Exp.

Eye

Res. (1990)

51, 519-529

Iris-derived Wlelanocytes Resembles Basic JEAN

PLOUET’AND

Contain Fibroblast DENIS

a ‘Growth Growth

Factor Factor

That

GOSPODAROWICZ

The University of California Medical Centre, Cancer Research Institute M- 7282, San Francisco, CA 94743, U.S.A. (Received 37 October 7989 and accepted in revised form 76 April 7990) A melanocytegrowth stimulatingfactor hasbeenpurified from bovine iris melanocytesand identified as beingcloselyrelatedto the basicform of fibroblastgrowth factor (bFGF).This conclusionwasbasedon the behavior of the melanocyte-derivedgrowth factor when submittedto heparin-sepharoseai%nity chromatography,as well as on its ability to cross-reactwith bFGFin radioimmuno-and radioreceptor assays.The ability of neutralizingbFGFantibodiesto blockcell proliferationin response to the melanocyte growth factor further confinnsthat it is closelyrelatedto bFGF.Sincemelanocytesexpressthe 3.7-kb and TO-kb bFGFtranscript, the possibilityexiststhat uncontrolled expressionof melanocyte-derivedbFGF could be involved in the malignant transformationof melanocytesinto melanomacells.

Key words: growth factors; melanocytes ; proliferation : differentiation : autocrine mechanisms. 1. Introduction

The melanocyte is a neural crest-derived cell that presents itself in culture as a ditrerentiated cell with well-defined biochemical marker : melanosome and tyrosinase. Normal melanocytes rarely undergo cel1 division in situ and cannot be replicated in vitro unless grown in the presence of growth-stimulating agents such as 12-tetradecanoyl phorbol-13-acetate (TPA) @singer and Marko, 1982), cholera toxin or a crude brain-derived fraction (Wilkins et al., 1985 ; Halaban et al., 1986). Upon transformation, melanocytes can develop into melanoma cells which are fast growing in situ and have a high metastatic potential. Various growth factors have been purifled from a number of established melanoma cell lines. These include: transforming growth factor-alpha (TGFcz) (Marquardt and Todaro, 1982), transforming growth factorbeta (TGFP), platelet-derived growth factor (PDGF) (Westermarketal., 1986),fibroblastgrowthfactor(FGF) (Halaban, Ghosh and Baird, 198 7), melanoma growth stimulatory activity (MGSA) (Richmond, Lawson, and

Nixon, 1982; Richmond et al., 1983), and a heparin binding growth factor (Ogata, Furukashi and Eisinger, 1987). TGFa, TGF/3, PDGF, FGF and MGSA have a11 been suggested as possible autocrine regulators of melanoma growth, and by inference have been implied in the control of melanocyte proliferation. The difficulties in culturing melanocytes have prevented, until now, the identification of their endogenous growth factors. These difficulties may be due in part to the fact that melanocyte cultures have, in the past. been initiated from tissuessuch as human

foreskin or nevi, which contain relatively few melanocytes, as compared to other cell types. Our attention has therefore focused on a tissue which, to the best of * For correspondence. 00144835/90/110519+ 37

11 %03.00/0

our knowledge, has never before been used for melanocyte culture - the anterior portion of the iris. Unlike other tissues, the anterior part of the iris is known to be densely populated by melanocytes derived from the neural crest (Hogan, Alvarado and Weddell, 19 7 1; YanofS and Fine, 19 75). As in the caseof skin-

derived melanocytes. iris melanocytes can give rise to fast growing melanoma upon transformation (Yanoff and Fine, 19 7 5). In the case of bovine irises, which are black, 90% of the bovine cell population derived from the anterior layer of the dorsal iris are melanocytes. Those cells, in contrast to skin-derived melanocytes, divide spontaneously in culture. This allowed us to grow the relatively large number of cells required for the biochemical and biological characterization of their autocrine growth factor.

2. Materials and Methods Reagents

Tissue culture media were obtained from Gibco (Grand Island, NY). Fetal calf serum (FCS) and calf serum (CS) were purchased from Hyclone Inc. (Logan, UT). Saline containing 005% trypsin, O-01 M sodium phosphate, and 002 % EDTA (STV) was obtained from Difco Laboratories (Detroit, MI), gentamicin from Schering (Kenilworth, NJ), and Fungizone from Squibb (Princeton, NJ). Tissue culture dishes were from Falcon Plastics (Oxnard, CA), for Nunc culture plates (530 cm2), which were obtained from Applied Scientific (San Francisco, CA). HeparinSepharose (HS) and protein A Sepharose were obtained from Pharmacia (Piscataway, NJ). Bovine pituitary bFGF and brain aFGF were purified as previously described (Gospodarowicz et al., 1984a: Bijhlen et al., 1985), and their homogeneity was determined by SDS-PAGE electro0 1990 AcademicPressLimited EER 51

520

J. PLOUET

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D. GOSPODAROWICZ

phoresis, amino acid composition, and amino acid sequence analysis. [‘251]bFGF was prepared as previously described (Neufeld and Gospodarowicz, 1986). High-density lipoproteins were purified from human plasma as previously described (Gospodarowicz et al., 1986). Nitrocellulose filters were purchased from Schleicher and Schuell (Keene, NH), and molecular size markers for RNA hybridization blot from Bethesda Research Laboratories (Gaithersburg, MD). The mtiltiprime DNA labeling kit and [32P]dCTP were purchased from Amersham (Arlington Heights, IL). Normal rabbit serum and sheep anti-rabbit gamma globulin were obtained from Antibodies Inc. (Davis, CA). Insulin, transferrin, poly-L-iysine, gelatin, Ponceau red and normal goat serum were purchased from Sigma (St Louis, MO). Biotinylated sheep anti rabbit antiserum and avidin-alkaline phosphatase were from Vector Laboratories (Burlingame, CA). 5-Bromo-4chloro-3-indolyl phosphate and nitro blue tetrazolium were purchased from Kierkegaard and Perry Laboratories (Gaithersburg, MD). Heparin sodium was purchased from Hepar Industries, Inc. (Franklin, OH) and HT 200 membrane from Gelman Sciences, Inc. (Ann Arbor, MI). Preparation of Melanocyte Cultures Eyes collected from freshly slaughtered steers were transported on ice to the laboratory. After being washed with ethanol, the corneas were dissected and the underlying iris was exposed. The anterior layer of the iris which contain melanocytes derived from the neural crest (Hogan et al., 1971) was gently scraped with a groove director or a cell scraper. Black tissue fragments which adhered to the scraper were then resuspended in a mixture of 1: 1 (v/v) low glucose, Dulbecco’s modiied Eagle’s medium (DMEM), F-12 medium supplemented with 10% calf serum, 2 mM 50 ,ug ml gentamicin-l, and @2 5 pugml glutamine, Fungizone-l. Aliquots were seeded in S-cm tissue culture dishes containing 5 ml of the same medium. Four to five days following seeding, the small tissue explants became tightly adhered to the dish. Cells containing melanosomes characteristic of melanocytes could be seen migrating out of the explant. The cells then began to proliferate rapidly, forming homogeneous, dark colored colonies. Homogeneous colonies exhibiting the characteristic morphology of melanocytes (Fig. 1) were then scraped from the dish using Pipetman P 200 and transferred into new tissue cultures dishes where cells were allowed to expand. Upon reaching confluence, cultures were trypsinized and passaged at a split ratio of 1: 10. Mitogenic Assays

Bovine adrenal cortex derived capillary endothelial (ACE) cells were isolated and maintained as previously described (Gospodarowicz et al., 1986). For mitogenic

FIG. 1. Morphology of iris melanocytes. Iris melanocytes were cultured in a mixture of 1: 1 (v/v) DMEM-F12 supplemented with 10% FCS, as described in Materials and Methods. Cells at confluency were passaged at a split ratio of l-10 in presence or absence of bFGF (1 ng ml-‘). A Primary culture: B, cells in their third passage and previously cultured in the presence of bFGF; C. cells in their third passage and previously cultured in the absence of bFGF.

assays, ACE cells were seeded at a density of 2 x 10” cells per 3 S-mm tissue culture dish, containing 2 ml of DMEM supplemented with 10% CS, 2 mM glutamine, 50 ,ug gentamicin ml-l, and 0.25 kg Fungizone ml-‘. When the activity of partially purified crude extract was tested on melanocytes, cells were maintained under serum-free conditions. Melanocytes were seeded at a density of 2 x lo4 cells per 35-mm tissue culture

IRIS

DERIVED

MELANOCYTE

GROWTH

FACTOR

dish containing 2 ml of growth medium. After cells had attached (ordinarily within 6 hr), media was removed, plates were washed once with PBS and 2 ml of a mixture of 1: 1 (v/v) of DMEM-F-12 supplemented with 2 mM glutamine, 50 ,ug gentamicin ml-‘, 02 5 ,ug Fungizone ml-‘, 5 pug lOP1Lg transferrin t-n-‘, insulin ml-l, and 500 yg of high density lipoproteins (HDL) protein ml-‘. bFGF or samples to be assessed were added every other day, and after various time intervals, cultures were trypsinized and cell density determined with a Coulter Counter. Preparation of Extracellular Matrix Produced by Irisderived Melanocytes

To prepare extracellular matrix-coated (ECM) substrata from iris derived melanocytes stock cultures of melanocytes were seeded on a gelatinized 12-well cluster plate. Gelatin was cross-linked to the surfaces of the wells, as described by Macklis. Sidman and Shine (198 S), and wells were coated with an aqueous solution (310 ~1) containing 60 yg gelatin and 1.3 ,ug carbodiimide. After 3 hr of incubation at room temperature, the wells were washed three times with water, then air dried and sterilized under UV irradiation for 2 hr. Immediately before use, the wells were washed with DORM supplemented with 10 Y0calf serum (CS), and stock cultures were then seeded at a density of 10 x lo4 cells per well. After 9-12 days in culture, the cells were removed from the extracellular matrix by the addition of 20 mM NH,OH in distilled water (Gospodarowicz et al., 1984b). The iris melanocyte derived ECM was washed five ties in phosphate buffered saline, then stored at 4°C until plating of the melanocytes for the proliferation assays.

521

buffer containing 0.6, 1.0 or 3.0 M NaCl, respectively (Neufeld et al.. 1987). The flow rate was 21 ml hr-*. Aliquots of the various fractions were diluted with 02 % gelatin in calcium- and magnesium-free PBS and tested for their abilities to stimulate cell proliferation. Radioimmunoassays

Basic fibroblast growth factor (FBF) or aFGF rabbit antisera were purified by protein A Sepharose tinity chromatography and the IgG fraction containing the anti bFGF or anti aFGF antibodies were used for RIA. Basic FGF RIA was performed as previously described (Neufeld et al., 1987). Briefly, either bFGF or experimental samples were added to tubes containing 0.1 ng [1251]bFGF and 1 ,ug of anti bFGF IgG fraction capable of binding 40-60% of the tracer. After 18-24 hr at room temperature, anti-rabbit IgG antibodies were added to the tubes, which were further incubated for 24 hr. The bound antigen was then precipitated by centrifugation (30 min, 1500 g, 4°C). The radioactivity present in the pellet was determined in a gamma counter. The sensitivity of the assay was 2 pg per tube. The interassay and intraassay variation coefficients were below 10%. The specificity of the antibody was determined as previously reported (Neufeld et al., 198 7), and aFGF at concentrations up to 10 ng per assay did not produce any displacement. For aFGF RIA [1251]aFGF (0.15 ng per tube) and rabbit anti-aFGF IgG capable of binding 25 % of the tracer were incubated in the presence or in the absence of aFGF or experimental samples, following a procedure essentially similar to the one used for bFGF RIA. The limit of sensitivity of the assay was 5 pg per tube. The cross-reactivity with bFGF was less than 1%. The interassay and intraassay variation coefficients were below 10%.

Purification of Cell Extracts

Iris cells grown to confluence in 530-cm2 plates were dissociated by exposure to STV. CS was added to neutralize trypsin, and the suspension was centrifuged (300 g, 5 min): the cell pellet was then suspended in PBS and after determination of the cell number, centrifuged (300 g, 5 min). The supernatant was discarded and the pellet was either extracted immediately or stored in liquid nitrogen. The pellet was resuspended in 2 ml of distilled water containing 0.5 % Triton X-100, and after incubation for 10 min at 4Y!, the suspension was aspirated into a 3-ml syringe and then repeatedly forced through a 25-gauge needle until the cells were lysed. An aliquot of 10 mM TrisHCl, pH 7.0, containing 3 M NaCl was added to obtain a final concentration of 04 M NaCl. The extract was then centrifuged (50000 g, 30 min, 4°C) and the supernatant, i.e. the crude extract, was applied to a heparin-Sepharose (HS) column (bed volume, O-5 ml) which had been pre-equilibrated at room temperature with 10 mM Tris-HCl, pH 7.0, containing 0.6 M NaCl. The column was sequentially eluted with the same

Immunoblot Analysis

Pituitary-derived bFGF (100 ng), HS-purified melanocyte extracts or molecular size markers were subjected to electrophoresis on 0.1% SDS-PAGE (15 % slab gels) and then transferred electrophoretically to BA 85 nitrocellulose filters (Towbin, Staehlin and Gordon, 19 79). The nitrocellulose filters were stained with Ponceau red and the positions of the molecular size markers were marked. The filters were then repeatedly washed with 50 mu Tris-HCl, pH 7.0, 0.5 M NaCl, 0.1% Tween 20, and incubated for 60 min at room temperature with the same buffer and polyclonal anti-bFGF antibodies (1 pug ml-‘). Control filters were not exposed to the antibodies. The filters were washed three times, probed for 30 min with a biotinylated anti-rabbit IgG, and then exposed for 30 min to an alkaline phosphatase-avidin complex, according to the instructions of the vendor. The iiiters were washed again and the alkaline phosphatase activity was visualized using 5-bromo-4-chloro-3indolyl phosphate and nitro-blue tetrazolium. 3 7-1

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522

Radioreceptor Assays In a fh-st attempt a conventional radioreceptor assay using purified membranes of BHK 21 cells (baby hamster kidney cell clone 2 1, Neufeld and Gospodarowicz, 1986) was used. Fifteen milligrams of membranes were mixed in a final volume of 80 ,ul binding buffer (F12 medium supplemented with 20 mM Hepes, pH 7.4, and 0.2% gelatin) with 0.5 ng of [*251]bFGF and various concentrations of either purified bFGF or experimental samples. After 30 min at room temperature, the membrane suspension was filtered on HT 200 membranes as described (Neufeld and Gospodarowicz, 1986). After extensive washing the radioactivity bound to the filter was counted in a gamma counter. In parallel experiments HS-purified melanocyte extracts were assayed for their FGF content in a radioreceptor assay using intact cultured melanocytes. Iris cells grown to near confluency in 24-well plates were rinsed three times with binding buffer supplemented with 1 pg ml heparin-’ and further incubated for 1 hr at 3 7°C. The cells were then rinsed with cold binding buffer and incubated with 0.25 ml of binding buffer containing 1 ,ug ml heparin-I, 0.5 ng of [1251]bFGF (1 x lo5 cpm) and various concentrations of either purified FGF or experimental samples. After 4 hr of incubation in the cold, wells were rinsed three times with cold PBS. Cells were lysed in 0.5 ml of 0.5 M NaOH and the cell lysate was counted in a gamma counter. Northern Blot Analysis Melanocytes were grown in 530-cm2 plates in the presence of serum supplemented medium. Confluent cultures were lysed in 5 M guanidinium thiocyanate, and poly adenylated mRNA was prepared as described (Maniatis, Fritsch and Sambrook, 1982). Poly adenylated mRNA was separated on 1.2 Y0 agarose-formaldehyde gels. After visualization of the 18 and 28s species, RNA was transferred to nitrocellulose (Thomas, 1980). To detect the bFGF gene transcripts, the filters were hybridized as previously described (Abraham et al., 1986) to a 32P-labeled 502 base pair fragment of the bovine cDNA clone hBB2. The filters were washed with 2 x SSC [150 mM NaCl, 15 mM Na, citrate (pH 7-O), 0.1 y0 SDS] at 65°C and then analyzed by autoradiography. 3. Results Our choice of the anterior iris, as a source of melanocytes, was based on the fortuitous observation that cornea1 endothelial cell cultures established from cornea which have accidentally come into contact with the anterior iris, often contained fast growing melanocyte colonies. This suggests that iris-derived melanocytes when accidentally placed in culture

AND

D. GOSPODAROWICZ

retained a growth potential not seen in their skin counterpart. Melanocytes were positively identified by the presence of melanosomes (Fig. 1). Cell colonies with morphology characteristic of melanocytes were isolated and expanded for large scale culture, as described in Materials and Methods. To assess whether or not melanocytes could contain a growth-promoting factor(s), cell extracts were prepared and tested on either ACE cells or melanocyte cultures. In both cases the cell extracts were capable of stimulating the proliferation of both cell types (unpubl. res.). This suggests the presence, in melanocytes, of growth-promoting factor(s) specific for either ACE cells or melanocytes or for both cell types. Since previous studies have shown that mitogenic factors for endothelial cells have a strong affinity for heparin (Shing et al., 1984, Gospodarowicz et al., 1984a; Klagsbrun and Shing, 1985), cell extracts were analyzed by HS affinity chromatography. The eluted fractions were tested for their mitogenic activity on ACE cells (Fig. 2). Most of the proteins in melanocyte extracts ( > 99 ‘$$ were either not retained by the column or eluted with 0.6 M NaCl. The unadsorbed material had little, if any, biological activity. Elution of the column with 1 M NaCl yielded

IO

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IM

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0

5

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I5

20

25

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FIG. 2. Heparin-Sepharose (HS) affinity chromatography of crude extracts prepared from melanocytes. The crude extracts (5.5 x lOa cells) were applied to an HS-affiity column, which had been prepared as described in Materials and Methods. Material bound to the column was eluted stepwise with 10 mM Tris HCI, pH 7.0, containing 0.6, 1 and 3 M NaCl. respectively. Aliquots of the eluted Factions were diluted 25-fold with calcium- and magnesium-free phosphate buffered saIine/0+2% gelatin. Aliquots of 10 ~1 were added every other day to adrenal cortex-derived capillary endothelial cells (ACE) that had been seeded at a density of 2 x lo4 cells per 35-mm tissue culture dish. The cells were counted after 4 days. All fractions eluted in the presence of 1 M and 3 M NaCl were also analyzed in an RIA specific for bFGF. Values are means of duplicate determinations which varied by less than 10%. Fractions 11-14 and fraction 17 were pooled and further analyzed.

IRIS

DERIVED

MELANOCYTE

GROWTH

523

FACTOR

a peak of bioactivity which accounted for about 2 % of the total bioactivity applied to the column. Elution with 3 M NaCl resulted in a major peak of bioactivity in which 50% of the original bioactivity was recovered. When the 1 M and 3 M NaCl fractions were analyzed with a RIA specific for bFGF, the 1 M NaCl fractions were not immunoreactive while the 3 M NaCl fractions had a major immunoreactive peak containing 20 ng of bFGF-like material per ,~l (Fig. 2). The 3 M NaCl HS-purified cell extract was then subjected to immunoblot analysis using specific antibFGF antibodies that do not recognize aFGF or other unrelated proteins. An immunoreactive band of M, 15000 was detected running below a standard of pituitary derived bFGF (Fig. 3, lanes A, B). This indicates that the purified cell extract contained material closely related or identical to bFGF, and with

FIG. 3. Immunoblot analysis of HS purified melanocytes cell extracts. Pituitary-derived bFGF (100 ng lane A) or 10 ~1 of purified cell extract (lane B) were electrophoresced on SDS-PAGE, transferred to nitrocellulose sheet and incubated with anti-bFGF polyclonal antibodies, as described in Materials and Methods. All lanes were subsequently exposed to goat anti-rabbit biotinylated antibody. Bound antibody was subsequently visualized as described in Materials and Methods. The MW markers used were phosphorylase B (97 400). bovine serum albumin (66 200). ovalbumin (42 700), carbonic anhydrase (3 1 000), soybean trypsin inhibitor (21 500). and lysozyme (14 400).

a MW similar to that of the truncated form of bFGF (des l-l 5) previously isolated from corpus luteum, adrenal cortex, and kidney extracts (Gospodarowicz, 1987). The bio- and immunoreactive 3 M NaCl peak fraction 17 was examined further by RIA. As seen in Fig. 4(A), 1 ,~l of the purified melanocyte cell extract contained 20 ng of bFGF-like immunoreactive material (final concentration, 20 pug ml-l). The total bFGF content

of the pooled fractions

15-19

was 15.5 pg.

Assuming a 50% recovery, the total bFGF present in 5.5 x lOa cells was 31 kg, which corresponds to 2 x lo6 molecules per cell. When the pooled bioactive 1 M NaCl fractions (1 l-14) were examined using an aFGF-specific RIA, 1 ,~l of the melanocyte cell extract contained 0.6 ng of a FGF-lie immunoreactive material [Fig. 4(B)]. The total amount of immunoreactive aFGF contained in the 1 M NaCl fractions was 1.26 pg. Assuming a 50% recovery, this value corresponds to 9 x lo4 aFGF copies per cell. When tested in an FGF radioreceptor binding assay [Fig. 5(A)] using BHK 21 membranes, 1 ,~l of the 3 M NaCl purified melanocyte cell extract contained 5.2 ng of bFGF-like material (final concentration, 5-2 pg ml-‘), while 1 yl of the 1 M NaCl purified melanocyte cell extract contained 0.2 ng of aFGF-like material (final concentration, 0.2 ,ug ml-l). When assayed on iris melanocytes [Fig. 5(B)], the 3 M NaCl did contain 3.1 pugml-’ of bFGF-like material, while the 1 M NaCl purified melanocyte cell extracts contained 0.15 yg ml-’ of a bFGF-like material. The ability of the 1 M and 3 M NaCl purified cell extracts to stimulate capillary endothelial cell proliferation was compared to that of native pituitaryderived bFGF [Fig. 6(A)]. Half-maximal stimulation of cell proliferation was achieved with 170 pg ml-’ of pituitary-derived bFGF and with 0.05 ,~l ml-’ of the 3 M NaCl purified cell extracts. This suggests that the 3 M NaCl purified cell extract contained 4.25 ,ug ml-’ of bFGF-like activity. In contrast, the 1 M NaCl purified cell extract had no significant mitogenic effect. This reflects the low concentration, as well as the low biological potency of aFGF compared to that of bFGF (Gospodarowicz, 198 7). That all of the bioactivity present in the 3 M NaCl purified cell extract was contributed by bFGF was confirmed by the demonstration that all of the bioactivity was neutralyzed by specific anti bFGF antibodies [Fig. 6(B)]. Control experiment showed that anti bFGF antibodies were able to block bFGF but not aFGF in stimulating cell proliferation. Since the mitogenic activity of the purified cell extract could be blocked by 80%. this indicates that bFGF account for most of its growth-promoting activity. Further proof that melanocytes can synthesize bFGF is also provided by blot hybridization analysis of total RNA with a 32P-labeled bFGF cDNA probe. As shown in Fig. 7, melanocytes contain the 7.0- and 3.7-kb transcripts

of the bFGF gene that are identical in size

524

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FIG. 4. Analysis of HS purified melanocyte cell extracts by (A) bFGF RIA or (B) aFGF RIA. The pooled 3 M and 1 M NaCl peak fractions obtained by HS affinity chromatography of melanocyte celI extracts were further examined by radioimmunoassay. [1251]FGF(100 pg per tube) and anti-FGF antibodies (1 fig per tube) were incubated with increasing volumes of the purified melanocyte cell extracts (A) or with increasing concentrations of FGF (0) as indicated. [1251]bFGFbinding is expressed as percent of [12Y]FGF binding in the absence of binding inhibitors and was determined as described under Materials and Methods. Values represent the means of duplicate determinations. Standard deviation was less than 10%.

bFGF 0.01 ---A 100 -

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FIG. 5. Analysis of HS purified melanocyte cell extracts by radioreceptor binding assay. A, [1251]bFGF (10” cpm per tube) and BHK 21 cell membranes (16 pg per tube) were incubated with increasing concentration of native bFGF (0) or increasing concentrations of the pooled 1 M NaCl (0) or 3 M NaCl (0) fractions. Specific [12Y]bFGF binding was then determined as described under Materials and Methods. Nonspecific [1261]bFGFbinding was determined in presence of 1 pg of unlabelled bFGF. Binding is expressed as percent of specific binding in the absence of binding competitors (controls). Values are the means of duplicate determinations. Standard error was less than 10%. B, Same as A, but confluent iris melanocyte cultures were used instead of BHK 21 cell membranes. aFGF (m), bFGF (0). 1 M NaCl (n), 3 M NaCl (a).

to those present in other cell types known

to express

the bFGF gene (Schweigerer et al., 1987, 1988). A 9.5kb transcript was also present. This hetereogeneity of mRNA transcripts has already been described

(Sternfeld et al., 1988) and may correspond to the fact that they are two alternative transcriptional start sites in the bFGF gene (Florklewicz and Sommer, 1989 ; Prats et al., 1989). Although the above data indicate that iris-derived melanocytes can produce mitogens closely related to the FGF family, and that among those bFGF seems to be the predominant species, they do not demonstrate that FGF is an autocrine growth factor for melanocytes. We therefore tested whether FGFs could act on melanocytes. As shown ln Fig. 8, both native bFGF and aFGF were capable of stimulating melanocyte proliferation with aFGF being tenfold less active. In order to determine whether or not the total mitogenic activity present in melanocytes responsible for their proliferation could be accounted for by basic and acidic FGFs, the effect of increasing concentrations of 1 M and 3 M NaCl heparln Sepharose (HS)-purified melanocyte cell extracts was compared to that of bFGF using melanocytes as target cells. The melanocytederived bFGF-like material present in the 3 M NaCl HSpurified cell extract was able to stimulate the proliferation of the same cell type from which it was derived (Fig. 8), and bioactivlty was equivalent to 5 ,ug of bFGF ml-‘. The 1 M NaCl pooled fractions were marginally active (equivalent to 03 pugml-’ of aFGF or 003 ,ug ml-’ of bFGF). This indicates that bFGF-lie material is the main mltogenic component present in melanocyte extracts. The bioactivity of the 3 M NaCl HS purified cell extract when tested on melanocytes was totally abolished by saturating concentrations of bFGF neutralizing antibodies, whereas that of 1 M NaCl HS was unaffected (data not shown). In addition, the growth promoting ability of extracellular matrices produced by iris derived melanocytes was decreased by 90% when pre-exposed to bFGF neutralizing antibodies (Fig. 9). This substrate has been shown by others to contain bFGF (Baird and Ling, 1987 : Vlodavsky et al., 198 7 ; Globus, Plouet and Gospodarowicz, 1989). The above set of data therefore indicate that bFGF not only can act as a self-

IRIS

DERIVED

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FIG. 7. Northern blot analysis of bFGF expression in irisderived melanocytes. Polyadenylated RNA (5 pg) prepared from melanocytes was separated on a 12% agarose gel containing 2.2 M formaldehyde. After being transferred to nitrocellulose, the RNA blot was hybridized to a O-5-kb bFGF probe and processed for autoradiography, as described in Materials and Methods. A Hind III digest of hDNA served as size marker. The position of the 3.7 and 7.0 kb as well as the position of the 18 and 28s species are shown.

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FIG. 6. EIfect of HS purified melanocyte cell extracts on the proliferation of capillary endothelial cells (A) and effect of anti-bFGF antibodies on the proliferation of capillary endothelial cells exposed to heparin Sepharose-purified melanocyte cell extracts (B). A. Increasing concentrations of 1 M NaCl(0) or 3 M NaCl(0) HS-purified cells extract were added every other day to AC8 cells that had been seeded at a density of 2 x 10” cells per 35mm dish, as described in Fig. 2. Their effects were compared to that of pituitary derived bFGF (0). Following trypsinization on day 4. the cells were counted in a Coulter Counter. Values represent the means of triplicate determinations. Standard deviation was less than 10%. B, ACE cells were seeded at a density of 2 x lo4 cells per 3S-mm well. Ten microlitres of 1 M NaCl(1 M) or 0.05 ~1 of 3 M NaCl peak fractions (3 M) were added or not every other day to the cells. Their effect was compared to that of 0.5 ng ml-’ of pituitary-derived bFGF (bFGF) or 10 ng ml-’ of brain-derived aFGF (aFGF) Anti-bFGF antibodies (10 ,ug ml-‘) was added ( + ) or not (-) as indicated, every other day. After 4 days in culture cells were trypsinixed and counted in a Coulter Counter. Values are means of triplicate determinations. Standard deviation was less than 10%.

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FGF (ng ml-’ ) FIG. 8. Effect of HS purified melanocyte cell extracts on the proliferation of melanocytes. Increasing concentrations of the pooled HS-purified 1 M NaCl (IJ) and 3 M NaCl fractions (0) were added every other day to melanocyte cultures initially seeded at a density of 2 x lo4 cells per 35-mm dish, as described in Materials and Methods. Their effect was compared to that of pituitary-derived bFGF (0) or brainderived aFGF (m). After 6 days in culture, cells were trypsinixed and counted in a Coulter Counter. Values represent the means of duplicate determinations. Standard deviation was less than 10%.

526

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Control Ab B-FGFAb A-FGFAb B-FGF

-

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L

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+ +

FIG. 9. Effect of iris-derived melanocytes ECM on the proliferation of iris-derived melanocytes. Well (22 mm diameter) coated with iris melanocytes derived ECM were exposed to Protein A-purified polyclonal antibodies specific for bFGF (BFGF Ab, 30 ,ug per well), anti bFGF antisera depleted of bFGF immunoneutralizing activities (Control Ab 3 ,ug per well), or protein A purified polyclonal antibodies specific for aFGF (AFGF Ab, 30 yg ml-l). After 16 hr of incubation of 3 7% the dishes were washed twice in DMEM containing 10% calf serum. Iris-derived melanocytes were seeded at 1 x 10” celis per well and maintained in the presence of medium supplemented with transferrin, insulin and HDL, as described in Materials and Methods. bFGF (BFGF, 1 ng ml-‘) was added every other day. After 6 days in culture the cells were trypsinized and counted using a Coulter counter.

stimulating (autocrine) growth factor for melanocytes, but also that the factors present in melanocyte extracts and which stimulate melanocyte proliferation are likely to be related in major part to bFGF and in minor part to aFGF.

4. Discussion Two populations of pigmented cells are present in the iris. One localized to the posterior surface of the iris is derived from neural epithelium and formed the iris pigmented epithelium while the other, localized to the anterior border surface of the iris and in its stroma, is derived from the neural crest. Depending on its density and pigmentation, these cells have a great deal to do with the color of the iris (Hogan et al., 1974). The present study established that iris melanocytes derived from the anterior border surface of the iris can easily be established in culture and identifies FGF as the main mitogenic species present in those cells. This is demonstrated by the chromatographic behavior of the growth factor(s) on HS, its ability to cross-react with bFGF and aFGF in either RIAs or radioreceptor assays, and its ability to stimulate the proliferation of bFGF-responsive target cells. Furthermore, all of the cell stimulatory activity is accounted for by the melanocyte-derived FGF, since all of this activity elutes from an HS affinity column at a salt concentration of 1 M and 3 M NaCl with little activity recovered in the O-6 M NaCl eluate.

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The identification of FGF-like activity in melanocytes was made possible by the ability of iris-derived melanocytes to proliferate spontaneously when maintained in vitro. This allowed the harvesting of the large number of cells needed for further biochemical and biological characterization of their mitogenic content. The rapid and vigorous proliferation of iris derived melanocytes contrasts with what has been reported for foreskinor nevi-derived melanocytes. These melanocytes seem to proliferate to only a limited extent even when exposed to a combination of TPA and cholera toxin or isobutyl methylxanthine (Eisinger and Marko, 1982). The ability of melanocytes derived from the iris to proliferate spontaneously, in vitro, could be due to the relative ease with which this cell type can be selected when one starts with the anterior part of the iris. In contrast, to select for skin melanocytes extensive enzymatic treatment as well as the use of immune resetting and Percoll gradient (Marco,. Houghton and Eisinger, 1982) or treatment with geneticin (Halaban and Altano, 1984) is required in order to eliminate contaminating cell types. The ability of iris melanocytes to proliferate vigorously correlates with its ability to synthesize large amounts of bFGF, on the average of 2 x lo6 copies per cell. This concentration is four fold that observed in pituitary derived folliculo-stellate cells, which until now, have been considered to have the highest in vivo concentration of FGF (Ferrara et al., 198 7). Of the two FGF species present in iris melanocytes, bFGF was the predominant form. It accounted for 98 % of the biological activity, while aFGF accounted for 2 %. It also accounted for 9 5 and 98 % of the binding activity in radioreceptor assays using either partially purified BHK 21 membranes of iris melanocytes. When results are computed in terms of mass, similar results are obtained with an excess of 22- to 25-fold more bFGF copies over aFGF copies present per cell. This ratio in favor of bFGF is similar to that reported for various other mesodermal or neuroectodermal cells which express bFGF as the major form of mitogen, with aFGF being the minor form (reviewed in Gospodarowicz et al., 1987). In the two cell types reported to preferentially synthesize aFGF-glioma and vascular smooth muscle cells (Liberman et al., 198 7 ; Winkles et al., 1988) later studies have shown that the predominant form present was in fact bFGF (Gospodarowicz et al., 1988; Gospodarowicz, 1990). The higher bFGF and aFGF concentration in melanocyte cell extract when tested by RIA as compared to bioactivity may reflect the fact that the RIA detects native as well as degraded FGF species which would no longer bind to FGF receptors. Signs that proteolytic cleavage of native bFGF occurred can be found in the fact that the major bFGF specie detected in melanocyte crude extract was the (des 1-15) bFGF. Lastly, the presence of bFGF transcript in melanocytes definitely suggest that melanocytes can produce this growth factor. In contrast, northern analysis of

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transcripts for aFGF were negative, mostly because the number of mRNA copies for aFGF are below our level of detection and would require more sensitive techniques, such as mRNA amplification (Rappolee et al., 1988). The ability of both bFGF and aFGF to stimulate the growth of melanocytes suggests that they could act as autocrine growth factors for that cell type. In addition, since FGFs have been shown to be differentiation factors, either shifting the pathway of differentiation of undifferentiated cells early in embryogenesis (Slack et al., 1987) or helping to maintain the proper phenotype of cultured cells (Gospodarowicz, Greenburg and Birdwell, 1978; Gospodarowicz et al., 1979), they could also be involved in melanogenesis. This point has not yet been determined. Acidic FGF was ten-fold less potent than bFGF, and we made no attempt in the present study to analyze whether the bioactivity of aFGF could be potentiated by heparin as seen with other cell types, such as BHK21 or human umbilical endothelial cells (reviewed in Gospodarowicz et al., 1987). Through its ability to stimulate angiogenesis, melanocyte-derived FGF could be involved in the wound healing process. It could also be involved in keratinocyte regeneration, since we have recently observed that FGF was a potent mitogen for that cell type (unpubl. res.). This is in agreement with earlier results that have shown that FGF was not only a mitogen for mesodermal or neuroectodermal derived cells, but for ectodermal cells, such as lens and cornea1 epithelial cells as well (Gospodarowicz et al., 1978, 1977), and this not only in vitro but also in vivo (Fredj-Reygrobellet et al., 1987). It is presently unknown whether skin-derived melanocytes also contain FGF. However, it is noteworthy that skin melanocytes have already been reported to respond to bFGF with increased cell proliferation (Halaban et al., 1987). Melanocytes have also been reported to respond to a crude brain extract preparation (named ECGF), which is now recognized to contain both aFGF and bFGF (Wilkins et al., 1985), and to placenta extracts (Halaban et al., 1988), from which bFGF has been purifled (Gospodarowicz et al., 1985). In addition, a factor identified either as bFGF (Halaban et al., 1988) or closely resembling bFGF (Ogata et al., 1987) by its chromatographic behavior on HS-affinity chromatography have been isolated from melanoma cells and reported to stimulate the proliferation of skin melanocytes or melanoma cells. Our data lead to two conclusions. First, both forms of FGF are active on melanocytes. Our results confirm, in part, the conclusion of Halaban et al. (198 7) that bFGF is mitogenic for melanocytes ; however, unlike these authors, we do not find significant synergism

between bFGF and dibutyryl CAMP (unpubl. res.). This most likely reflects the different origins of the melanocytes (iris vs. skin). Second, we observed that aFGF is mitogenic on melanocytes, while it was previously reported that aFGF (also called ECGF)had no activity

for that cell type (Halaban et al., 1987).

These

differences could either be attributed to intrinsic difference between skin and iris melanocytes or more likely, may reflect the use of different factor preparations. The major result of the present work is that bFGF acts as an autocrine growth factor for iris

derived melanocytes. Whether this also applies to skinderived melanocytes can only be establishedwhen we are able to grow that cell type with the same easeas iris melanocytes. This should allow for the analysis of the expression of both FGF genesin skin melanocytes. Transfection experiments using vectors carrying

either the normal bFGF or aFGF gene have shown that uncontrolled expression of FGFs could lead to cell transformation and tumor formation (Neufeld et al., 1988; Jaye et al., 1988). In the case of primary melanoma and metastatic melanoma exposure to

antisense oligonuclotides targeted against three different sets of bFGF mRNA inhibit cell proliferation and colony formation in soft agar (Becker, Meier, and Herlyn, in press). This strongly suggests that uncontrolled FGF expression in iris-derived melanocytes could be a contributing factor toward the transformation of that cell type into melanoma.

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Iris-derived melanocytes contain a growth factor that resembles basic fibroblast growth factor.

A melanocyte growth stimulating factor has been purified from bovine iris melanocytes and identified as being closely related to the basic form of fib...
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