Affiliations of authors: M. Ozen, D. Giri, F. Ropiquet, M. Ittmann, Department of Pathology, Baylor College of Medicine, Houston, TX, and Houston Department of Veterans Affairs Medical Center; A. Mansukhani, Department of Microbiology, New York University School of Medicine, NY.
Correspondence to: Michael Ittmann, M.D., Ph.D., Research Service, Houston Department of Veterans Affairs Medical Center, 2002 Holcombe Blvd., Houston, TX 77030 (e-mail: mittmann{at}bcm.tmc.edu).
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ABSTRACT |
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INTRODUCTION |
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Autocrine expression of FGF6 (3) by prostate cancer cells has been identified in 40% of human prostate cancers in vivo, and the majority of prostate cancers overexpress FGF8 (46). Increased expression of FGFR-1 is present in poorly differentiated human prostate cancers in vivo (7,8). Autocrine expression of FGFs and expression of FGF receptors have been reported in all of the commonly used prostate cancer cell linesi.e., PC-3, DU145, and LNCaP (911). Prostate cancers express appropriate receptors to respond individually to these FGFs (3,7,8,10, 12). However, FGFs have a variety of biologic effects in vivo in different contexts, including enhancement of proliferation, motility, and angiogenesis and inhibition of apoptosis, so that the effect of the observed alterations in FGF signaling in prostate cancer may be complex.
To determine the role of FGF signaling in prostate cancer cells, we disrupted FGF receptor signaling by expression of a dominant-negative (DN) FGFR-1 (DN FGFR-1) protein in human prostate cancer cell lines and assessed the effects on cell viability by measuring cell cycle parameters.
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MATERIALS AND METHODS |
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Construction of recombinant adenoviruses.
A recombinant adenovirus containing FGFR-1 DN cDNA was constructed by homologous recombination as described previously (16). Briefly, the DN FGFR-1 cDNA was cloned into an adenovirus shuttle vector, pAVS6. The pAVS6 FGFR-1 DN plasmid was then cotransfected with pJM17 plasmid, which carries the adenoviral genome, into the 293 cell line. The E1-defective recombinant adenovirus (FGFR DN adenovirus) was produced by homologous recombination between pAvS6-FGFR-1 DN plasmid and pJM17 in 293 cells. The E1 function is supplied by the 293 cells, which contain an integrated copy of the E1 gene, allowing the cells with E1-defective recombinant adenovirus to express the replication-deficient adenovirus. Adenoviral plaques were screened for the presence of FGFR-1 DN sequences with the use of primers 5'-TTCCAGTACTCTTGGATCGG-3' and 5'-AGGTACGATGAGACCCGCACC-3'. A positive plaque was amplified and titered with the use of 293 cells. The control adenoviruses were identical, except that they carried either -galactosidase (LacZ)- or green fluorescent protein (GFP)-coding sequences.
Cell culture and infection of cells. Cell lines were infected with adenovirus in the presence of a minimal volume of infection medium (RPMI-1640 medium supplemented with 2% FBS), incubated for 1 hour at 37 °C in a shaker, and transferred to an incubator (37 °C, 5% CO2) after the addition of RPMI-1640 medium supplemented with 10% FBS. Preliminary experiments were carried out in all three cell lines with variable concentrations of GFP adenoviruses (10104 virus/cell). The presence of bright green fluorescence was assessed after 24 hours by fluorescence microscopy. To achieve greater than 70% infection by this criterion required 50 plaque-forming units (pfu) per cell for LNCaP and 2 x 103 pfu per cell for DU145; these ratios were, therefore, used in subsequent experiments. PC-3 required greater than 104 pfu per cell and thus were not used for subsequent experiments.
Proliferation and colony-formation assays. Cells (5 x 104) were plated in triplicate in 35-mm dishes, allowed to attach to the dish, and infected as described above. Attached cells were then trypsinized and counted with the use of a Coulter counter after 24, 48, and 72 hours for the proliferation assay. For colony-formation assays, 103 cells were infected by incubation with adenovirus in 0.3 mL of infection medium and plated on 10-cm dishes. Cells were incubated at 37 °C and supplied with fresh medium every 3 days. After 12 days, they were fixed with 10% formalin and stained with crystal violet, and the colonies visible to the naked eye were counted.
Cell cycle analysis. Cells (2 x 105) were plated in 100-mm dishes and infected with adenoviral vectors as described above, refed, and incubated in growth medium for 24, 48, and 72 hours. Both floating and attached cells were harvested and stained with propidium iodide for DNA cell cycle analysis following a standard protocol as described elsewhere (17). DNA content was measured by use of a flow cytometer (Epics XL-MCI; Beckman Coulter, Miami, FL), and cell cycle analysis was performed with the use of Multi Cycle for Windows version 3.0 software (Phoenix Flow Systems, San Diego, CA). Cell cycle-blocking experiments were carried out by adding 5 µg/mL of aphidocolin (Sigma Chemical Co., St. Louis, MO) to the culture medium, which leads to arrest at the G1/S checkpoint. After 16 hours, the cells were infected with FGFR DN or GFP adenovirus as described above in the presence of aphidocolin and then maintained for a further 16 hours in the presence of aphidicolin. The medium was removed, and cells were refed with medium without aphidicolin and collected for flow cytometry and determination of cell number at 6, 24, and 48 hours after release. Control cells were maintained in aphidicolin and collected after 48 hours for determination of cell number.
Chromosome preparations and mitotic index. Approximately 80% confluent cell cultures were used for chromosome preparations following standard techniques described previously (18). Briefly, cells were treated with 0.04 µg/mL of Colcemid for 20 minutes, trypsinized, treated with 0.06 M potassium chloride for 20 minutes, fixed in 3 : 1 methanolacetic acid, centrifuged at 1100g for 5 minutes at room temperature, and dropped onto wet slides. Slides were stained with Giemsa, and at least 500 cells from each sample were counted for mitotic index and G2-interphase analysis. G2/S cells were characterized by a visibly enlarged nucleus, while G0/G1 nuclei were of the normal size. Dead cell fragments were identified as having nuclei considerably smaller than normal.
Immunoprecipitation and western blot analysis.
Prostate cancer cells were plated at 1 x 106 cells per 10-cm dish and infected with adenoviruses as described above. The cells were collected at each time point and lysed in lysis buffer (i.e., 20 mM TrisHCl [pH 8.0], 2 mM EDTA, 1 mM dithiothreitol, 0.1% Nonidet P-40, 250 mM NaCl, 2 µg/mL aprotinin, 2 µg/mL leupeptin, 2 µg/mL benzamidine, and 1 mM phenylmethanesulfonyl fluoride) and cleared by centrifugation for 10 minutes in a microcentrifuge at 4 °C at 12 000g. For assessment of FGFR-1 phosphorylation, cells were treated with 10 ng/mL of recombinant FGF2 (R&D Systems, Minneapolis, MN) for 10 minutes before cell lysis. Protein concentration was determined by use of a BioRad protein assay (BioRad Laboratories, Hercules, CA). Polyclonal anti-FGFR-1 antibody (sc-15; Santa Cruz Biotechnology, Santa Cruz, CA) (4 µg of antibody to 800 µg of cell lysate) was added to each sample for 2 hours at 4 °C for each immunoprecipitation. The immune complexes were precipitated by incubation with 25 µL of protein A/G Sepharose (Pierce Chemical Co., Rockford, IL) for 2 hours at 4 °C. The beads were then washed in buffer containing 10 mM HEPES (pH 7.4), 25 mM NaCl, and 1 mM dithiothreitol (DTT). The washed beads were then boiled in sample buffer and centrifuged at 12 000g for 10 minutes at room temperature, and the supernatant was subjected to 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE). The resolved proteins were electrotransferred to nitrocellulose membranes and then blocked with a 5% solution of fat-free milk in phosphate-buffered saline containing 0.5% Tween 20 (PBST). The membrane was then incubated with 500 ng/mL of antiphosphotyrosine antibody (PY20; Transduction Laboratories, Lexington, KY) at 4 °C overnight. The membranes then were washed with PBST and were treated with appropriate secondary antibodies. The antigenantibody reaction was visualized by use of an enhanced chemiluminescence (ECL) assay (Amersham Life Science Inc., Arlington Heights, IL) and exposure to ECL film (Amersham Life Science Inc.). To detect cyclin B1 by western blot analysis, membranes were incubated with 400 ng/mL of mouse monoclonal antibody (sc-245; Santa Cruz Biotechnology) overnight at 4 °C and developed as described above. Control antibody was an anti--actin monoclonal antibody (A531; Sigma Chemical Co.) at a 1 : 5000 dilution. Each lane contained 30 µg of cell lysate protein.
cdc2 kinase assay. One milligram of the cell lysate protein was used for each cdc2 kinase assay. One microgram of mouse monoclonal anti-cdc2 antibody (sc-54; Santa Cruz Biotechnology) was added to each sample overnight at 4 °C in a rotator. The immune complexes were precipitated by incubation with 25 µL of protein A/G Sepharose for 1 hour at 4 °C. Sepharose beads were collected by centrifugation at 12 000g for 10 minutes at 4 °C and washed four times in wash buffer containing 20 mM HEPES, 25 mM NaCl, and 1 mM DTT. The pellet was resuspended in protein kinase assay buffer (i.e., 50 mM TrisHCl [pH 7.5], 10 mM MgCl2, and 1 mM DTT). Two micrograms of histone H1 protein (Roche Diagnostics, Indianapolis, IN) and 1 µL of premix containing 3.3 µCi of [32P]adenosine triphosphate (ATP) (4500 Ci/mmol) in protein kinase assay buffer containing 1 mM ATP were added to each sample, which was then incubated at 30 °C for 30 minutes. Each sample was boiled in sample buffer and centrifuged for 30 seconds at 12 000g at room temperature, and supernatants were analyzed by SDSPAGE in a 12% gel. The dried gel was then subjected to autoradiography.
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RESULTS |
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For the determination of the biologic effect of FGF receptor expression in human prostate cancer, DN constructs of FGFR-1 and FGFR-2 were transfected into the three commonly used prostate cancer cell lines (LNCaP, DU145, and PC3) and cells were selected in hygromycin. Both DN constructs consist of the extracellular and transmembrane domains of the corresponding FGF receptor, with a truncation leading to loss of the kinase domain. As described previously, both of these constructs function as DN inhibitors of FGF receptor signaling (13). The DN receptors were both cloned in the pCEP4 vector, which contains a hygromycin resistance gene. Shown in Fig. 1, after selection in hygromycin, only rare colonies were present in any of the three cell lines transfected with either the FGFR-1 or the FGFR-2 DN receptor, whereas numerous colonies were present in control cells transfected with pCEP4 vector control. Inhibition of colony formation by transfection of DN FGF receptor constructs was greater than 99%. To confirm that the effect seen was the result of expression of the DN FGF receptor and not a disruption of expression of the hygromycin resistance gene, we cotransfected the cell lines with the DN receptor constructs and a 2 : 1 excess of plasmid containing a neomycin resistance gene (pSV2Neo). Transfected cells were then selected in G418. Cells transfected with the DN FGF receptors had a 75%90% reduction in colonies compared with cells transfected with pSV2 Neo and control pCEP4 (data not shown), confirming that the effect was due to the DN receptor expression.
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For the examination of the effect of the DN FGFR-1 on cell cycle progression, LNCaP and DU145 cells were infected with FGFR DN adenovirus or control LacZ virus and flow cytometry was performed after propidium iodide staining of cells collected 24, 48, or 72 hours after infection. Compared with cells infected with control LacZ virus, both cell types showed a marked increase in the percentage of cells in G2/M by 24 hours after infection accompanied by a decrease in the fraction of cells in G1/G0 (Fig. 4). At 48 hours after infection, there was a continued increase in the percentage of G2/M cells accompanied by accumulation of dead-cell debris. By 72 hours, there was a marked accumulation of dead-cell fragments in the DN FGFR-1-infected cultures of both cell types (data not shown).
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The key regulator of the G2 to M transition is the accumulation of cyclin Bcdc2 kinase complexes that are then activated by dephosphorylation by cdc25 during the G2/M transition (19). We, therefore, evaluated the effect of FGFR-1 DN receptor expression on these key regulatory molecules. Infection of LNCaP cells with FGFR DN-expressing adenovirus led to an accumulation of cyclin B1 by as early as 6 hours after infection (Fig. 7, A). However, as seen in Fig. 7
, B, there was a marked decrease in the cdc2 kinase activity (as measured by in vitro kinase assay with histone H1 as a substrate) in cells infected with FGFR DN virus when compared with lacZ-infected controls. Thus, although cyclin B1 accumulates in FGFR DN-infected cells, there appears to be a failure to accumulate active cyclin Bcdc2 complexes.
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DISCUSSION |
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Many aspects of the underlying mechanisms by which the DN FGF receptor leads to G2 arrest have yet to be elucidated. We have demonstrated that the DN FGFR-1 receptor decreases FGFR-1 activity. However, there are four types of FGF receptors, and all three prostate cancer cell lines express multiple types of FGF receptor. Transphosphorylation between different forms of FGF receptors has been demonstrated (22), so that it is likely that the DN FGFR-1 receptor also inhibits the activity of these other receptors. The observation that stable transfection of the DN FGFR-2 construct also led to a profound inhibition of colony formation is consistent with this idea. However, proof of this hypothesis is difficult, given the complex interactions among multiple FGF receptors expressed at variable levels in different cell lines and the variable affinities of currently available antibodies for each receptor. It is also possible that the DN FGF receptors might interact with proteins other than FGF receptors and that some of the observed phenotypes may be the result of such interactions.
The mechanism by which G2 arrest is induced by FGFR-1 DN receptor expression is unclear. We have demonstrated a decrease in cdc2 kinase activity in FGFR-1 DN-expressing cells, and since cdc2 kinase activity is essential for the G2/M transition (19), this decreased activity is assumed to be critical in the observed G2 arrest. The exact mechanism by which cdc2 kinase activity is decreased is not known, but it is not the result of decreased expression of cyclin B1, because this critical regulator of cdc2 is increased in treated cells. Further investigations of the multiple factors controlling cdc2 activity are necessary to determine the mechanism leading to G2 arrest.
We have found that disruption of FGF signaling is lethal for all three prostate cancer cell lines tested. By contrast, infection with similar amounts of FGFR DN adenovirus had only a slight effect on primary prostatic epithelial cells (data not shown). This inability of the FGFR DN adenovirus to induce G2 arrest and cell death could be a result of differences between primary cells and cancer cell lines in the levels of expression of the different FGF receptor types or could reflect a fundamental difference in the dependence of the cancer cells on autocrine FGF stimulation. However, not all of the cell lines are dependent on FGF receptor activity, because clones expressing FGFR DN receptors were easily established in NIH3T3 cells with the use of these same vectors (13). A pancreatic cancer cell line expressing an FGFR-1 DN construct has been established (23), and Yayon et al. (24) were able to establish a similar melanoma cell line. However, using four other melanoma cell lines, Yayon et al. (24) were not able to establish cell lines expressing the DN construct, implying that there may be a strong selection against expressing the FGFR-1 DN construct in these cells. Given the evidence that autocrine stimulation by FGFs may play an important role in melanoma (25), we are currently investigating the effect of FGFR-1 DN adenovirus infection in melanoma cell lines. The effect of FGF DN receptors in other malignancies in which FGF receptor activation is thought to play a role will also need to be evaluated.
In summary, there is abundant evidence of increased FGF receptor signaling in prostate cancer based on analysis of animal models, human tissues, and human prostate cancer cell lines. In addition to the known effects of FGFs on cell proliferation, motility, and angiogenesis, which may all promote prostate cancer progression, we now have evidence that FGFs may act as essential survival factors as well. Small molecule inhibitors of FGF receptor signaling are already undergoing phase I clinical trials as cancer therapy (26), and our data support the potential for these agents as treatments of prostate cancer. Our finding that loss of FGF signaling leads to G2 arrest suggests that FGF-receptor signaling antagonists might act synergistically with other cancer therapies leading to G2 arrest, such as radiotherapy or treatment with alkylating agents. Indeed, it has been shown that FGF2 inhibits radiation-induced apoptosis in some contexts (27) so that blocking the FGF signal transduction pathway may enhance radiation-induced cell death. Further work is needed to evaluate these therapeutic possibilities and to understand the mechanism by which loss of FGF receptor signaling leads to G2 arrest and cell death in prostate cancer.
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NOTES |
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We would like to thank the following people from the Baylor College of Medicine: Karen Schmidt for skilled technical assistance, Drs. JoAnn Trial and Terry Timme for flow cytometry and data analysis assistance, and Dr. Marco Marcelli for advice on preparation of adenovirus constructs. We also thank Dr. Sen Pathak of The University of Texas M. D. Anderson Cancer Center for helpful discussion regarding the cytogenetic data.
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Manuscript received February 9, 2001; revised September 13, 2001; accepted October 3, 2001.
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