from experiments performed at two different times were very similar and were therefore combined and analyzed together (Table 1). All 20 control mice had large tumors in the tibias and surrounding muscles (median weight of bone tumors = 2.5 g [interquartile range {IQR} = 1.43.3 g]) of the injected leg, and all 20 mice had lymph node metastases. In mice treated with paclitaxel, tumor incidence was 17 of 20, the median weight of the tumors was 1.8 g (IQR = 0.32.5 g), and all 17 of the mice with tumors had lymph node metastases. In mice treated with STI571, tumor incidence was 10 of 20, the median weight of the tumors was 1.8 g (IQR = 0.72.3 g), and all 10 of the mice with tumors had lymph node metastases. In mice treated with the combination of STI571 and paclitaxel, tumor incidence was 7 of 20, the median weight of the tumors was 0.6 g (IQR = 0.11.5 g), and five of the seven mice with tumors had lymph node metastases.
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To determine the extent of osteolysis in the different groups, we performed digital radiography on the hind legs of three mice that were randomly selected from each treatment group in each of the two independent experiments on weeks 3 and 4 of treatment. Mice in the control group and mice treated with paclitaxel alone developed lytic bone lesions as early as week 3 of the study. Mice treated with STI571 alone or with STI571 plus paclitaxel also had bone lesions at the same point in time, but those lesions were smaller than lesions in mice in the control or paclitaxel-only groups and displayed a lesser degree of osteolysis (data not shown). At the termination of the study (i.e., during week 6 of treatment), digital radiography was performed on all mice. Fig. 2 shows representative images of the injected hind legs of mice from the four treatment groups. We observed severe destruction (lysis) of the injected tibias of control mice and mice treated with paclitaxel alone. By contrast, mice that received oral STI571 or STI571 plus paclitaxel had relatively intact tibias (Fig. 2
). Thus, treatment with the combination of STI571 plus paclitaxel was associated with a substantial delay in the development and progression of osteolytic lesions.
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In all 80 mice, PC-3MM2 cells that were implanted in the tibia grew progressively, lysed the bone, and then grew into the surrounding muscles. We collected tumor specimens from the bone and the surrounding muscles of all 80 mice and processed them for histologic and immunohistochemical analysis. Tumors from mice treated with STI571 plus paclitaxel contained prominent necrotic zones. Immunohistochemistry using antibodies specific for PDGF B, PDGF-R, activated PDGF-R
, bFGF, VEGF, and IL-8 demonstrated striking differences in the abundance of these proteins between tumor cells growing in the bone and tumor cells growing in the surrounding muscle (Fig. 3
). Specifically, tumor cells growing adjacent to bone expressed high levels of PDGF, PDGF-R, activated PDGF-R, and IL-8, whereas tumor cells growing in the muscle expressed low levels of these proteins. These regional differences in the expression of these proteins (high in tumor cells growing adjacent to the bone and low in tumor cells growing in the muscle) were observed in all 54 of the mice that bore tumors. Tumor cells adjacent to the bone also expressed higher levels of VEGF and bFGF than tumor cells in the surrounding muscles, although not to the same extent as was observed for the levels of the other proteins. The abundance of PDGF, PDGF-R, bFGF, VEGF, and IL-8 in tumor cells from mice treated with paclitaxel, STI571, or STI571 plus paclitaxel was similar to the abundance of those proteins in tumor cells from control mice. In all mice, only tumor cells that grew adjacent to bone expressed PDGF-R. Immunostaining with antibodies specific for tyrosine-autophosphorylated PDGF-R demonstrated that PDGF-R expressed on the surface of tumor cells adjacent to bone was phosphorylated in control mice and in mice treated with paclitaxel, whereas PDGF-R expressed on the surface of tumor cells in the surrounding muscles was not. By contrast, PDGF-R on the tumor cells adjacent to the bones in mice treated with STI571 or STI571 plus paclitaxel did not react with this antibody, even though PDGF-R was expressed by these cells, suggesting that STI571 inhibits the phosphorylation of PDGF-R, not only in vitro but also in vivo (Fig. 4
).
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The mechanism underlying the smaller tumor size among mice treated with STI571 could reflect a decrease in tumor cell division, an increase in tumor cell apoptosis, or both. We therefore determined the number of tumor cells that expressed PCNA, a marker of cell proliferation, and that had DNA strand breaks characteristic of apoptotic cells (i.e., TUNEL-positive cells) in PC-3MM2-derived tumors from all mice with tumors in the four treatment groups (Fig. 5 and Table 2
).
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The number of apoptotic cells in tumors growing adjacent to bone or in the muscle of mice in the four different treatment groups was determined by the TUNEL assay. The mean numbers of TUNEL-positive cells in tumors growing adjacent to bone in control, paclitaxel-treated, STI571-treated, and paclitaxel plus STI571-treated mice were seven cells (95% CI = 5 to 9 cells), 35 cells (95% CI = 25 to 46 cells), 31 cells (95% CI = 22 to 40 cells), and 75 cells (95% CI = 62 to 88 cells), respectively (Table 2). The mean number of TUNEL-positive cells in tumors growing in the muscle of control, paclitaxel-treated, STI571-treated, and paclitaxel plus STI571-treated mice was eight cells (95% CI = 6 to 9 cells), 31 cells (95% CI = 22 to 40 cells), seven cells (95% CI = 5 to 10 cells), and 35 cells (95% CI = 24 to 45 cells), respectively (Table 2
). The 2 x 2 factorial analysis revealed that mice treated with paclitaxel (alone and in combination with STI571) had statistically significantly more TUNEL-positive cells in bone (P<.001) and muscle (P<.001) lesions compared with control and STI571-treated mice. By contrast, treatment with STI571 (alone and combination with paclitaxel) was associated with statistically significantly more TUNEL-positive cells only in bone lesions (P<.001) compared with control mice or mice treated with only paclitaxel. The combination therapy, STI571 plus paclitaxel, was not associated with synergistic effects in the bone (P = .21) or muscle (P = .47) lesions.
Double Immunofluorescence Staining for CD31/PECAM-1 (Endothelial Cells) and PDGF-R or TUNEL (Apoptotic Cells)
In the next set of experiments, we used double immunofluorescence staining to examine 1) whether tumor-associated endothelial cells express PDGF-R and 2) the effects of the various treatments on the induction of apoptosis in tumor-associated endothelial cells. Endothelial cells within PC-3MM2-derived tumors in bone expressed PDGF-R on their surface, whereas endothelial cells in PC-3MM2-derived tumors in muscle did not (Fig. 6). We base this conclusion on our results from immunofluorescence double-labeling for CD31 (red) and PDGF-R (green), which, when colocalized, stained PDGF-R-expressing endothelial cells in the bone lesions of control and treated mice yellow. Immunofluorescence double-labeling of tumors for CD31 expression (red) and TUNEL positivity (green) revealed colocalization (yellow staining) in endothelial cells within bone lesions of mice treated with STI571 plus paclitaxel (Fig. 6
, A). Only mice treated with paclitaxel alone or with STI571 displayed TUNEL-positive endothelial cells in muscle tumors (Fig. 6
, B). Endothelial cells in uninvolved bones (contralateral leg) of mice treated with STI571 plus paclitaxel did not express PDGF-R on their surface and did not undergo apoptosis in any of the treatment groups (Fig. 6
, A, bottom row).
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Effects of In Vivo Treatments on Microvessel Density
In the last set of experiments, we examined whether the observed increase in apoptosis of endothelial cells in bone lesions of mice treated with paclitaxel, STI571, or STI571 plus paclitaxel was associated with a decrease in vascularization. To do so, we immunostained tumors excised from mice in the various treatment groups with an antibody to CD31 to detect endothelial cells and then captured the images of 10 randomly chosen 0.159-mm2 microscope fields per tumor and used those images to determine the mean number of immunostained microvessel structures per field, which we used as a measure of microvessel density (Table 2). In bone lesions from control mice and mice treated with paclitaxel, STI571, or STI571 plus paclitaxel, microvessel density (number of CD31-positive cells/field) was 56 (95% CI = 42 to 70), 39 (95% CI = 27 to 51), 35 (95% CI = 23 to 46), and 19 (95% CI = 10 to 27), respectively (Table 2
). In muscle lesions from control mice, mice treated with paclitaxel, STI571, or STI571 plus paclitaxel, microvessel density (in units of CD31-positive cells/field) was 54 (95% CI = 43 to 64), 41 (95% CI = 30 to 52), 51 (95% CI = 37 to 66), and 38 (95% CI = 26 to 49), respectively (Table 2
). The 2 x 2 factorial analysis revealed that mice treated with paclitaxel (alone and in combination with STI571) had statistically significantly lower microvessel density in bone lesions (P = .0018) and muscle lesions (P = .019) than control and STI571-treated mice. Mice treated with STI571 (alone and in combination with paclitaxel) had statistically significantly lower microvessel density in bone lesions (P<.001) but not in muscle lesions (P = .92) than mice treated with water or paclitaxel alone. The combination treatment with paclitaxel plus STI571 was not associated with a synergistic effect on microvessel density in either bone (P = .73) or muscle (P = .91) lesions. These results suggest that STI571 induces apoptosis only in endothelial cells that express PDGF-R and, moreover, that STI571 sensitized the tumor-associated endothelial cells to paclitaxel-mediated cytotoxicity.
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DISCUSSION |
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The progressive growth of many human carcinomas, including those of the prostate (4649), ovary (50), lung (51), colon (52), stomach (53), esophagus (54), and breast (55,56), and gliomas (5759), choriocarcinomas (60), melanomas (61), soft tissue tumors (62), and acquired immunodeficiency syndrome-related Kaposis sarcomas (63), has been associated with expression of PDGF-R or the PDGF-R and its ligand (4963). Our results closely agree with those of previous studies that showed that inhibition of PDGF-stimulated tyrosine phosphorylation of PDGF-R with the small molecule N-[4-(trifluoromethyl)phenyl]5-methylisoxazole-4-carboxamide is associated with inhibition of the growth of human tumors in nude mice (34,50).
Our detailed histologic and immunohistochemical analyses revealed that robust expression of PDGF and PDGF-R was restricted to PC-3MM2 cells growing adjacent to the mouse bone. The bone microenvironment has high levels of transforming growth factor-beta (TGF-), which plays an important role in homeostatic processes of the bone, such as resorption and repair (64,65). In response to injury, TGF-
is expressed in bone and regulates expression of other growth factors, such as EGF (66,67), PDGF (68), and their receptors (69). Such associations between the expression of TGF-
and PDGF and PDGF-R could explain our finding that only those PC-3MM2 cells growing adjacent to the bone expressed high levels of PDGF and PDGF-R. It is also possible that PDGF and PDGF-R are preferentially expressed by tumor cells growing near bone tissue because both are constitutive factors in bone marrow; thus, their expression in tumor cells could reflect the responses of those cells to a bone-specific milieu (3436). In this regard, our finding that PDGF-R was expressed on endothelial cells within tumor lesions in the bone but not in the muscle, was striking. Specifically, we found that autophosphorylated PDGF-R was expressed on tumor-associated endothelial cells within the bone lesions but not on tumor-associated endothelial cells in muscle, which were located only 23 mm away from the bone lesions. This differential expression is likely caused by the production of PDGF by tumor cells growing adjacent to the bone but not by those growing at a distance from the bone (Fig. 3
). Because mice treated with STI571 plus paclitaxel had increased apoptosis in receptor-positive tumor cells (Fig. 5
) and endothelial cells (Fig. 6
, A), the expression of activated PDGF-R in tumor-associated endothelial cells, but not in endothelial cells within the uninvolved bone, provides an attractive target for specific antivascular therapy.
The differences in protein expression levels between tumor cells growing adjacent to bone tissue and those growing in the muscle were not limited to PDGF and PDGF-R. We also found that tumor cells adjacent to the bone expressed higher levels of the proangiogenic factors bFGF, IL-8, and VEGF than tumor cells growing in the muscle. These results suggest that angiogenesis in PC-3MM2 cell-derived lesions growing in the musculature could be caused by the presence of sufficient levels of proangiogenic factors other than PDGF, such as bFGF, IL-8, VEGF, IL-1, or TNF- (14,17,18).
Treatment of mice with STI571 plus paclitaxel was associated with a decrease in the incidence and size of tumors in the bone and musculature compared with mice treated with water or paclitaxel alone. We found that mice treated with STI571 plus paclitaxel had statistically significantly fewer dividing tumor cells in bone lesions than mice treated with paclitaxel alone or with water. PDGF directly depolymerizes microtubules during the initiation of DNA synthesis and cell division (70,71). STI571 inhibits PDGF-mediated PDGF-R autophosphorylation and hence stabilizes microtubules in the target cells, a process similar to the mechanism of action of paclitaxel, i.e., lowering the critical concentration of tubulin monomers for polymerization and promoting tubulin assembly into distinct microtubule bundles with stability against depolymerization (72). Thus, combining the two drugs produces additive therapeutic effects.
The progressive growth and metastasis of neoplasms is dependent on the development of vasculature (i.e., angiogenesis) (6,11,12). Endothelial cells can respond to a variety of signals, including those mediated by bFGF, VEGF, EGF, and PDGF (12). Endothelial cell function, proliferation, and survival depend on the expression of specific receptors to these and other factors (1416), and inhibition of the interactions of these factors with their receptors can lead to endothelial cell apoptosis (7376). Destruction of vasculature within neoplasms is known to produce necrosis of actively growing tumors (1116). Hence the induction of apoptosis in tumor-associated endothelial cells (within the bone lesions) produces regression of the tumors.
In summary, we have shown that human prostate cancer cells growing in the bones of nude mice expressed high levels of PDGF and that both tumor cells in the bone and the tumor-associated endothelial cells expressed activated PDGF-R. Systemic treatment with STI571 plus paclitaxel but not with either drug alone was associated with the statistically significant induction of apoptosis in the tumor-associated endothelial cells and in the tumor cells themselves, which was associated with the inhibition of tumor growth and, consequently, the preservation of bone structure. By contrast, endothelial cells in normal bone were not affected by systemic treatment with STI571 plus paclitaxel, presumably because they did not express PDGF-R. The authors of a recent multi-institutional phase II study (35) involving the PDGF-R inhibitor SU101, which was used as a single agent to treat patients with hormone-refractory prostate cancer, recommended further clinical studies with other PDGF-R inhibitors. Our data, however, clearly show that clinically significant therapy of prostate cancer bone metastasis in mice was achieved by the combined administration of STI571 plus paclitaxel. A heterogeneous disease such as prostate cancer requires multimodal therapy, and the translation of these findings to the clinical reality is currently underway.
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H. Uehara and S. J. Kim contributed equally to this work.
Supported in part by Cancer Center Support Core grant CA16672 and Specialized Projects of Oncology Research Excellence (SPORE) in Prostate Cancer grant CA90270 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services.
We thank Walter Pagel for critical editorial review and Lola López for expert assistance with the preparation of this manuscript.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1 Landis SH, Murray T, Bolden Wingo PA. Cancer statistics 1999. Cancer 1999;49:831.
2 Soh S, Kattan MW, Berkman S, Wheeler TM, Scardino PT. Has there been a recent shift in the pathological features and prognosis of patients treated with radical prostatectomy? J Urol 1997;157:22128.[Medline]
3 Barrettoni BA, Carter JR. Mechanisms of cancer metastasis to bone. J Bone Joint Surg Am 1986;68A:30812.[Medline]
4 Jacobs SC. Spread of prostatic cancer to bone. Urology 1983;21:33744.[Medline]
5 Petrylak DP, MacArthur RB, OConnor J, Shelton G, Judge T, Balog J, et al. Phase I trial of docetaxel with estramustine in androgen-independent prostate cancer. J Clin Oncol 1999;17:95867.
6 Smith DC, Esper P, Strawderman M, Redman B, Poenta KJ. Phase II trial of oral estramustine, oral etoposide, and intravenous paclitaxel in hormone-refractory prostate cancer. J Clin Oncol 1999;17:166471.
7 Fidler IJ. Critical factors in the biology of human cancer metastasis: twenty-eighth GHA Clowes Memorial Award Lecture. Cancer Res 1990;50:61308.[Abstract]
8 Fidler IJ. Modulation of the organ microenvironment for the treatment of cancer metastasis. J Natl Cancer Inst 1995;87:158892.[Medline]
9 Fidler IJ, Radinsky R. Genetic control of cancer metastasis. J Natl Cancer Inst 1990;82:1668.[Medline]
10 Paget S. The distribution of secondary growths in cancer of the breast. Lancet 1889;1:5713.
11 Folkman MJ. The role of angiogenesis in tumor growth. Semin Cancer Biol 1992;3:6571.[Medline]
12 Auerbach W, Auerbach R. Angiogenesis inhibition: a review. Pharmacol Ther 1994;63:265311.[CrossRef][Medline]
13 Bouck N, Stellmach V, Hsu SC. How tumors become angiogenic. Adv Cancer Res 1996;69:13574.[Medline]
14 Dvorak HF. Tumors: wounds that do not heal. N Engl J Med 1986;315:16509.[Medline]
15 Fidler IJ. Angiogenic heterogeneity: regulation of neoplastic angiogenesis by the organ microenvironment. J Natl Cancer Inst 2001;93:10401.
16 Folkman J. Seminars in Medicine of the Beth Israel Hospital, Boston. Clinical applications of research on angiogenesis. N Engl J Med 1995;333:175763.
17 Rak J, Filmus J, Kerbel RS. Reciprocal paracrine interactions between tumour cells and endothelial cells: the angiogenesis progression hypothesis. Eur J Cancer 1996;32A:243850.[CrossRef]
18 Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 1996;86:35364.[Medline]
19 Ullrich A, Schlessinger J. Signal transduction by receptors with tyrosine kinase activity. Cell 1990;61:20312.[Medline]
20 Yarden Y, Ullrich A. Growth factor receptor tyrosine kinases. Annu Rev Biochem 1988;57:44378.[CrossRef][Medline]
21 Kaplan DR, Martin-Zanca D, Parada LF. Tyrosine phosphorylation and tyrosine kinase activity of the trk proto-oncogene product induced by NGF. Nature 1991;350:15860.[CrossRef][Medline]
22 Ross P, Raines EW, Bowen-Pope DF. The biology of platelet-derived growth factor. Cell 1986;46:1559.[Medline]
23 Heldin CH, Westermark B. Mechanism of action and in vivo role of platelet-derived growth factor. Physiol Rev 1999;79:12831316.
24 Schiffer CA. Signal transduction inhibition: changing paradigms in cancer care. Semin Oncol 2001;28:349.
25 Xie J, Aszterbaum M, Zhang X, Bonifas JM, Zacchary C, Epstein E, et al. A role of PDGFR-beta in basal cell carcinoma proliferation. Proc Natl Acad Sci U S A 2001;98:92559.
26 Fuma K, Papanicolaou V, Juhlin C, Rastad J, Akerstrom G, Heldin CH, et al. Expression of platelet-derived growth factor alpha-receptors on stromal tissue cells in human carcinoid tumors. Cancer Res 1990;50:74853.[Abstract]
27 Liu YC, Chen SC, Chang C, Leu CM, Hu CP. Platelet-derived growth factor is an autocrine stimulator for the growth and survival of human esophageal carcinoma cell lines. Exp Cell Res 1996;228:20611.[CrossRef][Medline]
28 Bornfeldt KE, Raines EW, Nakano T, Graves LM, Krebs EG, Ross R. Insulin-like growth factor-I and platelet-derived growth factor-BB induce directed migration of human arterial smooth muscle cells via signaling pathways that are distinct from those of proliferation. J Clin Invest 1994;93:126674.[Medline]
29 Plate KH, Breier G, Farrell CL, Risau W. Platelet-derived growth factor receptor-beta is induced during tumor development and upregulated during tumor progression in endothelial cells in human gliomas. Lab Invest 1992;67:52934.[Medline]
30 Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, et al. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 1997;91:23141.[Medline]
31 Zha J, Harada H, Yang E, Jockel J, Korsmeyer SJ. Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 1433 not BCL-X(L). Cell 1996;87:61928.[Medline]
32 Druker BJ, Tamura S, Buchdunger E, Ohno S, Segal GM, Fanning S, et al. Effects of a selective inhibitor of the Ab1 tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med 1996;2:5616.[Medline]
33 McCormick F. New-age drug meets resistance. Nature 2001;412:2812.[CrossRef][Medline]
34 Carroll M, Ohno-Jones S, Tamura S, Buchdunger E, Zimmermann J, Lydon NB, et al. CGP57148, a tyrosine kinase inhibitor, inhibits the growth of cells expressing BCR-ABL, TEL-ABL, and TEL-PDGF-R fusion proteins. Blood 1997;90:494752.
35 Ko YJ, Small EJ, Kabbinavar F, Chachoua A, Taneja S, Reese D, et al. A multi-institutional phase II study of SU101, a platelet-derived growth factor receptor inhibitor, for patients with hormone-refractory prostate cancer. Clin Cancer Res 2001;7:8005.
36 Chott A, Sun Z, Morganstern D, Pan J, Li T, Susani M, et al. Tyrosine kinases expressed in vivo by human prostate cancer bone marrow metastases and loss of the type I insulin-like growth factor receptor. Am J Pathol 1999;155:12719.
37 Chackal-Roy M, Niemeyer C, Moore M, Zetter BR. Stimulation of human prostatic carcinoma cell growth by factors present in human bone marrow. J Clin Invest 1989;84:4350.[Medline]
38 Buchdunger E, Cioffi CL, Law N, Stover D, Ohno-Jones S, Druker BJ, et al. Abl protein-tyrosine kinase inhibitor STI571 inhibits in vitro signal transduction mediated by c-kit and platelet-derived growth factor receptors. J Pharmacol Exp Ther 2000;295:13945.
39 Pettaway CA, Pathak S, Greene G, Ramirez E, Wilson MR, Killion JJ, et al. Selection of highly metastatic variants of different human prostate carcinomas utilizing orthotopic implantation in nude mice. Clin Cancer Res 1996;2:162736.[Abstract]
40 Morikawa K, Walker SM, Nakajima M, Pathak S, Jessup JM, Fidler IJ. Influence of organ environment on the growth, selection, and metastasis of human colon carcinoma cells in nude mice. Cancer Res 1988;48:686371.[Abstract]
41 Solorzano CC, Baker CH, Tsan R, Traxler P, Cohen P, Buchdunger E, et al. Optimization for the blockade of the epidermal growth factor receptor signaling for therapy of human pancreatic carcinoma. Clin Cancer Res 2001;7:256372.
42 Mori S, Sawai T, Teshima T, Kyogoku M. A new decalcifying technique for immunohistochemical studies of calcified tissue, especially applicable to cell surface marker demonstration. J Histochem Cytochem 1988;36:1114.[Abstract]
43 Baker CH, Solorzano CC, Fidler IJ. Blockade of vascular endothelial growth factor receptor signaling for therapy of metastatic human pancreatic cancer. Cancer Res 2002;62:19962003.
44 Neter J, Wasserman W, Kutner MH, editors. Applied linear statistical models, 4th ed. New York (NY): McGraw-Hill; 1996.
45 Fidler IJ, Yano S, Zhang RD, Fujimaki T, Bucana CD. The seed and soil hypothesis: vascularization and brain metastasis. Lancet Oncol 2002;3:537.[CrossRef][Medline]
46 Fudge K, Bostwick DG, Stearns ME. Platelet-derived growth factor A and B chains and the alpha and beta receptors in prostatic intraepithelial neoplasia. Prostate 1996;29:2826.[CrossRef][Medline]
47 Pirtskhalaishvili G, Nelson JB. Endothelium-derived factors as paracrine mediators of prostate cancer progression. Prostate 2000;44:7787.[CrossRef][Medline]
48 Sitaras NM, Sariban E, Bravo M, Pantazis P, Antoniades HN. Constitutive production of platelet-derived growth factor-like proteins by human prostate carcinoma cell lines. Cancer Res 1988;48:19305.[Abstract]
49 Shawver LK, Schwartz DP, Mann E, Chen H, Tsai J, Chu L, et al. Inhibition of platelet-derived growth factor-mediated signal transduction and tumor growth by N-[4-(trifluoromethyl)-phenyl] 5-methylisoxazole-4-carboxamide. Clin Cancer Res 1997;3:116777.[Abstract]
50 Henriksen R, Funa K, Wilander E, Backstrom T, Ridderheim M, Oberg K. Expression and prognostic significance of platelet-derived growth factor and its receptors in epithelial ovarian neoplasms. Cancer Res 1993;53:45504.[Abstract]
51 Antoniades HN, Galanopoulos T, Neville-Golden J, OHara CJ. Malignant epithelial cells in primary human lung carcinomas co-express in vivo platelet-derived growth factor (PDGF) and PDGF receptor mRNAs and their protein products. Proc Natl Acad Sci U S A 1992;89:39426.[Abstract]
52 Lindmark G, Sundberg C, Glimelius B, Phalman L, Rubin K, Gerdin B. Stromal expression of platelet-derived growth factor alpha-receptor and platelet-derived growth factor B-chain in colorectal cancer. Lab Invest 1993;69:6829.[Medline]
53 Chung CK, Antoniades HN. Expression of c-sis/platelet-derived growth factor B, insulin-like growth factor I, and transforming growth factor alpha messenger RNAs and their respective receptor messenger RNAs in primary human gastric carcinomas: in vivo studies with in situ hybridization and immunohistochemistry. Cancer Res 1992;52:34539.[Abstract]
54 Yoshida K, Kuniyashu H, Yasui W, Kitadai Y, Toge T, Tahara E. Expression of growth factors and their receptors in human esophageal carcinomas: regulation of expression by epidermal growth factor and transforming growth factor-alpha. J Cancer Res Clin Oncol 1993;119:4017.[Medline]
55 Seymour L, Dajee D, Bezwoda WR. Tissue platelet-derived growth factor (PDGF) predicts for shortened survival and treatment failure in advanced breast cancer. Breast Cancer Res Treat 1993;26:24752.[Medline]
56 Yi B, Williams PJ, Niewolna M, Wang Y, Yoneda T. Tumor-derived platelet-derived growth factor-BB plays a critical role in osteoclastic bone metastasis in an animal model of human breast cancer. Cancer Res 2002;62:91723.
57 Hermanson M, Funa K, Hartman M, Claesson-Welsh L, Heldin CH, Westermark B, et al. Platelet-derived growth factor and its receptors in human glioma tissue: expression of messenger RNA and protein suggests the presence of autocrine and paracrine loops. Cancer Res 1992;52:32139.[Abstract]
58 Nister M, Claesson-Welsh L, Eriksson A, Heldin CH, Westermark B. Differential expression of platelet-derived growth factor receptors in human malignant glioma cell lines. J Biol Chem 1991;266:1675563.
59 Nister M, Libermann TA, Betsholtz C, Pettersson M, Claesson-Welsh L, Heldin C, et al. Expression of messenger RNAs for platelet-derived growth factor and transforming growth factor-alpha and their receptors in human malignant glioma cell lines. Cancer Res 1988;48:39108.[Abstract]
60 Holmgren L, Flan F, Larsson E, Ohlsson R. Successive activation of the platelet-derived growth factor beta receptor and platelet-derived growth factor B genes correlates with genesis of human choriocarcinoma. Cancer Res 1993;53:292741.[Abstract]
61 Krasagakis K, Garba C, Orfanos CE. Cytokines in human melanoma cells: synthesis, autocrine stimulation and regulatory functionsan overview. Melanoma Res 1993;3:42533.[Medline]
62 Wang J, Coltera D, Gown AM. Cell proliferation in human soft tissue tumors correlate with platelet-derived growth factor B chain expression: an immunohistochemical and in situ hybridization study. Cancer Res 1994;54:5604.[Abstract]
63 Sturzl M, Roth WK, Brockmeyer NH, Zietz C, Speiser B, Hofschneider PH. Expression of platelet-derived growth factor and its receptor in AIDS-related Kaposi sarcoma in vivo suggests paracrine and autocrine mechanisms of tumor maintenance. Proc Natl Acad Sci U S A 1992;89:7046 50.[Abstract]
64 Tashjian AH, Voelkel EF, Lazzaro M, Singer FR, Roberts AB, Derynck R, et al. Alpha and beta human transforming growth factors stimulate prostaglandin production and bone resorption in cultured mouse calvaria. Proc Natl Acad Sci U S A 1985;82:45358.[Abstract]
65 Karsdal MA, Fjording MS, Foged NT, Delaisse JM, Lochter A. Transforming growth factor-beta-induced osteoblast elongation regulates osteoclastic bone resorption through a p38 mitogen-activated protein kinase- and matrix metalloproteinase-dependent pathway. J Biol Chem 2001;276:393508.
66 Boerner P, Resnick RJ, Racker E. Stimulation of glycolysis and amino acid uptake in NRK-49F cells by transforming growth factor-beta and epidermal growth factor. Proc Natl Acad Sci U S A 1985;82:13503.[Abstract]
67 Saha D, Datta PK, Sheng H, Morrow JD, Wada M, Moses HL, et al. Synergistic induction of cyclooxygenase-2 by transforming growth factor-beta1 and epidermal growth factor inhibits apoptosis in epithelial cells. Neoplasia 1999;1:50817.[CrossRef][Medline]
68 Soory M, Virdi H. Implications of minocycline, platelet-derived growth factor, and transforming growth factor-beta on inflammatory repair potential in the periodontium. J Periodontol 1999;70:113643.[Medline]
69 Matsubara H, Moriguchi Y, Mori Y, Masaki H, Tsutsumi Y, Shibasaki Y, et al. Transactivation of EGF receptor induced by angiotensin II regulates fibronectin and TGF-beta gene expression via transcriptional and post-transcriptional mechanisms. Mol Cell Biochem 2000;212:187201.[CrossRef][Medline]
70 Yoon SY, Tefferi A, Li CY. Cellular distribution of platelet-derived growth factor, transforming growth factor-beta, basic fibroblast growth factor, and their receptors in normal bone marrow. Acta Haematol 2000;104:1517.[CrossRef][Medline]
71 Thyberg J. The microtubular cytoskeleton and the initiation of DNA synthesis. Exp Cell Res 1984;155:18.[Medline]
72 Schiff PB, Fant J, Horwitz SB. Promotion of microtubule assembly in vitro by taxol. Nature 1979;277:6657.[Medline]
73 Syridopoulos I, Brogi E, Kearney M, Sullivan AB, Cetrulo C, Isner M, et al. Vascular endothelial growth factor inhibits endothelial cell apoptosis induced by tumor necrosis factor-alpha: balance between growth and death signals. J Mol Cell Cardiol 1997;29:132130.[CrossRef][Medline]
74 Gerber HP, Dixit V, Ferrera N. Vascular endothelial growth factor induces expression of the antiapoptotic proteins Bcl-2 and A1 in vascular endothelial cells. J Biol Chem 1998;273:133136.
75 Nor JE, Christensen J, Mooney DJ, Polverini PJ. Vascular endothelial growth factor (VEGF)-mediated angiogenesis is associated with enhanced endothelial cell survival and induction of Bcl-2 expression. Am J Pathol 1999;154:37584.
76 Watanabe Y, Dvorak HV. Vascular permeability factor/vascular endothelial growth factor inhibits anchorage-disruption-induced apoptosis in microvessel endothelial cells by inducing scaffold formation. Exp Cell Res 1997;233:3409.[CrossRef][Medline]
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