Journal of Neuroscience Research 27:689-696 (1990)

Basic Fibroblast Growth Factor: A Potential Autocrine Regulator of Human Glioma Cell Growth J.L. Gross, R.S. Morrison, K. Eidsvoog, W.F. Herblin, P.L. Kornblith, and D.L. Dexter Medical Products Department, E.I. du Pont de Nemours and Co., Inc., Wilmington, Delaware (J.L.G., K.E., W.F.H., D.L.D.), R.S. Dow Neurological Sciences Institute and Comprehensive Cancer Center, Portland, Oregon (R.S.M.), and Department of Neurosurgery, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, New York (P.L.K.)

Basic fibroblast growth factor (bFGF) is a potent mitogen and angiogenic factor. bFGF is expressed by a variety of solid human tumors and has been implicated as an autocrine regulator of tumor growth. Different solid tumor lines including glioma, colon carcinoma and melanoma were examined for intracellular immunoreactive bFGF, high- and lowaffinity bFGF receptors and mitogenic response to bFGF when grown in chemically defined medium. All tumor lines contained significant levels of bFGF. In addition, ail tumor lines contained subsets of five forms of immunoreactive bFGF, as well as 0.68-20 X lo6 low affinity bFGF binding sites (Kd = 15-300 nM). Most, but not all lines exhibited high affinity bFGF receptors (Kd = 2 5 4 0 pM). Glioma cell lines were distinguished by expressing the highest levels of bFGF protein as well as the most high-affinity receptors for bFGF. Furthermore, glioma cell lines were the only tumor type mitogenically responsive to bFGF. These results indicate that glioma cells express high levels of this potent mitogen and angiogenic factor relative to human colon carcinoma and melanoma cells. The expression of bFGF and bFGF receptors by glioma cells may be related to abnormal growth and neoplastic progression in these tumors. Key words: basic fibroblast growth factor, brain tumor, glioblastoma, autocrine growth factor, glial cells, neoplastic progression

INTRODUCTION Glial cell-derived neoplasms (gliomas) comprise the vast majority of nervous system tumors in humans (Zulch, 1986; Kornblith et al., 1987). Most of these neoplasms are derived from cclls of thc astrocyte lineage. Patients with malignant gliomas normally exhibit very poor postoperative survival times despite improve0 1990 Wiley-Liss, Inc.

ments in surgery, radiation and chemotherapy. Elucidating the regulatory pathways controlling glioma growth provides a logical basis for developing new curative or palliative therapies. Fibroblart growth factors are multifunctional polypeptides initially characterized for their mitogenic properties (Rifkin and Moscatelli, 1989). The FGFs increase the growth of many cell types in vitro, including glial cells (Morrison and de Vellis, 1981; Pettmann et al., 1982), and they stimulate blood vessel growth (Shing et al., 1985; Folkman and Klagsbrun, 1987). The potential importance of FGF in tumor biology is supported by the observation that several recently described oncogenes discovered in human tumors encode proteins structurally related to the FGF family of growth factors (Delli-Bovi et al., 1987; Taira et al., 1987; Smith et al., 1988; Zhan ct al., 1988; Finch et al., 1989; Marics et al., 1989). Furthermore, elevated levels of basic and acidic FGF can induce altered cell growth and phenotypic transformation (Blam et al., 1988; Neufeld et al., 1988; Rogelj et al., 1988; Sasada et al., 1988; Dotto et al., 1989). Cytogenetic analyses of malignant human gliomas demonstrate the presence of multiple chromosomal abnormalities (Shapiro, 1986). Many of these chromoromal abnormalities may relate to alterations in the expression of growth factors or growth factor receptors. Members of the fibroblast growth factor (FGF) family represent a likely source of tumor growth modifiers. There is now evidence to support a role for bFGF in the development and progression of malignant brain tumors as well. Glioma cells express bFGF mRNA (Murphy et

Received August 29, 1990; revised September 20, 1990: accepted September 20, 1990. Address reprint requests to Dr. K.S. Morrison, R.S. Dow Neurological Sciences Institute and Comprehensive Cancer Center, Portland, OR 97209.

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al., 1990; Takahashi et al., 1990), bFGF protein (Morrison et al,, 1990; Paulus et al., 1990) and bFGF rcceptors (Morrison et al., 1990) representing the cellular machinery necessary for an autocrine pathway. bFGF stimulates glioma proliferation in culture (Westphal et al., 1988; Morrison et al., 1990) and glioma growth in soft agar (Morrison et al., 1990) a property associated with ncoplactic transformation. Tn addition. elevated levels of bFGF mRNA have been detected in acoustic neuromas and astrocytomas relative to meningiomas (Murphy et al., 1989). Finally, gliomas express at least one other member of the FGF family, acidic FGF (Libermann et al., 1987). In the present rcport, we provide evidence that human glioma cells can be distinguished from several other human tumor types on the basis of their expressing significantly higher levels of bFGF, high-affinity receptors for bFGF, and responding mitogenically to bFGF.

MATERIALS AND METHODS Materials were obtained from the following sources: Na I2'I was from New England Nuclear (100 mCi/ml in dilute NaOH); heparin-agarose, selenium, transferrin, hydrocortisone, and insulin were from Sigma; goat antimouse antibody was from Bio-Rad; Cetal calf serum and Ham's F- 12/Dulbecco's modified eagle medium (DMEM) (1:1) were from GIBCO. CHAPS detergent and protease inhibitors were from Boehringer Mannheim Biochemicals. Human recombinant bFGF (hr-bFGF) was generously provided by Synergen Inc. (Boulder, CO).

tained in serum-free medium for another 24 hr. Cells were then converted to chemically defined media (CDM), Ham's F-l2/Dulbecco's Modified Eagle Medium (DMEM) ( l : l ) , with 10 pgiml insulin, 5 pg/ml transferrin, 30 nM selenium, and 50 nM hydrocortisone. Varying concentrations (dose response) of hr-bFCF were added at the time of convcrsion to CDM (designated day 0) and on days 3 and 5 . On the sixth day after convcrsion to CDM, cells were washed twice with PBS and trypsinized (0.25% in PBS). Cell number was determined using a Coulter counter (Coulter Electronics, Hialeah, FL).

Receptor Binding Assay hr-bFGF was iodinated using lactoperoxidase and purified by gel filtration and affinity chromatography (heparin agarose) as previously described (Morrison et al., 1990). SNB-19 cells were plated in 24-well cluster dishes at a density of 1 X lo5 cellsiwell (2 cm2). The cells were maintained for 48 hr in scrum-supplemented medium (20% fetal bovine serum). bFGF binding was measured as described by Moscatclli (1987) using a single concentration of ['2s11-hr-bFGF and multiple concentrations of unlabeled hr-bFGF. Nonspecific binding was determined by the administration of 3 p M unlabeled hrbFGF. The binding data were analyzed by nonlinear regression techniques and are displayed in the format of Scatchard (1 949). Receptor number was normalized to the cell number measured at the same time as the binding. bFGF Extraction Confluent cultures were trypsinized and pelleted. Pellets were resuspended at 2 X lo7 cells/ml and sonicated in buffer with 2 M NaCl, 0.1% 3-3-cholamidopropyl-dimethylammonio- 1-propanesulfonate (CHAPS) detergent, and the following protease inhibitors: 400 nM leupeptin, 1 pgiml PMSF and 1 pg/ml of antipain, elastinol, bestatin, pepstatin, p-APMSF. After centrifugation to remove nuclei and membranes (IOOKxg), the supernatants were diluted 1:6 in buffer (10 mM Trisi HCl, pH 7.0, 0.1 M NaCl) and applied to heparin agarose columns. Proteins including bFGF were eluted with a step wash of 3 M NaCl. Samples were analyzed quantitatively and qualitatively by dot blot and Western gel analysis respectively (Morrison et al., 1990).

Cell Culture Human glioma lines (SNB-19, SNB-75. SNB-56, and SNB-78) were maintained as previously described (Cross et al., 1988). Rat C6 glioma lines and HCT-I5 human colon carcinoma lines were from the American Type Culture Collection and were cultured as recommended. Clone A and DLD-2 human colon carcinoma lines werc cultured as previously described (Dexter et al., 1982). The human melanoma RPMI-7272 line and the B- 16 murine melanoma cells were kindly provided by Drs. Daniel Rifkin (New York University Medical Center, NY) and Mary Neville (E.I. duPont de Nemours, Wilmington, DE) and were propagated in RPMI media containing 10% heat-inactivated fetal bovine serum (FBS). The rat RT2 glioma line (Powers et al., 1987) Immunoblotting and Western Blotting was cultured in the same media. Crude cell lysates, heparin-enriched fractions, and hr-bFGF were serially diluted and applied to a 96-well Cell Proliferation Assay dot-blot apparatus containing nitrocellulose. Residual Cells were plated at 8 x 103/cm2in serum-supple- protein binding sites were blocked by incubation in mented media. Within 18-20 hr postplating, cells were blocking buffer. The paper was incubated with murine washed with phosphate-buffered saline (PBS) and main- monoclonal anti-hr-bFGF antibody (DE6) (Reilly et al.,

bFCF in Human Glioma TABLE I. Growth Response of Tumor Cell Lines to hr-bFGF*

3 ,

691 I

% Change in cell no.

Cell line SNB-I9 SNB-75 SNB-56 SNB-78 DON C6 Clone a HCT- 1 5 DLD-2 RPMI-7272 B-16

Cell type

over CDM

Human glioma Human glioma Human gliorna Human glioma Human glioma Rat glioma Human colon carcinoma Human colon carcinoma Human colon carcinoma Human melanoma Mouse melanoma

+38. +34, +41 +27, +38. f.54, + 5 0 +47, t41, +49 +41, + I 9 + 44 + 1.59 + 12 -46, -15,o NT" -41. -15 - 10

20

*hr-bFGF (560 pM) was added to tumor cell lines as described under Materials and Methods. Cells were trypsinized and counted 6 days after conversion to CDM plus purified hr-bFGF. Thrce dishes were used per treatment. Data arc cxprcssed as the percent of change in cell nurnbcr cornparcd to cell number in CDM alone. Each number represents the results of a single experiment. "NT. not tested.

0.1

0.01

1

10

100

1000

bFGF (pM)

,

1

B

VI h

15v

ce w

fz

10-

'1-

6

0

1989) at 0.2 pg/ml in blocking buffer. After washing, the nitrocellulose sheets were treated with peroxidaseconjugated goat antimouse antibody and developed in buffer containing the substrate 4-chloro- 1-naphthol (Morrison et al., 1990). The hr-bFGF standard curve was linear from 5-50 ng bFGF, whereas the lower limit of detection was approximately 0.3-0.6 ng hr-bFGF. For Western blot analysis, extracts were combined with 3 x reducing sample buffer and separated on a 12.5% polyacrylamide gel in the presence of SDS. The separated proteins were transferred electrophoretically to Immobilon (NEN). Immunodetection was performed as described above. The density of each immunoreactive band was measured using a scanning dcnsitometer (Molecular Dynamics) and compared with hr-bFGF.

RESULTS The addition of purified hr-bFGF to cultured human and rat glioma cell lines maintained in CDM significantly enhanced the number of cells measured 6 days after treatment compared to growth in CDM alone (Table I). In marked contrast to glioma cells, tumor cells derived from melanomas or colon carcinomas did not reach greater cell densities in the presence of hr-bFGF. In fact, bFGF had an inhibitory effect on the growth of the two melanoma lines tested. The influence of hr-bFGF on glioma cell growth was dose-dependent and saturable, suggesting interaction with specific bFCF receptors (Fig. 1A,B). As shown for a rat C6 glioma line (Fig. IB), the concentration of hr-bFGF required for half-maximal stimulation of cell

CDM

Fig. 1 . Growth response of SNB-56 cells (A) and C6 cells (B) as a function of hr-bFGF concentration. The concentration range was 0.6-1660 pM. Cells were plated and maintained as described under Materials and Methods. Six days after conversion to CDM and growth factor addition, cells were trypsinized and counted. Quadruplicate dishes were used per treatment. Data are expressed as the average number of cells per dish. The range in cell number was

Basic fibroblast growth factor: a potential autocrine regulator of human glioma cell growth.

Basic fibroblast growth factor (bFGF) is a potent mitogen and angiogenic factor. bFGF is expressed by a variety of solid human tumors and has been imp...
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