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

Abstract

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.

Ciglitazone negatively regulates CXCL1 signaling through MITF to suppress melanoma growth

“…25 Blocking antibodies to either CXCL1 or CXCR2 inhibit melanoma cell growth showing the key role of the CXCL1/CXCR2-signaling pathway in melanoma development. 26,27 In addition to its autocrine role, CXCL1 has been shown to have an important paracrine role by regulating angiogenesis during tumor development including melanoma. 28,29 First, we show that ciglitazone negatively regulates mRNA and protein CXCL1 levels.…”

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

“…Immunocytochemical localization of MGSA was performed using the FB2AH7 monoclonal antibody to MGSA as previously described [3,7]. The control mouse IgM antibody was to a-lipotechoic acid.…”

“…We also examined intracellular MGSA protein by immunocytochemistry using the FB2AH7 monoclonal antibody to MGSA previously described [7].…”

Enhanced tumor-forming capacity for immortalized melanocytes expressing melanoma growth stimulatory activity/growth-regulated cytokine β and γ proteins

“…Two other MGSA/GRO genes (␤ and ␥) have since been cloned that encode highly related proteins binding to the same receptors as the MGSA/GRO␣ gene product (Haskill et al, 1990). Blocking antibodies to either MGSA/GRO␣ (Lawson et al, 1987) or its receptor, CXCR2 (Norgauer et al, 1996), have been shown to inhibit the growth of melanoma cells in vitro. These data demonstrate that in the melanoma model, MGSA/GRO can act as an autocrine growth factor.…”