Normal Human Melanocytes That Express a bFGF Transgene Still Require Exogenous bFGF for Growth In Vitro
1998; Elsevier BV; Volume: 110; Issue: 5 Linguagem: Inglês
10.1046/j.1523-1747.1998.00166.x
ISSN1523-1747
Autores Tópico(s)melanin and skin pigmentation
ResumoThe expression of basic fibroblast growth factor (bFGF) has been implicated as an important factor in the development of malignant melanoma. The timing of this expression suggests that bFGF plays a role early in melanoma tumor progression. Benign nevi produce bFGF, and cells cultured from these lesions show a loss of dependence on exogenous bFGF for growth. We have examined the effects of constitutive bFGF expression on the in vitro growth requirements of normal human melanocytes. bFGF was overexpressed in normal human epidermal melanocytes through genomic insertion of a human bFGF cDNA in a retroviral vector. These melanocytes produced the 18 kDa bFGF isoform as well as the higher molecular weight isoforms. The bFGF was not released into the culture medium, but it was present in the cell nucleus. The bFGF produced by these cells was mitogenic for 3T3 fibroblasts and therefore possessed functional activity; however, melanocytes producing bFGF had the same appearance and growth patterns as those infected with control virus or uninfected melanocytes. Expression of bFGF did not confer independence from the exogenous mitogen, nor would these cells form colonies in a soft-agar medium. These results indicate that expression of bFGF alone is not enough to cause aberrant growth of normal human melanocytes. The expression of basic fibroblast growth factor (bFGF) has been implicated as an important factor in the development of malignant melanoma. The timing of this expression suggests that bFGF plays a role early in melanoma tumor progression. Benign nevi produce bFGF, and cells cultured from these lesions show a loss of dependence on exogenous bFGF for growth. We have examined the effects of constitutive bFGF expression on the in vitro growth requirements of normal human melanocytes. bFGF was overexpressed in normal human epidermal melanocytes through genomic insertion of a human bFGF cDNA in a retroviral vector. These melanocytes produced the 18 kDa bFGF isoform as well as the higher molecular weight isoforms. The bFGF was not released into the culture medium, but it was present in the cell nucleus. The bFGF produced by these cells was mitogenic for 3T3 fibroblasts and therefore possessed functional activity; however, melanocytes producing bFGF had the same appearance and growth patterns as those infected with control virus or uninfected melanocytes. Expression of bFGF did not confer independence from the exogenous mitogen, nor would these cells form colonies in a soft-agar medium. These results indicate that expression of bFGF alone is not enough to cause aberrant growth of normal human melanocytes. α-melanocyte stimulating hormone basic fibroblast growth factor normal human epidermal melanocytes The aberrant expression of basic fibroblast growth factor (bFGF/FGF-2) in malignant melanoma has been implicated as an important factor in the development of this cancer (Halaban et al., 1988aHalaban R. Kwon B.S. Ghosh S. Delli Bovi P. Baird A. bFGF as an autocrine growth factor for human melanomas.Oncogene Res. 1988; 3: 177-186PubMed Google Scholar). bFGF is a pleiotropic growth factor that stimulates cell division in many cell types of mesodermal and neuroectodermal origin (Burgess and Maciag, 1989Burgess W.H. Maciag T. The heparin-binding (fibroblast) growth factor family of proteins.Annu Rev Biochem. 1989; 58: 575-606Crossref PubMed Google Scholar). It also has functions in vertebrate development and angiogenesis, and it promotes the survival and differentiation of neurons. There are four isoforms of bFGF (18, 22, 23, and 24 kDa), which are all synthesized from the same mRNA via alternative translational start codons (Florkiewicz and Sommer, 1989Florkiewicz R.Z. Sommer A. Human basic fibroblast growth factor gene encodes four polypeptides: Three initiate translation from non-AUG codons.Proc Natl Acad Sci USA. 1989; 86: 3978-3981Crossref PubMed Scopus (446) Google Scholar; Bugler et al., 1991Bugler B. Amalric F. Prats H. Alternative initiation of translation determines cytoplasmic or nuclear localization of basic fibroblast growth factor.Mol Cell Biol. 1991; 11: 573-577Crossref PubMed Scopus (304) Google Scholar). The three higher molecular weight isoforms contain a nuclear localization sequence, and bFGF has been identified in the nucleus of several different cell types (Florkiewicz et al., 1991Florkiewicz R.Z. Baird A. Gonzalez A.-M. Multiple forms of bFGF. differential nuclear and cell surface localization.Growth Factors. 1991; 4: 265-275Crossref PubMed Scopus (196) Google Scholar). These higher molecular weight isoforms seem to have different biologic functions than the 18 kDa isoform, because overexpression of particular isoforms in fibroblasts or endothelial cells elicits distinct transforming effects (Couderc et al., 1991Couderc B. Prats H. Bayard F. Amalric F. Potential oncogenic effects of basic fibroblast growth factor requires cooperation between CUG and AUG-initiated forms.Cell Regulation. 1991; 2: 709-718PubMed Google Scholar; Bikfalvi et al., 1995Bikfalvi A. Klein S. Pintucci G. Quarto N. Mignatti P. Rifkin D.B. Differential modulation of cell phenotype by different molecular weight forms of basic fibroblast growth factor: possible intracellular signaling by the high molecular weight forms.J Cell Biol. 1995; 129: 233-243Crossref PubMed Scopus (190) Google Scholar). Although it is not produced by normal human melanocytes, bFGF is strongly mitogenic for these cells (Halaban et al., 1987Halaban R. Ghosh S. Baird A. bFGF is the putative natural growth factor for human melanocytes.In Vitro Cell Devel Biol. 1987; 23: 47-52Crossref PubMed Scopus (211) Google Scholar). bFGF produced by keratinocytes is thought to be a natural mitogen for melanocytes in the basal epidermis, and bFGF is an essential component of the growth medium for cultured melanocytes (Halaban et al., 1988bHalaban R. Langdon R. Birchall N. et al.Basic fibroblast growth factor from human keratinocytes is a natural mitogen for melanocytes.J Cell Biol. 1988; 107: 1611-1619Crossref PubMed Scopus (423) Google Scholar). Expression of bFGF mRNA and protein is detected in almost 100% of melanoma tumors in situ (Scott et al., 1991Scott G. Stoler M. Sarkar S. Halaban R. Localization of basic fibroblast growth factor mRNA in melanocytic lesions by in situ hybridization.J Invest Dermatol. 1991; 96: 318-322Abstract Full Text PDF PubMed Google Scholar; Reed et al., 1994Reed J.A. McNutt N.S. Albino A.P. Differential expression of basic fibroblast growth factor (bFGF) in melanocytic lesions demonstrated by in situ hybridization. Implications for tumor progression.Am J Pathol. 1994; 144: 329-336PubMed Google Scholar; Ueda et al., 1994Ueda M. Funasaka Y. Ichihashi M. Mishima Y. Stable and strong expression of basic fibroblast growth factor in naevus cell naevus contrasts with aberrant expression in melanoma.Br J Dermatol. 1994; 130: 320-324Crossref PubMed Scopus (25) Google Scholar). bFGF mRNA expression is also detected in most cell lines derived from metastatic melanomas (Albino et al., 1991Albino A.P. Davis B.M. Nanus D.M. Induction of growth factor RNA expression in human malignant melanoma: markers of transformation.Cancer Res. 1991; 51: 4815-4820PubMed Google Scholar). Inhibition of bFGF synthesis or function in melanoma cell lines by anti-sense oligonucleotides or by internalized neutralizing antibodies against bFGF leads to growth suppression (Halaban et al., 1988aHalaban R. Kwon B.S. Ghosh S. Delli Bovi P. Baird A. bFGF as an autocrine growth factor for human melanomas.Oncogene Res. 1988; 3: 177-186PubMed Google Scholar; Becker et al., 1989Becker D. Meier C.B. Herlyn M. Proliferation of human malignant melanomas is inhibited by antisense oligodeoxynucleotides targeted against basic fibroblast growth factor.EMBO J. 1989; 8: 3685-3691Crossref PubMed Scopus (244) Google Scholar). Studies that have demonstrated the presence of bFGF in nonmalignant nevi suggest that its expression may be an important event early in melanoma tumor progression. Moderate to high levels of bFGF expression have been detected in dysplastic nevi, and there is disagreement over its expression in common acquired nevi (Scott et al., 1991Scott G. Stoler M. Sarkar S. Halaban R. Localization of basic fibroblast growth factor mRNA in melanocytic lesions by in situ hybridization.J Invest Dermatol. 1991; 96: 318-322Abstract Full Text PDF PubMed Google Scholar; Reed et al., 1994Reed J.A. McNutt N.S. Albino A.P. Differential expression of basic fibroblast growth factor (bFGF) in melanocytic lesions demonstrated by in situ hybridization. Implications for tumor progression.Am J Pathol. 1994; 144: 329-336PubMed Google Scholar). In either case, bFGF appears to be expressed most strongly or exclusively by cells from the junctional component of the nevus, although one contrary observation has recently been reported (Wang et al., 1996Wang Y. Rao U. Mascari R. et al.Molecular analysis of melanoma precursor lesions.Cell Growth Differ. 1996; 7: 1733-1740PubMed Google Scholar). Melanocytic cells cultured from nevi also express bFGF in vitro. Nevus cells show a loss of dependence on exogenous bFGF for growth in culture, although they are still dependent on other mitogens that are necessary to support the growth of normal melanocytes (Mancianti et al., 1993Mancianti M.L. Gyorfi T. Shih I.M. et al.Growth regulation of cultured human nevus cells.J Invest Dermatol. 1993; 100: 281S-287SAbstract Full Text PDF PubMed Scopus (28) Google Scholar). Strong support for the idea that bFGF expression provides autocrine growth stimulus in melanoma cells came from experiments performed by Dotto et al., 1989Dotto G.P. Moellman G. Ghosh S. Edwards M. Halaban R. Transformation of murine melanocytes by basic fibroblast growth factor cDNA and oncogenes and selective suppression of the transformed phenotype in a reconstituted cutaneous environment.J Cell Biol. 1989; 109: 3115-3128Crossref PubMed Scopus (136) Google Scholar, who characterized the transforming effects of bFGF expression in murine melanocytes. Overexpression of bFGF in a murine melanocyte cell line led to loss of dependence on phorbol ester for growth. The cells also adopted an undifferentiated phenotype, as they switched from a dendritic to a fibroblastic cell shape and ceased pigment production; however, it is difficult to draw direct inferences about the effects of bFGF expression in normal human melanocytes from these experiments. The murine melanocytes used in these experiments were already independent of certain mitogens necessary for the long-term culture of human melanocytes. They did not require external inducers of adenylate cyclase and they did not have an absolute requirement for bFGF. Also, the cell line used in these experiments had undergone numerous passages in culture, and the cells had an abnormal karyotype, indicating that they may have already been partially transformed. Although the expression of bFGF seems to promote the growth of melanoma cells, its mechanism of action, especially in cells from earlier stages of progression, remains undefined. We have created a model system in which to study whether expression of bFGF in normal melanocytes is sufficient to reproduce the in vitro phenotype of cells from the early stages of melanoma tumor progression. In particular we wanted to determine whether expression of bFGF in these cells caused loss of normal growth control by conferring independence from exogenous bFGF. We have expressed bFGF in normal human epidermal melanocytes through transfer of a human bFGF cDNA in an amphotropic murine retrovirus. Expression of bFGF in these cells did not render them independent of the exogenous mitogen. The melanocytes that produced bFGF did not exhibit other malignant phenotypes as compared with melanocytes that were infected with control virus or normal uninfected melanocytes. The bFGF produced by these melanocytes was biologically active, because it stimulated DNA synthesis in 3T3 cells. The bFGF also localized to the nucleus of melanocytes, as has been reported in other cell types. These results indicate that constitutive expression of bFGF alone is not sufficient to reproduce the in vitro phenotype of cells from early melanocytic lesions. Normal human epidermal melanocytes (NHEM) from neonatal foreskins were obtained from Clonetics (San Diego, CA). Melanocyte basal medium consisted of four parts MCDB 153 (Sigma, St. Louis, MO) to one part Leibovitz’s L-15 medium (Gibco/BRL, Gaithersburg, MD) with 2 mM Ca+2, 5 μg insulin per ml, 0.5 μg hydrocortisone per ml, and 25 μg gentamicin per ml (Herlyn et al., 1988Herlyn M. Mancianti M.L. Jambrosic J. Bolen J.B. Koprowski H. Regulatory factors that determine growth and phenotype of normal human melanocytes.Exp Cell Res. 1988; 179: 322-331Crossref PubMed Scopus (83) Google Scholar). For routine culture, basal medium was supplemented with 1 ng recombinant human bFGF per ml (Promega, Madison, WI), 10 ng 12-O-tetradecanoyl-phorbol-13-acetate (TPA, Calbiochem, San Diego, CA) per ml; 1% fetal calf serum (Gemini Bio-Products, Calabasas, CA); and either 6.5 μg bovine pituitary extract (Collaborative Biomedical Products, Bedford, MA) per ml or 10 ng α-melanocyte stimulating hormone (α-MSH, Sigma) per ml. The primary melanoma cell line WM1650 was kindly provided by Dr. Meenhard Herlyn (Department of Molecular and Cellular Biology, The Wistar Institute, Philadelphia, PA), and was cultured in the same medium as normal melanocytes. The metastatic melanoma cell line MelJuSo was given to us by Dr. Judith Johnson (Institut fur Immunologie, Munich, Germany). Cultures of PA317 retrovirus packaging cells and the mouse 3T3tk– cell line from which it was derived were obtained from Dr. R. Eisenman (Fred Hutchinson Center for Cancer Research, Seattle, WA). Swiss 3T3 mouse fibroblasts came from the laboratory of Dr. Daniel Straus (University of California, Riverside, CA). MelJuSo melanoma cells and all mouse cell lines were maintained in Dulbecco’s modified Eagle’s medium with 5% fetal calf serum. A human bFGF cDNA (kindly donated by Dr. R. Florkiewicz, Prizm Pharmaceuticals, San Diego, CA) was cloned into pLXSN (Miller and Rosman, 1989Miller A.D. Rosman G.J. Improved retroviral vectors for gene transfer and expression.BioTechniques. 1989; 7: 980-990PubMed Google Scholar), which contains the modified proviral components of Moloney murine leukemia virus (Mann et al., 1983Mann R. Mulligan R.C. Baltimore D. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus.Cell. 1983; 33: 153-159Abstract Full Text PDF PubMed Scopus (1116) Google Scholar). The virus also carries a neo gene that confers resistance to the antibiotic G418, allowing for selection of cells with integrated virus. This construct was transfected via calcium phosphate transfection into the amphotropic packaging cell line PA317 (Miller and Buttimore, 1986Miller A.D. Buttimore C. Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production.Mol Cell Biol. 1986; 6: 2895-2902Crossref PubMed Scopus (1145) Google Scholar). G418 resistant PA317 cell clones were screened for virus production by slot blot analysis: RNA extracted from viral supernatants was blotted onto nylon filters and hybridized with a 32P-labeled human bFGF cDNA fragment followed by autoradiography. The titer of virus from clones producing substantial amounts of viral RNA was determined on 3T3tk– mouse fibroblasts. Viral supernatants were harvested from confluent flasks of producer cells after incubation in Iscove’s medium (Irvine, Santa Ana, CA) with 10% fetal calf serum for 24 h, and were passed through 0.2 μm filters before use. Melanocyte cultures at 25–50% confluence were incubated with viral supernatant with 8 μg polybrene (Sigma) per ml for 3 h, followed by refeeding with nonselective medium. Infected melanocyte cultures were allowed to grow for 7–10 d before addition of selective medium containing 500 μg G418 (Geneticin, Gibco-BRL) per ml, and colonies grew out of selection over the next 20–30 d. From each infection, all resulting clones were collected as mass populations for use in subsequent experiments. Retrovirus that contains only the neo gene was used to create control populations of melanocytes. The packaging cell lines that produce the bFGF virus and the neo-only virus yield supernatants with titers (on 3T3tk– cells) of 107 and 108 G418 resistant colony forming units (cfu) per ml, respectively. RNA was prepared from melanocyte cultures by the method of Chomczynski and Sacchi, 1987Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63147) Google Scholar, and analyzed by northern blot as described elsewhere (Thayer et al., 1990Thayer M.J. Lugo T.G. Leach R.J. Fournier R.E.K. Regulation of chimeric phosphoenolpyruvate carboxykinase genes by the trans-dominant locus TSE1.Mol Cell Biol. 1990; 10: 2660-2668Crossref PubMed Scopus (7) Google Scholar). Filters were hybridized with a 32P-labeled human bFGF cDNA fragment (Abraham et al., 1986Abraham J.A. Whang J.L. Tumolo A. Mergia A. Friedman J. Gospodarowicz D. Fiddes J.C. Human basic fibroblast growth factor: nucleotide sequence and genomic organization.EMBO J. 1986; 5: 2523-2528Crossref PubMed Scopus (480) Google Scholar) to detect bFGF mRNA. Filters were stripped and re-probed with a DNA fragment of rat 18s rRNA as a loading control. Cell lysates for western blot analysis were prepared and partially purified over heparin-Sepharose (Pharmacia Biotech, Piscataway, NJ) as described in Florkiewicz et al., 1991Florkiewicz R.Z. Baird A. Gonzalez A.-M. Multiple forms of bFGF. differential nuclear and cell surface localization.Growth Factors. 1991; 4: 265-275Crossref PubMed Scopus (196) Google Scholar. Heparin binding proteins were eluted directly into electrophoresis sample buffer. The partially purified lysates were separated by electrophoresis on a 15% sodium dodecyl sulfate-polyacrylamide gel, and were electroblotted onto a nitrocellulose membrane in 25 mM AMPSO (3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid) (Sigma) and 20% methanol. A monoclonal antibody against bovine bFGF (anti-bovine bFGF type 2, Upstate Biotechnology, Lake Placid, NY) was utilized to detect bFGF, using anti-mouse IgG secondary antibody conjugated to horseradish peroxidase (Sigma) followed by visualization with a chemiluminescent substrate (ECL, Amersham Life Science, Bucks, U.K.). Assays to measure cellular proliferation rates were conducted over a 21 d period. Cells were plated into 24 well plates at 104 cells per well in standard growth medium. Wells were precoated with 1% gelatin before plating. The next day, designated day 0, the medium was replaced with experimental medium consisting of melanocyte basal medium plus 0.75% fetal calf serum and 10 ng α-MSH per ml, with or without 1 ng recombinant human bFGF per ml. Cells from triplicate wells per condition were trypsinized and counted by hemacytometer on day 0 and at 7 d intervals thereafter. The short-term mitogenic response to bFGF was assayed by measuring DNA synthesis. Again, 104 cells per well were plated in 24 well plated precoated with 1% gelatin. Cells were plated in standard growth medium and allowed to attach for 24 h, at which time the medium was replaced with melanocyte basal medium plus 1 mg bovine serum albumin per ml, with or without addition of 1 ng bFGF per ml, 10 ng TPA per ml, and 10 ng α-MSH per ml. Cells were incubated for 4 d with a change of medium on day 3 and addition of 5 μCi [3H]thymidine per ml (80–90 Ci per mMol, Amersham) during the last 12 h. The cells were then trypsinized and harvested onto glass fiber filters (Halaban et al., 1987Halaban R. Ghosh S. Baird A. bFGF is the putative natural growth factor for human melanocytes.In Vitro Cell Devel Biol. 1987; 23: 47-52Crossref PubMed Scopus (211) Google Scholar), followed by washing with deionized water. After drying, the amount of radioactivity retained on each filter was measured by scintillation counting. Results were averaged from triplicate wells per condition. The assay for growth in soft agar was performed as described previously (Lugo and Witte, 1989Lugo T.G. Witte O.N. The BCR-ABL oncogene transforms Rat-1 cells and co-operates with v-myc..Mol Cell Biol. 1989; 9: 1263-1270Crossref PubMed Google Scholar), but using melanocyte basal medium supplemented with 1 ng bFGF per ml, 10 ng α-MSH per ml, and 10% fetal calf serum. The mitogenic activity in conditioned medium and cell extract was determined for cultures plated at 2.5 × 105 cells in 25 cm2 flasks. Conditioned medium was prepared by incubating melanocyte cultures in serum-free Iscove’s medium plus antibiotics for 24 h. Conditioned medium was cleared of cell debris by centrifugation at 200 ×g for 10 min. To prepare cell extracts, cells were trypsinized after removal of conditioned medium and washed once with serum-free Iscove’s medium. The cells were then resuspended in 0.5 ml serum-free medium and sonicated on ice using two 20 s pulses. The extract was clarified by centrifugation at 13,500 ×g for 10 min at 4°C. The total protein concentration of cell extracts was determined by Bradford assay (Bradford, 1976Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding.Anal Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215608) Google Scholar). Mitogenic activity was determined on Swiss 3T3 cells plated at 104 cells per well in 24 well plates, which were rendered quiescent by incubation in serum-free medium for 24 h. 3T3 cells were exposed to 100 μl of cell extract per well, or were refed with conditioned medium, followed by incubation for 24 h with addition of 5 μCi [3H]thymidine per ml during the last 5 h. The 3T3 cells were then harvested and processed as described above. Cells were plated on glass coverslips and allowed to attach for 48 h. The cells were fixed by treatment with 3% paraformaldehyde for 8 min at room temperature followed by permeablization in methanol at –20°C for 5 min. Fixed cells were blocked with 5% goat serum in PHEM buffer, which consists of 60 mM PIPES (piperazine-N,N′-bis[2-ethanesulfonic acid]), 25 mM HEPES (N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]), 10 mM ethyleneglycol-bis(b-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, and 2 mM MgCl2 at pH 6.9. All antibody incubations were done in PHEM buffer. The cells were incubated with a monoclonal antibody to bovine bFGF (Upstate Biotechnology) for 2 h at room temperature and then washed with three changes of buffer. An anti-mouse IgG secondary antibody conjugated to fluorescein isothiocyanate was then applied to cells for 1 h, followed by washing and microscopic observation under ultraviolet illumination. An amphotropic, nonreplicative retrovirus carrying a human bFGF cDNA was constructed in order to produce constitutive bFGF expression in normal melanocytes. NHEM from neonatal foreskins were infected with the bFGF virus (NHEM-FGF) or the neo-only virus (NHEM-Neo). A typical infection yielded 5–10 G418 resistant colonies of NHEM-FGF and 20–60 colonies of NHEM-Neo. All colonies from each infection were collected as mass populations for expansion and used in subsequent experiments. Two independent populations of both NHEM-FGF and NHEM-Neo were used for experiments. Northern blot analysis revealed high levels of bFGF mRNA in NHEM-FGF whereas uninfected melanocytes produced no detectable message Figure 1a; the single mRNA species detected in NHEM-FGF was consistent with transcription of the bFGF cDNA controlled by the viral vector promoter. Production of the multiple bFGF protein isoforms in NHEM-FGF was confirmed by western blot Figure 1a. The in vitro growth characteristics of NHEM-FGF and NHEM-Neo were examined. In order to distinguish any effects from the number of population doublings undergone by these cells during selection, uninfected melanocytes from the same original culture, which had undergone no more than six population doublings, were also included as controls in these experiments (NHEM). To determine whether expression of bFGF allowed NHEM-FGF to grow in culture independently of exogenous bFGF, the cell growth rate was determined in medium with and without 1 ng bFGF per ml Figure 2a. NHEM-FGF would not divide in the absence of bFGF in the culture medium. No growth was detected after 3 wk in cultures that lacked bFGF, whereas cultures with bFGF grew to high density. As expected, NHEM and NHEM-Neo would also not grow in the absence of bFGF. The growth rates of NHEM-FGF and NHEM-Neo were approximately equal in medium containing bFGF, whereas uninfected melanocytes had a significantly faster growth rate Figure 2a. WM160, a cell line derived from a primary human melanoma, grew to densities equivalent to those of NHEM-FGF and NHEM-Neo, but in the absence of exogenous bFGF Figure 2a. This cell line produces endogenous bFGF as determined by western blot (not shown). A similar effect was seen in short-term assays for growth stimulation by bFGF. We also measured the response of these cells to two other melanocyte mitogens: α-MSH and TPA. Short-term assays were carried out in defined medium in which the response to various mitogens was measured by [3H]thymidine uptake. Stimulation by these mitogens of two independent populations of NHEM-FGF, as well as control populations, is illustrated in Figure 3. Exogenous bFGF stimulated DNA synthesis in NHEM-FGF as readily as it did in NHEM-Neo and uninfected melanocytes. The presence of 10 ng α-MSH per ml enhanced the response to bFGF in a synergistic fashion, as reported previously (Herlyn et al., 1988Herlyn M. Mancianti M.L. Jambrosic J. Bolen J.B. Koprowski H. Regulatory factors that determine growth and phenotype of normal human melanocytes.Exp Cell Res. 1988; 179: 322-331Crossref PubMed Scopus (83) Google Scholar). NHEM-FGF was also stimulated by 10 ng TPA per ml. In experiment 2 Figure 3, the level of [3H]thymidine uptake was higher in all treatments for NHEM-Neo, and the level of incorporation in basal medium was proportionately higher as well; however, NHEM-FGF and NHEM-Neo control cells showed the same overall pattern of response to bFGF, α-MSH, and TPA. Because NHEM-FGF still required exogenous bFGF for growth, it was probable that these cells were not exporting bFGF produced from the transgene into the extracellular medium. In order to assess this, conditioned media and cell extracts from NHEM-FGF and uninfected melanocytes were analyzed for mitogenic activity by measuring their ability to stimulate DNA synthesis in 3T3 cells (Table 1). Neither fraction from NHEM contained any significant mitogenic activity. The cell extract of NHEM-FGF was highly stimulatory for 3T3 cells, whereas the conditioned medium from these cells contained only a small amount of mitogenic activity, which was probably due to bFGF escaping from dead or damaged cells. This assay was repeated three times, and in each case most of the mitogenic activity produced by cells carrying the bFGF transgene appeared to remain intracellular. This suggests that normal melanocytes cannot effectively export bFGF. This experiment also confirms that the bFGF produced by NHEM-FGF is biologically active.Table 1Analysis of transformed phenotypes in experimental melanocyte cultures and melanoma cell linesCell lineSoft-agar PE(%)bPlating efficiency in soft agar, expressed as macroscopically visible colonies formed per 100 cells plated.Mitogenic activity (cpm)aMitogenic activity was determined on 3T3 cells. Results shown are from a representative experiment in which cell extract or conditioned medium from melanocytes was added to cultures of 3T3 cells and DNA synthesis was measured by [3H]thymidine uptake, expressed as cpm. SD is given in parentheses. [3H]Thymidine incorporation of control 3T3 cells that received no additions was 1750 ± 444 cpm.Cell extractConditioned mediumNHEMndcnd, not determined.1.0 × 104(4.0 × 103)1 × 104(3.5 × 103)NHEM-Neo<0.001ndndNHEM-FGF 2.0ndnda Mitogenic activity was determined on 3T3 cells. Results shown are from a representative experiment in which cell extract or conditioned medium from melanocytes was added to cultures of 3T3 cells and DNA synthesis was measured by [3H]thymidine uptake, expressed as cpm. SD is given in parentheses. [3H]Thymidine incorporation of control 3T3 cells that received no additions was 1750 ± 444 cpm.b Plating efficiency in soft agar, expressed as macroscopically visible colonies formed per 100 cells plated.c nd, not determined. Open table in a new tab NHEM-FGF cells were examined for other characteristics of malignant transformation. The morphology of NHEM-FGF, NHEM-Neo, and uninfected melanocytes was similar under phase-contrast microscopy Figure 4. The virally infected melanocytes had a more variable morphology than NHEM, with a tendency to form more dendrites; however, both NHEM-FGF and NHEM-Neo remained primarily bipolar. The expression of bFGF did not appear to affect the pigmentation of NHEM-FGF as determined by visual examination of cell pellets. There was no evidence that NHEM-FGF had an extended lifespan in culture, when compared with NHEM-Neo. There is evidence that loss of anchorage independence is a trait acquired early in tumor progression in melanocytes (Herlyn et al., 1985Herlyn M. Thurin J. Balaban G. et al.Characteristics of cultured human melanocytes isolated from different stages of tumor progression.Cancer Res. 1985; 45: 5670-5676PubMed Google Scholar). We tested the ability of NHEM-FGF cells to grow in a soft-agar medium. Neither NHEM-FGF nor NHEM-Neo could form colo
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