Characterization of the role of melanoma growth stimulatory activity (MGSA) in the growth of normal melanocytes, nevocytes, and malignant melanocytes

Melanoma growth stimulatory activity (MGSA) was originally described as an endogenous growth factor for human melanoma cells. To test the hypothesis that an MGSA autocrine loop is responsible for the partial freedom from growth control observed in nevocytes and melanoma cells, MGSA growth response and MGSA mRNA/protein levels were examined in these cells compared with normal melanocytes. As a single agent, or in combination with other factors, MGSA stimulated the growth of normal human epidermal melanocytes as well as other growth promoters for melanocytes. Nevocytes were not as responsive to exogenous MGSA as melanocytes. MGSA mRNA was minimal or not detected in cultured normal melanocytes, although the protein was present when the cells were cultured in the presence of serum/growth factors and absent when serum/growth factors were omitted. In contrast, MGSA mRNA was constitutively expressed in the absence of exogenous growth factors in cultures established from benign intradermal and dysplastic nevi and melanoma lesions in different stages of tumor progression. Nevus cultures contained immunoreactive MGSA protein in the presence of serum but were negative or only faintly positive in the absence of serum. Melanoma cell lines were positive for MGSA protein in both the presence and the absence of serum. Thus, continued expression of both MGSA mRNA and MGSA protein in the absence of exogenous hormones or serum factors may correlate with partial freedom from growth control exhibited by malignant melanocytes.     

Recombinant expression, biochemical characterization, and biological activities of the human MGSA/gro protein

Melanoma growth stimulatory activity (MGSA) is a mitogenic protein secreted by Hs294T melanoma cells that corresponds to the polypeptide encoded by the human gro gene. The MGSA/gro cDNA has been expressed in mammalian cells and the secreted recombinant factor has been purified. Biochemical and biological characterization shows that the recombinant protein is identical with the natural protein and is devoid of posttranslational glycosylation, sulfation, and phosphorylation. The two C-terminal amino acids are proteolytically removed from the mature recombinant MGSA, indicating a length of 71 instead of the predicted 73 amino acids. The recombinant MGSA is mitogenically active on the Hs294T melanoma cells. The purified MGSA competes with interleukin 8 for binding to neutrophil receptors and exhibits neutrophil chemotactic activity equivalent to that of interleukin 8.     

Expression and secretion of gro/MGSA by stimulated human endothelial cells.

Melanoma growth stimulatory activity factor (MGSA) is a polypeptide which was initially isolated from Hs294 human melanoma cells. Its sequence is identical to the deduced amino acid sequence of the human gro cDNA, isolated from a human tumor cell line. MGSA stimulates the proliferation of malignant melanoma cells, but its function for normal cells has not been defined. Here we report that human umbilical vein endothelial cells are capable of synthesizing and secreting MGSA. The expression and secretion of MGSA are strongly induced by factors often involved in inflammation such as IL-1, TNF, LPS and thrombin. The induction of MGSA mRNA is dose and time dependent and is independent of new protein synthesis. This stimulation could be mimicked by TPA, suggesting that the action could be mediated through activation of protein kinase C. Furthermore, addition of MGSA to the endothelial cell cultures induces gro/MGSA gene expression, implying that an autocrine mechanism exists. Our data suggest that the protein encoded by gro/MGSA mRNA may play a role in inflammation and exert its effects on endothelial cells in an autocrine fashion.     

Growth factor modulation of melanoma growth stimulatory activity mRNA expression in human malignant melanoma cells correlates with cell growth

This report demonstrates that the expression of melanoma growth stimulatory activity (MGSA) mRNA can be modulated in a positive fashion in the Hs294T human melanoma cell line by PDGF and MGSA. There is close correlation between MGSA expression and the pattern of cell growth in Hs294T cells.     

Preparation of a monoclonal antibody to a melanoma growth-stimulatory activity released into serum-free culture medium by Hs0294 malignant melanoma cells

Autostimulatory growth factors may contribute to the ability of malignant cells to escape normal growth controls. We have previously shown that Hs0294 human malignant melanoma cells release into culture medium an acid-soluble, heat-stable, trypsin-sensitive, autostimulatory monolayer mitogen which can be purified from acetic acid extracts of conditioned medium by gel filtration, reverse-phase high-performance liquid chromatography, and preparative electrophoresis. The majority of this melanoma growth-stimulatory activity (MGSA) resides in a 16-Kd moiety, though bioactivity is also associated with 24-26 and less than 14-Kd forms of MGSA (Richmond and Thomas: J Cell Physiol 129:375, 1986). In order to further characterize this growth factor, monoclonal antibodies were prepared against a partially purified preparation of the autostimulatory melanoma mitogen. Monoclonal antibody clones were selected based on supernate inhibition of 3H-thymidine incorporation in serum-free Hs0294 melanoma cultures. One of these, termed FB2AH7, slows, but does not completely block, the growth of Hs0294 cells in serum-free medium in a dose-dependent manner. This antibody does not slow the growth of normal rat kidney fibroblasts, which neither produce nor require this mitogen, in either serum-free medium or medium containing 0.8% calf serum. This monoclonal antibody also blocks the mitogenic effects of partially purified preparations of this melanoma growth stimulatory activity (MGSA) on both Hs0294 cells and normal rat kidney fibroblasts. The FB2AH7 antibody has been demonstrated to bind MGSA by Western blot and by immunoprecipitation procedures. Western blot analysis of reverse-phase high-performance liquid chromatography purified growth factor demonstrated that FB2AH7 antibody binds to the 16-Kd and approximately 13-14-Kd forms of MGSA. FB2AH7 antibody can be used in immunoprecipitation experiments to bind the approximately 13-16-Kd forms of MGSA. The specificity of the binding of FB2AH7 antibody for MGSA but not other growth factors has been demonstrated in a modified dot blot assay. These data thus support the hypothesis that MGSA is an autostimulatory melanoma mitogen distinct from other growth factors.     

Purification of melanoma growth stimulatory activity

The Hs0294 human malignant melanoma cell line produces a monolayer mitogen that stimulates the serum free growth of low-density cultures of Hs0294 cells. This report describes the purification of that mitogen, termed MGSA for melanoma growth stimulatory activity, from serum-free conditioned medium from the Hs0294 cultures. MGSA has been purified from acetic acid extracts of lyophilized conditioned medium by gel filtration, reverse-phase high-pressure liquid chromatography (RP-HPLC), and preparative electrophoresis, resulting in a greater than 400,000-fold purification. MGSA bioactivity resides in acid- and heat-stable polypeptides of high and low molecular weight (24-28 kd and less than 14-16 kd). However, the majority of the activity is reproducibly associated with the approximately 16-kd moiety eluting from RP-HPLC at approximately 35% acetonitrile. Reduction with dithiothreitol or B-mercaptoethanol results in a loss of biological activity but does not convert the 24-28-kd moieties to the less than 14-16-kd forms of MGSA. 125I-MGSA that has been purified by preparative electrophoresis (16 kd) specifically binds to Hs0294 melanoma cells and retains 100% of the growth-stimulatory activity. The 16-kd MGSA stimulates the proliferation of Hs0294 cells at concentrations of 0.3-30 pM. The electrophoretic mobility of MGSA is also unaltered by the preparative electrophoresis procedure, further demonstrating that this procedure does not alter the biochemical integrity of the growth factor. Purified MGSA does not enable anchorage-independent growth of normal rat kidney (NRK) cells and is therefore different from the previously described transforming growth factors. The amino acid composition of MGSA differs from that of other previously described growth factors. These data demonstrate that MGSA represents a separate class of growth factors with biological and biochemical properties different from other growth factors.     

Melanoma growth stimulatory activity: isolation from human melanoma tumors and characterization of tissue distribution

Melanoma growth stimulatory activity (MGSA) is an acid and heat stable, auto-stimulatory growth factor which was first isolated from culture medium conditioned by the Hs294T human melanoma cell line. In this report, we describe the purification of MGSA from acid ethanol extracts of Hs294T tumors grown in nude mice using a series of Bio-Gel P30, reverse phase-high performance liquid chromatography and heparin-sepharose steps. This modified procedure provides a 10-fold improved yield of MGSA over previously published procedures. Purified MGSA-stimulated melanoma cell growth in both 3H-thymidine and cell number assays over a concentration range of 0.06 to 6 ng/ml. The MGSA bioactivity was primarily associated with fractions which exhibited molecular weights of 16 and 13-14 Kd based upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-1), transforming growth factor-beta (TGF beta), and epidermal growth factor (EGF) in combination with TGF beta did not stimulate 3H-thymidine incorporation in Hs294T cells under the conditions used for MGSA bioassay. Monoclonal antibody to MGSA was used to screen melanoma and benign nevus cultures as well as fixed sectioned tissue for MGSA. The majority of the melanoma cultures were MGSA positive, while most nevus cultures were MGSA negative. However, when fixed sectioned tissue was screened for MGSA immunoreactivity, melanoma tissue was MGSA positive and three-fourths of the benign nevi were MGSA positive. In addition, epidermal keratinocytes and several tissues exhibiting proliferative disorders contained immunoreactive MGSA. These data suggest that MGSA may be a normal regulator of growth and that the microenvironment of the cell may regulate both production of MGSA and response to MGSA.     

Cloning and expression of cDNA encoding human basic fibroblast growth factor

A cDNA encoding human basic fibroblast growth factor (bFGF) was isolated from a human foreskin fibroblast cDNA library. The cDNA, 4 kilobases in size, had a coding sequence, 5' and 3' untranslated regions, and a poly(A) chain. Isolation of additional cDNA clones that had a short 3' untranslated region suggested the presence of multiple mRNA forms. By Northern blot analysis, at least five bFGF mRNA species were detected in cultured fibroblast cells. Transfection of the cDNA to COS cells resulted in the detection of mitogenic activity in the culture medium of the transfected cells, suggesting that a part of the synthesized protein might be secreted from cells despite its unusual short signal sequence.     

Characterization of two high affinity human interleukin-8 receptors.

Interleukin 8 (IL-8) and melanocyte growth-stimulatory activity/gro (MGSA) are structurally related proinflammatory cytokines that are chemoattractants and activators of neutrophils. Recently, cDNA clones encoding a high affinity IL-8 receptor (IL-8R-A) and a "low affinity" IL-8 receptor (IL-8R-B) have been isolated from human cDNA libraries. These two receptors have 77% amino acid identity and are members of the G protein-coupled superfamily of receptors with seven transmembrane domains. We have expressed these two receptors in mammalian cells and find that in this system both receptors bind IL-8 with high affinity (Kd approximately 2 nM). The receptor affinities differ for MGSA, however. IL-8R-A binds MGSA with low affinity (Kd approximately 450 nM); IL-8R-B binds MGSA with high affinity (Kd approximately 2 nM). The transfected cells respond to ligand binding with a transient increase in the intracellular Ca2+ concentration. A Ca2+ response is found for IL-8R-A following the binding of IL-8; no response is found for MGSA. A Ca2+ response for IL-8R-B follows the binding of both ligands. Blot hybridization with oligonucleotide probes specific for the two receptors shows that mRNA for both receptors is present in human neutrophils. Analysis of IL-8 and MGSA binding data on neutrophils as well as Ca2+ response and desensitization data shows that the presence of these two IL-8 receptors on the cell surface can account for the profile of these two ligands on neutrophils.     

Epidermal growth factor-like transforming growth factor. I. Isolation, chemical characterization, and potentiation by other transforming factors from feline sarcoma virus-transformed rat cells

An acid-stable transforming growth factor (TGF) that interacts with epidermal growth factor (EGF) receptors and is structurally related to EGF was isolated from serum-free culture fluids of Snyder-Theilen feline sarcoma virus-transformed rat embryo (FeSV-Fre) cells. Purification of this EGF-like TGF (eTGF) was achieved by molecular filtration chromatography and successive reverse-phase high pressure liquid chromatography steps on octadecyl support eluted with acetonitrile and 1-propanol gradients, respectively. Rat eTGF consists of a 7.4-kD single polypeptide chain that co-migrates with biological activity in dodecyl sulfate-polyacrylamide electrophoresis gels. Like preparations of a related TGF from human melanoma cells (Marquardt, H., and Todaro, G.J. (1982) J. Biol. Chem. 257, 5220-5225), but unlike EGF from rat, human, or mouse, rat eTGF has phenylalanine and lacks methionine. However, the sequence of the first 30 amino acid residues in rat eTGF is H2N-Val-Val-Ser-His-Phe-Asn-Lys-Cys-Pro-Asp-Ser-His-Thr-Gln-Tyr-Cys-Phe-His-Gly - Thr-(x)-Arg-Phe-Leu-Val-Gln-Glu-Glu-(Lys)-(Lys)-, which is significantly (20% and 28%) homologous to the NH2-terminal region of mouse EGF and human EGF, respectively. In addition to eTGF, molecular filtration chromatography of acid-soluble extracts from medium conditioned by FeSV-Fre cells resolved a 14-kD transforming factor(s) apparently devoid of intrinsic mitogenic activity but able to elicit a strong anchorage-independent growth response in the presence of eTGF or EGF. These results show that: 1) a 7.4-kDa TGF structurally and functionally related to EGF has been isolated from FeSV-Fre cells and 2) the full anchorage-independent growth-promoting activity of medium conditioned by FeSV-Fre cells is due to the coordinate action of at least two types of factors, the 7.4-kDa eTGF and a second 14-kDa transforming factor(s).     

The murine transforming growth factor-beta precursor

Transforming growth factor-beta (TGF-beta) is a homodimeric polypeptide which can act, often in cooperation with other growth factors, as a mitogenic factor for a variety of cells. TGF-beta can also exert growth inhibitory activity on many other cell lines. We have isolated cDNAs coding for the murine TGF-beta cDNA precursor. The deduced amino acid sequence localizes the 112-amino acid long TGF-beta monomer to the C terminus of the precursor. Two areas of the precursor exhibit a marked degree of homology to the human counterpart. One of these regions comprises the mature TGF-beta monomer, while the other corresponds to the NH2 terminus of the precursor and suggests an important biological function for this area. Northern hybridization results identify a major 2.5-kilobase TGF-beta mRNA and several minor TGF-beta mRNA species.     

The endogenous fibroblast growth factor-2 antisense gene product regulates pituitary cell growth and hormone production

Basic fibroblast growth factor (bFGF; FGF-2) is one of 19 related members of a growth factor family with mitogenic and hormone-regulatory functions. In Xenopus laevis oocytes, a 1.5-kb FGF-2 antisense (GFG) RNA complementary to the third exon and 3'-untranslated region (UTR) of FGF-2 mRNA has been implicated in FGF-2 mRNA editing and stability. The human homolog has been cloned, and we localized this gene by yeast artificial chromosome (YAC), somatic cell, and radiation hybrid panels to the same chromosomal site as FGF-2 (chromosome 4, JO4513 adjacent to D4S430), confirming this as a human endogenous antisense gene. The full-length GFG antisense RNA encodes a 35-kDa protein, which is highly homologous with the MutT family of antimutator nucleosidetriphosphatases (NTPases). We show that human pituitary tumors express FGF-2 and its endogenous antisense partner GFG. While normal pituitary expresses GFG but not FGF-2, pituitary adenomas express FGF-2 and have reduced levels of GFG; aggressive and recurrent adenomas expressed more FGF than GFG mRNA. To examine the effects of this antisense gene in the pituitary, we transfected the pituitary-derived GH4 mammosomatotroph cell line with constructs encoding the full-length human GFG cDNA. Transiently and stably transfected cells expressed the 35-kDa GFG protein that was localized to the cytoplasm. These cells exhibited enhanced PRL expression as documented by transiently transfected PRL-luciferase reporter assay and by endogenous PRL protein. GFG expression in these cells did not alter endogenous FGF-2 expression but increased the proportion of the higher molecular mass 22-kDa form of GH. Moreover, GFG expression inhibited cell proliferation as shown by [(3)H]thymidine incorporation, proliferating cell nuclear antigen (PCNA) nuclear staining, and cell cycle analysis. We conclude that the GFG-encoded protein has divergent hormone-regulatory and antiproliferative actions in the pituitary that are independent of FGF-2 expression. GFG represents a novel mechanism involved in restraining pituitary tumor cell growth while promoting hormonal activity.     

Mouse and human GTPBP2, newly identified members of the GP-1 family of GTPase

We earlier identified the GTPBP1 gene which encodes a putative GTPase structurally related to peptidyl elongation factors. This finding was the result of a search for genes, the expression of which is induced by interferon-gamma in a macrophage cell line, THP-1. In the current study, we probed the expressed sequence tag database with the deduced amino acid sequence of GTPBP1 to search for partial cDNA clones homologous to GTPBP1. We used one of the partial cDNA clones to screen a mouse brain cDNA library and identified a novel gene, mouse GTPBP2, encoding a protein consisting of 582 amino acids and carrying GTP-binding motifs. The deduced amino acid sequence of mouse GTPBP2 revealed 44.2% similarity to mouse GTPBP1. We also cloned a human homologue of this gene from a cDNA library of the human T cell line, Jurkat. GTPBP2 protein was found highly conserved between human and mouse (over 99% identical), thereby suggesting a fundamental role of this molecule across species. On Northern blot analysis of various mouse tissues, GTPBP2 mRNA was detected in brain, thymus, kidney and skeletal muscle, but was scarce in liver. Level of expression of GTPBP2 mRNA was enhanced by interferon-gamma in THP-1 cells, HeLa cells, and thioglycollate-elicited mouse peritoneal macrophages. In addition, we determined the chromosomal localization of GTPBP1 and GTPBP2 genes in human and mouse. The GTPBP1 gene was mapped to mouse chromosome 15, region E3, and human chromosome 22q12-13.1, while the GTPBP2 gene is located in mouse chromosome 17, region C-D, and human chromosome 6p21-12.     

The genes for basic and acidic fibroblast growth factors are on different human chromosomes

Basic and acidic fibroblast growth factor (FGF) are related both structurally and functionally. A bovine basic FGF cDNA and a human acidic FGF genomic fragment were used as hybridization probes in Southern blot analysis of DNAs isolated from a panel of 30 mouse-human cell hybrids. The gene encoding basic FGF was assigned to human chromosome 4, and the gene for acidic FGF to human chromosome 5. The two growth factors which are presumed to have a common evolutionary ancestor are therefore not linked. A HindIII restriction fragment length polymorphism was detected for human basic FGF.