Introduction

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Chemotherapeutic agents can elicit a number of cellular responses including growth arrest and activation of apoptosis, or programmed cell death. Apoptosis is initiated through a complex signal transduction network that is only partially understood. Many cellular factors can alter the response of the cell to chemotherapeutic agents by modulating this response-signaling pathway. Signals from the extracellular environment may also be important in regulating an apoptotic response, as certain growth factors and cytokines can down-regulate the apoptotic response to chemotherapy drugs and decrease the sensitivity of cells to these agents (Borsellino et al., 1995; Grothey et al., 1999).

Basic fibroblast growth factor (bFGF/FGF-2) belongs to a family of pleiotropic cytokines that function in the normal physiology and pathology of many tissues (Szebenyi and Fallon, 1999). bFGF is also a potent angiogenic factor in that it acts as both a mitogen and chemoattractant for endothelial cells (Gospodarowicz et al., 1979). Extracellular bFGF binds to high-affinity and low-affinity sites on the cell surface. A family of four receptor-tyrosine kinases comprise the high-affinity binding sites for bFGF as well as other members of the FGF family (Johnson and Williams, 1993). Each of the four receptors has multiple splice variants, which have different extracellular domains and ligand binding specificities. bFGF also binds to heparan sulfate-containing proteoglycans with lower affinity, and interaction with these sites is thought to be necessary for binding and activation of the high-affinity receptors (Spivak-Kroizman et al., 1994). Binding of bFGF to high-affinity receptors leads to intracellular propagation of a mitogenic signal through activation of phospholipase C and the ras-raf-MAP kinase pathway (Mohammadi et al., 1991; Kouhara et al., 1997). For neural cells and smooth muscle cells, bFGF is a survival factor and it is necessary for the maintenance of these cells in culture. bFGF mediates this effect by inhibiting apoptosis (Fox and Shanley, 1996; Ohgoh et al., 1998).

Because of its ability to act as a survival factor in some differentiated cells, bFGF recently has been investigated as a factor that might rescue cells from apoptosis induced by chemotherapy drugs and DNA-damaging agents. Elevated levels of intracellular bFGF correlate with resistance to fludarabine in chronic lymphocytic leukemia (Menzel et al., 1996). Furthermore, overexpression of a bFGF cDNA in immortal mouse embryo fibroblasts (NIH 3T3) can result in resistance to a variety of agents, including etoposide, 5-fluorouracil, and N-(phosphonacetyl)-L-aspartate (Huang et al., 1994; Wieder et al., 1997). Expression in NIH 3T3 cells of a chimeric bFGF containing a signal peptide for secretion results in constitutive activation of FGF receptors through an autocrine loop and blocks apoptosis induced by treatment with cisplatin (Shaulian et al., 1997). Overexpression of bFGF is also associated with resistance to cisplatin in a human bladder cancer cell line (Miyake et al., 1998). Moreover, the addition of exogenous bFGF to endothelial cells inhibits apoptosis induced by DNA damage from ionizing radiation, both in vitro and in mice (Fuks et al., 1994). On the other hand, recent evidence suggests that both overexpressed and exogenous bFGF can enhance apoptosis in MCF7 breast tumor cells exposed to cisplatin, etoposide, or 5-fluorouracil (Wang et al., 1998; Fenig et al., 1999).

To understand how bFGF might affect the response to chemotherapeutic agents, it is necessary to elucidate the signal transduction pathways involved. We have begun this analysis in NIH 3T3 cells by studying the effect of exogenous bFGF on induction of apoptosis by cisplatin, a DNA cross-linking agent used in the treatment of many cancers. The cellular factors that render cells more sensitive or resistant to cisplatin are not entirely known, but probably include p53 status (Hawkins et al., 1996) and nucleotide excision repair machinery (Fan et al., 1995), and might also include survival factors such as bFGF (Shaulian et al., 1997). However, we found that cells pretreated with bFGF were more sensitive to cisplatin-induced apoptosis compared with cells not treated with bFGF. This effect was time-dependent and reversible, and required higher concentrations of bFGF than those needed to stimulate DNA synthesis in NIH 3T3 cells. Finally, the effect of bFGF on apoptosis was not reversed by suramin, a relatively nonspecific inhibitor of FGF receptor-mediated signaling. Therefore, bFGF appeared to enhance cisplatin-induced apoptosis in NIH 3T3 cells by a mechanism that might not be mediated by classical high-affinity FGF receptor pathways.

	Materials and Methods

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Cell Culture and Cell Proliferation Assays. NIH 3T3 cells were obtained from Dr. M. Gottesman (National Cancer Institute, Bethesda, MD) and were routinely carried in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum (CS), 100 units/ml penicillin, and 100 µg/ml streptomycin.

Apoptosis Assays. Cisplatin (Sigma) stock solutions were prepared in saline at 1 mg/ml and were stored at room temperature protected from light. Cells (6 × 10⁵) were seeded in 10-cm plates and allowed to attach overnight. The next day, the cells were refed with standard growth medium containing bFGF or PBS carrier only and incubated for 24 h. The cells then were exposed to 10 µg/ml cisplatin in standard growth medium (±bFGF) for 12 h, after which the cisplatin medium was removed and replaced with fresh medium without cisplatin, maintaining the presence or absence of bFGF. When suramin was used, it was added simultaneously with bFGF and was maintained whenever bFGF was present. Cells were harvested either 18 or 24 h after the end of the cisplatin treatment. Floating and adherent cells were collected from duplicate plates for each treatment (adherent cells were trypsinized) and pelleted together. The cell pellet was resuspended in 0.5 ml of growth medium and added dropwise to 5 ml of cold 1% paraformaldehyde and incubated for 15 min on ice. The cells then were washed once with PBS, resuspended in 5 ml of 70% ethanol, and stored at 20°C for 3 to 5 days before TUNEL (terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling) analysis. A single plate of control cells not treated with cisplatin was run in parallel with cisplatin-treated cells in every experiment and was harvested on the second day after plating, before the cells became confluent.

Colony Formation. A total of 1 × 10⁵ cells were seeded in 10-cm plates in standard growth medium and were allowed to attach overnight. The cells were pretreated with 10 ng/ml bFGF or PBS carrier only for 24 h and then were exposed to different concentrations of cisplatin (±bFGF) for 12 h. The cisplatin was washed away and the cells from each treatment were trypsinized and replated into triplicate 6-cm plates at 450 cells per plate. The plates were incubated for 7 days in the maintained presence or absence of bFGF and then were stained with 0.5% methylene blue in 50% ethanol. Prism Graph Pad software (version 2.0) was used to determine IC₅₀ values.

	Results

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Pretreatment with bFGF Enhances Cisplatin-Induced Apoptosis. We investigated whether bFGF might affect cisplatin-induced apoptosis in NIH 3T3 cells. Cells were incubated for 24 h in the presence or absence of bFGF, then treated with 10 µg/ml cisplatin for 12 h, and finally refed with growth medium (±bFGF) lacking cisplatin for 24 h. Floating and attached cells were harvested and analyzed by flow cytometry for DNA content by PI staining and for DNA fragmentation by TUNEL assay. Apoptosis is a rapid process, and cells may quickly progress from a normal DNA content that stains TUNEL-positive to degraded DNA of sub-G₁ content. Therefore, the total percentage of apoptotic cells was quantified as the percentage of cells with sub-G₁ DNA content plus the percentage of TUNEL-positive cells in the G₁-G₂/M range (Table 1).

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Cisplatin-induced apoptosis in NIH 3T3 cells is enhanced by pretreatment with bFGF. The amount of apoptosis was measured by TUNEL assay in cells not exposed to cisplatin or bFGF (A), or in cells pretreated for 24 h with carrier only (B) or 10 ng/ml bFGF (C) and subsequently exposed to 10 µg/ml cisplatin for 12 h. Cisplatin-treated cells were harvested and fixed at 24 h post-treatment. TUNEL positivity is shown on the y-axis, and PI staining on the x-axis. For quantitation purposes, cells in the R3 (upper right) region were taken as TUNEL positive and cells in the R4 (left) region were taken as having sub-G1 DNA content. Shown below are the PI histograms illustrating the cell cycle distribution in each sample.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Dose response and reversibility of the enhancement of cisplatin-induced apoptosis by bFGF. NIH 3T3 cells were pretreated as indicated and then exposed to 10 µg/ml cisplatin for 12 h, and analyzed for apoptosis as in Fig. 1. A, cells were pretreated with increasing concentrations of bFGF for 24 h before exposure to cisplatin. In this experiment, the cells were harvested 18 h after cisplatin exposure. The bars represent the total amount of apoptosis (TUNEL-positive plus sub-G1) detected with each treatment. The percentages for each population are given below the bars. B, cells were pretreated with or without 10 ng/ml bFGF for 48 h, or were pretreated with 10 ng/ml bFGF for 24 h and then rinsed with PBS and incubated without bFGF for another 24 h. Apoptosis was determined as in A.

Pretreatment with bFGF Sensitizes NIH 3T3 Cells to the Cytotoxic Effects of Cisplatin. The dose of cisplatin used to measure the effect of bFGF on apoptosis was lethal for both bFGF-treated and untreated cells. We used this high dose to assess changes in the apoptotic response to a high level of DNA damage. To determine whether bFGF also enhanced cytotoxicity to lower concentrations of cisplatin, we performed a colony-forming assay on NIH 3T3 cells after exposure to varying cisplatin concentrations, with or without prior incubation in bFGF. As shown in Fig. 3, pretreatment with bFGF for 24 h resulted in increased sensitivity to cisplatin. The IC₅₀ for a 12-h exposure to cisplatin was 0.1 µg/ml in bFGF-treated cells, and 0.3 µg/ml in cells not treated with bFGF. This assay was also performed twice using a 1-h exposure to higher doses of cisplatin; in these experiments, pretreatment with bFGF led to a 2.1- and 2.6-fold decrease in the IC₅₀ of cisplatin, relative to cells not treated with bFGF (data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Dose response to cisplatin. Subconfluent plates of cells were pretreated with or without 10 ng/ml bFGF for 24 h, and then were exposed to various concentrations of cisplatin for 12 h. The cells then were replated at low density and allowed to form colonies in the continued presence or absence of bFGF. Data points represent the average number of colonies formed from triplicate plates, expressed as the percent survival relative to 100% in the absence of cisplatin (with and without bFGF). Error bars indicate S.E.M.

bFGF at 10 ng/ml Induces Specific Cellular Changes Not Associated with Lower Concentrations of bFGF. Previous studies have shown that bFGF can alter the morphology of certain cell types ( Kato and Gospodarowicz, 1985; Kalman et al., 1999). We also noticed that NIH 3T3 cells acquired a distinct morphology after a 24-h incubation in the presence of bFGF, before cisplatin exposure. Analysis by light microscopy revealed that bFGF-treated cells tended to be smaller and rounder than the typical flat morphology of NIH 3T3 cells (Fig. 4). bFGF-treated cells also had an increased number of highly refractile cells and produced more dendritic processes than untreated cells. Induction of the altered morphology occurred with the same dose response to bFGF as the enhancement of cisplatin-induced apoptosis (Table 2). Very little change in morphology relative to untreated cells was observed at 1 ng/ml bFGF, whereas the change saturated at 10 ng/ml. These changes in cellular morphology were also reversible within 24 h, following the same trend as the enhancement of cisplatin-induced apoptosis (data not shown).

View larger version (133K): [in this window] [in a new window]   Fig. 4.   Morphology of NIH 3T3 cells treated with 10 ng/ml bFGF. Cells (3 × 105) were seeded in 10-cm plates and allowed to attach overnight before treating without (A) or with (B) bFGF for 24 h. Photographs were taken by using phase contrast microscopy at an original magnification of 100×. The open arrow indicates a cell that is considered to be refractile, and the solid arrows indicate dendritic processes.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Cell proliferation and 3H-thymidine incorporation in response to bFGF treatment. A, quiescent NIH 3T3 cells were treated with increasing concentrations of bFGF, or were placed back into 10% CS, and 3H-thymidine incorporation was measured as described in Materials and Methods. Bars represent the average amount of radioactivity retained on filters from triplicate wells, ± S.E.M., expressed as cpm. B, 1 × 104 NIH 3T3 cells were plated in DMEM + 10% CS, treated with 0, 1, or 10 ng/ml bFGF, and then collected and counted over the course of 4 days. Values represent the average number of cells from triplicate plates ± S.E.M. C, quiescent NIH 3T3 cells were treated with 0.5 ng/ml bFGF, with or without 100 µM suramin (sur). Control cells received neither bFGF nor suramin. 3H-thymidine incorporation was measured as in A. **P < .01 versus 0.5 ng/ml bFGF as determined by two-tailed t test.

Suramin Does Not Inhibit the Effect of bFGF on Apoptosis or Morphology. The disparity between the concentrations of bFGF required for mitogenesis and for enhancement of apoptosis suggested that these two effects may be initiated by different signaling pathways. Mitogenic signaling occurs through the well characterized high-affinity FGF receptors (Johnson and Williams, 1993). To establish whether activation of the high-affinity FGF receptors was necessary for the enhancement of cisplatin-induced apoptosis, we attempted to block the effect of bFGF with suramin. Suramin is a small, polyanionic compound that acts as a relatively nonspecific inhibitor of FGF receptor-ligand interactions (Yayon and Klagsbrun, 1990). At a concentration of 100 µM, suramin reduced by 83% the ³H-thymidine incorporation stimulated by 0.5 ng/ml bFGF (Fig. 5C), suggesting that this concentration of suramin was able to block FGF receptor-mediated signaling.

View larger version (34K): [in this window] [in a new window]   Fig. 6.   The bFGF-enhanced accumulation of TUNEL-positive cells after cisplatin exposure is not reversed by suramin. NIH 3T3 cells were pretreated for 24 h with medium only (A), 10 ng/ml bFGF (B), 100 µM suramin (C), or both bFGF and suramin (D). The cells then were exposed to 10 µg/ml cisplatin for 12 h and harvested 24 h after the cisplatin was removed. The scatter plots were analyzed as in Fig. 1. TUNEL-positive (R3), sub-G1 (R4), and the total percentage of apoptotic cells (% APOP) are given to the right of each plot.

	Discussion

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The addition of exogenous bFGF to the growth medium of NIH 3T3 cells produced a strong and reproducible enhancement of cisplatin-induced apoptosis (Table 1 and Fig. 1). The addition of bFGF also significantly reduced the dose-dependent survival of cells exposed to increasing concentrations of cisplatin (Fig. 3), and therefore directly increased the sensitivity of NIH 3T3 cells to this chemotherapy drug. Exogenous bFGF did not produce any cytotoxic effects in the absence of cisplatin, and it had the expected stimulatory effect on cell growth at both low and high concentrations (Fig. 5B). Enhancement of cisplatin-induced apoptosis was observed at bFGF concentrations of 10 ng/ml and higher (Fig. 2A), whereas stimulation of ³H-thymidine incorporation occurred at 0.5 ng/ml (Fig. 5A). The concentrations of bFGF that sensitized cells to cisplatin also produced a distinct shift in the cellular morphology of NIH 3T3 cells (Table 2 and Fig. 4), and both this morphology shift and the drug sensitization were reversible with similar kinetics (Fig. 2B and data not shown).

Other studies have examined the effects of bFGF on the sensitivity of cells to chemotherapy drugs, with varying results. Most of these studies have used expression of bFGF from a transfected cDNA. Overexpression of bFGF increases the survival of NIH 3T3 cells treated with etoposide or 5-fluorouracil (Wieder et al., 1997), and also provides resistance to the DNA damaging agent PALA (Huang et al., 1994). Shaulian et al. (1997) found that NIH 3T3 cells expressing a bFGF transgene encoding a signal peptide are resistant to cisplatin. In contrast, recent studies indicate that bFGF sensitizes MCF-7 breast tumor cells to apoptosis induced by cisplatin, etoposide, and 5-fluorouracil, whether bFGF is expressed from a transgene or added to the culture medium (Wang et al., 1998; Fenig et al., 1999; Maloof et al., 1999).

The ability of bFGF to sensitize or protect cells may depend on the cell type used, because bFGF sensitization previously has only been reported with MCF-7 cells. However, our current results suggest that bFGF can also sensitize NIH 3T3 cells to cisplatin. We similarly have observed sensitization to cisplatin cytotoxicity with exogenous bFGF added to MCF-7, SKOV3, and A2780 cell lines, and we have found bFGF-related sensitization of NIH 3T3 cells to doxorubicin and UV light (our unpublished results). Therefore, it does not appear that simple cell line differences or drug differences can account for the differential effects of bFGF. The recent reports that MCF-7 cells can be sensitized to chemotherapy with either transfected or exogenous bFGF suggest that it is not simply the mode of bFGF delivery that accounts for differential responses to bFGF (Wang et al., 1998; Fenig et al., 1999; Maloof et al., 1999). However, we cannot rule out the possibility that some cell types (such as NIH 3T3 cells) are protected by transfected bFGF but sensitized by exogenous bFGF.

Indeed, the overexpression of bFGF may affect the cellular response to chemotherapeutic agents differently than does the stimulation of cells with exogenous bFGF. Expression of bFGF from a full-length cDNA leads to the production of multiple intracellular isoforms of bFGF derived from the initiation of translation at alternative start codons on the same mRNA (Florkiewicz and Sommer, 1989). The 18-kDa isoform, initiated from a primary AUG start codon, remains localized in the cytoplasm, whereas three high molecular weight isoforms (22, 23, and 24 kDa) initiated from alternative upstream CUG start codons translocate to the nucleus, where they are thought to elicit distinct biological activities (Bikfalvi et al., 1995). The overexpression of 24-kDa bFGF alone provides resistance to ionizing radiation in HeLa cells, whereas overexpression of 18-kDa bFGF alone provides no resistance (Cohen-Jonathan et al., 1997). Therefore, expression of the high-molecular-weight bFGF isoforms may affect signal transduction pathways other than those activated by extracellular 18-kDa bFGF, and these different pathways may have different effects on the response of cells to DNA damaging agents.

We found that bFGF sensitized NIH 3T3 cells to cisplatin-induced apoptosis only at concentrations of 10 ng/ml and higher (Fig. 2). The shift in the cellular morphology of NIH 3T3 cells followed a similar dose dependence (Table 2). These concentrations of bFGF were significantly higher than those needed to stimulate ³H-thymidine incorporation and cell growth (Fig. 5). Also, suramin, which inhibits bFGF from binding to and activating the high-affinity FGF receptor, prevented bFGF stimulation of DNA synthesis but did not affect the enhancement of cisplatin-induced apoptosis (Figs. 5 and 6). These data suggest that the signal by which bFGF sensitizes NIH 3T3 cells to cisplatin might be received and propagated through a separate receptor or signal transduction pathway than that which stimulates mitogenesis.

It is not clear how high concentrations of bFGF might act independently of "classic" FGF signaling, and at this point, we can only speculate as to how this signal is received. The heparan sulfate proteoglycans (HSPGs) constitute the low-affinity cell-surface receptors for bFGF, and it is conceivable that they might act as mediators of bFGF activity at higher concentrations. bFGF binds to HSPGs with a K_d of approximately 1 to 2 nM (Moscatelli, 1987), and we have observed enhancement of cisplatin-induced apoptosis and changes in cellular morphology at a bFGF concentration of 5 to 10 ng/ml, or about 0.3 to 0.6 nM.

The syndecans (syndecan 1-4) are one group of transmembrane HSPGs that function in adhesion to the extracellular matrix, and it has been demonstrated recently that these low-affinity receptors are capable of transmitting extracellular signals into the cytoplasm. Syndecan-4 is expressed in mouse fibroblasts and is necessary for the assembly of focal adhesions and cellular spreading in these cells (Saoncella et al., 1999). Interestingly, the addition of 10 to 30 ng/ml bFGF to NIH 3T3 cells causes loss of phosphorylation from serine-183 in the cytoplasmic tail of syndecan-4 (Horowitz and Simons, 1998). The cytoplasmic tail has been shown to interact with signaling factors such as protein kinase C and the GTP-binding protein Rho (Saoncella et al., 1999). Also of interest in light of our observed effect of bFGF on NIH 3T3 cell morphology is the recent report that exogenous bFGF triggers distinct process outgrowth in astrocytes in culture; the effect is dependent on c-Ha-Ras and is blocked by Rac1 and RhoA, but the cell-surface mediators of this effect have not been studied (Kalman et al., 1999).

Another possibility is that at high concentrations bFGF competes for the binding of an unknown factor(s) to HSPGs at the cell surface or in the extracellular matrix. Many growth factors and cytokines bind to HSPGs, and such binding is necessary for high-affinity receptor binding and activation by some cytokines that are not members of the FGF family (Cook et al., 1995). There may be a factor(s) in serum or the extracellular matrix that has the potential to modulate the cell's susceptibility to apoptosis. If bFGF were to compete for the binding of an antiapoptotic factor to HSPGs, and thereby inhibit binding of this factor to its high-affinity receptor, then cells could be rendered more susceptible to apoptosis. We also cannot rule out an effect of high concentrations of bFGF on down-regulating high-affinity FGF receptors and/or its downstream signaling components that would otherwise confer a protective effect against cisplatin and other apoptotic signals.

Regardless of the mode of signal transduction, high concentrations of bFGF clearly sensitize NIH 3T3 cells to cisplatin, as demonstrated by an enhancement of cisplatin-induced apoptosis and a dose-dependent reduction in survival. A better understanding of how bFGF works to modulate apoptosis might yield answers as to why this factor protects cells under some conditions and sensitizes cells under other conditions. This knowledge could provide valuable insights into what role bFGF might play in the response of tumors to chemotherapy.