B16F10 Murine Melanoma Cells Express interleukin-2 and a Functional lnterleukin-2 Receptor

Abstract

In this study, we analyzed interleukin-2 (IL-2) and IL-2 receptor (IL-2R) expression in murine B16F10 melanoma and studied the effect of recombinant IL-2 (rIL-2) on the proliferation of these cells. Flow cytometry analysis revealed the presence of the IL-2R α subunit in B16F10 melanoma, with a mean posi-tivity rate of 30%. Using confocal microscopy, the expression of this chain could be visualized on the surface of B16F10 cells and in intracellular compartments when the cells were permeabilized with ethanol. In addition to the α subunit, the IL-2R β subunit was also expressed in B16F10 cells as shown by reverse transcription and polymerase chain reaction analysis. The functionality of the IL-2R on B16F10 cells was shown by the fact that cell proliferation increased dose-dependently with the addition of rIL-2 to the culture medium. We also detected expression of the IL-2 gene in B16F10 cells. In Northern blot assays, a typical band of 0.9 kb corresponding to IL·2 mRNA was observed, although supernatants from B16F10 cultures had no detectable IL-2 activity. Furthermore, the addition of neutralizing antibody (anti-IL-2) to cell cultures had no effect on cell proliferation. From these results, we concluded that an IL-2 signalling system is present in murine B16F10 melanoma cells and that IL-2 favors B16F10 cell proliferation, suggesting a role for this cytokine in the tumoral activity of these cells.

Comparison of four methods for the purification and refolding of human interleukin‐2–mouse granulocyte/macrophage colony‐stimulating factor fusion protein

“…For FDC‐P1 cells, the culture medium was supplemented with 10% WEHI‐3 cell‐conditioned medium (WEHI‐3CM). B16F10 cells (IL‐2 receptor‐positive [12] and derived from C57BL/6 mouse melanoma cells, A.T.C.C. CRL‐6475) were stored in our laboratory and maintained in RPMI 1640 medium (Gibco) supplemented with 10% FBS and 50 units/ml penicillin/streptomycin.…”

Serum-Soluble IL-2 Receptor and IL-6 Levels in Patients with Melanoma

“…Among these cytokines, interleukin-2 (IL-2) is of special importance due to its paradoxical effects on tumor growth as described in different reports. On the one hand, IL-2 is used in immunotherapy o f metastatic melanoma and kid ney cancer [1,2], while on the other it has been shown that B16F10 murine melanoma cells express the IL-2/IL-2 receptor (IL-2R) system and that IL-2 has a proliferative effect both in vitro [3] and in vivo [4] on these cells. Other researchers have reported that the IL-2/IL-2R system is also present in different human melanoma cell lines [5][6][7][8], suggesting the possible implication o f IL-2 in the tumor activity o f these cells.…”

Cyclosporin A Upmodulates the α-Subunit of the Interleukin-2 Receptor and the Metastatic Ability of Murine B16F10 Melanoma Cells

“…Isolation of mRNA was performed as previously described [18]. After extraction and quantification, 2 Ìg of poly A + RNA was blotted onto Nylon filters (Schleicher & Schuell, Dassel, Germany), using a Minifold II blotting apparatus (Schleicher & Schuell SRC 072/0).…”

“…However, the expression of the IL-2/IL-2R system has been reported in several tumor cells of nonhematopoietic origin [12][13][14][15][16][17]. In recent experiments, we have demonstrated that B16F10 melanoma cells express IL-2 and IL-2 receptor mRNA, and that the proliferation and metastatic potential of melanoma cells increases upon treatment with IL-2 in vitro [18]. Moreover, to study the modulation of IL-2 mRNA expression, we have treated B16F10 cells with CsA as an inhibitor of the IL-2 gene.…”