Androgen receptors in human melanoma cell lines IIB-MEL-LES and IIB-MEL-IAN and in human melanoma metastases.

The presence and characteristics of androgen receptors (ARs) have been described by our group in one human melanoma cell line. We have now investigated their presence in two other human melanoma cell lines, IIB-MEL-LES and IIB-MEL-IAN, as well as in biopsies from human metastatic melanoma. Scatchard analysis revealed a single binding component for both cell lines, the apparent dissociation constant obtained being 15 nM, with a binding capacity of 280 fmol/mg total cell protein, for IIB-MEL-LES cells and 14 nM, with a binding capacity of 206 fmol/mg total cell protein for IIB-MEL-IAN cells. When specificity was assessed, not only androgen and anti-androgen but also non-androgenic compounds were able to compete for [3H]R1881 binding, as seen before. When immunocytochemistry of IIB-MEL-LES and IIB-MEL-IAN cells was performed for ARs, both cell lines were deeply stained in the nucleus, whereas no staining was found for oestrogen or progesterone receptors. Every specimen of melanoma metastases tested for the presence of ARs was deeply stained, and in the majority the intensity of the staining was high. Several hormones and anti-hormones were tested for their ability to affect cell proliferation. In both cell lines, testosterone, dihydrotesterone, oestradiol and progesterone significantly stimulated cell proliferation, and this was reversed by hydroxyflutamide, bicalutamide or tamoxifen.

Identification and characterization of a 14 kDa immunosuppressive protein derived from IIB-MEL-J, a human melanoma cell line.

The causes of decreased immune competence in melanoma patients as well as in other cancer patients are incompletely understood. The identification of the factor(s) responsible for this behaviour remains elusive. The present report demonstrates that an immunosuppressive activity (ISA), manifested in vitro as an inhibition of proliferation of human peripheral blood lymphocytes (PBL) induced both by phytohemagglutinin (PHA) or the cytokine IL-2, was exhibited by serum-free conditioned medium (SFCM) from the human melanoma cell line IIB-MEL-J, as well as by two other melanoma cell lines, IIB-MEL-LES and IIB-MEL-IAN, established in our laboratory. The ISA was found to be exerted by a protein, which co-eluted with serum albumin in anionic exchange Mono-Q, gel filtration chromatography and Blue Sepharose columns. It showed a molecular weight (Mw) of 14 kDa when separated from albumin traces by means of a Sephacryl S 200 column. It is not recognized by a pan-specific anti-transforming growth factor-beta (TGF-beta) antibody as determined by Western blots assays performed on the SFCM. The immunosuppressive factor (ISF) is secreted by IIB-MEL-J cell line in soluble from and in very scarce amounts, non-detectable by polyacrylamide gel electrophoresis (PAGE). This characteristic difficults its obtention in adequate quantities to sequentiation. Since this inhibitory factor may have a role in protecting melanoma tumors from attack by the host immune system, preparative isolation will be attempted. But we consider these results only as preliminary one's.

Biologic, immunocytochemical, and cytogenetic characterization of two new human melanoma cell lines: IIB-MEL-LES and IIB-MEL-IAN.

Two human melanoma cell lines, derived from metastases of two patients with epithelioid malignant amelanotic melanomas, and designated IIB-MEL-LES and IIB-MEL-IAN, have been established. Both cell lines have been in continuous culture over 2 years and were propagated continuously for 85 and 75 serial passages, respectively. Morphologically, IIB-MEL-LES is composed predominantly of spindle shaped cells, whereas IIB-MEL-IAN grows as a monolayer of cuboid and stellate shaped cells with many rounded cells in suspension. Immunocytochemical studies revealed that both cell lines express S-100 protein, vimentin, and GD3 and GD2 gangliosides but are negative for keratin and collagen. Both cell lines express HLA class I and HLA-DR antigens in variable proportions. The MAGE-1 gene is expressed only by the IIB-MEL-IAN cell line, as revealed by PCR analysis. Cytogenetic analysis of both cell lines revealed abnormal karyotypes; the modal chromosome numbers of IIB-MEL-LES and IIB-MEL-IAN were 48 and 81, respectively. IIB-MEL-LES cells presented rearrangements in chromosomes 1, 14 and X, gains in chromosomes 10, 20, and 21 losses in chromosomes 15 and Y. The most frequent markers observed in IIB-MEL-IAN cells were 7q+, 10p+, 2p+, i(6p), 2q+, and 10q-. Clonal gains were observed in chromosomes 12 and 21, whereas losses were seen in chromosomes 1, 2, 3, 4, 6, 7, 11, and 17. Both cell lines were capable of forming colonies in soft agar and developed tumors when transplanted into nude mice, reproducing and maintaining the characteristics of the original tumors. These cell lines and their xenografts appear to provide useful systems for studying the biology, genetics and histogenesis of human malignant melanoma and could be utilized for the development of melanoma vaccines.

Atypical androgen receptor in the human melanoma cell line IIB-MEL-J.

To evaluate the presence of androgen receptors in the human melanoma cell line IIB-MEL-J, a Scatchard plot analysis was performed. Cells in culture revealed a single binding component with an apparent dissociation constant (KD) at 37 degrees C of 11 nM and a binding capacity of 326 fmol/mg protein when measured with [3H]-R1881. Competition analysis revealed an atypical relaxation of specificity, since not only androgen (testosterone, dihydrotestosterone [DHT], R1881) and antiandrogen (hydroxy-flutamide [OH-FLU]) competed for [3H]-R1881 binding, but also estradiol, progesterone, and cortisol at 500-fold excess concentration. Binding of [3H]-estradiol and [3H]-R5020 in the absence of unlabeled DHT were completely suppressed in its presence. Immunohistochemistry of androgen receptor with a monoclonal antibody showed that nuclei were vigorously stained. Different doses of flutamide (FLU) and OH-FLU tested on cultured IIB-MEL-J cells in the presence of serum inhibited significantly cell proliferation in a dose-dependent manner. When cells were incubated with 10 nM DHT and 1% charcoal-adsorbed serum, a significant stimulation of growth that was observed was inhibited by 4 microM OH-FLU. DHT stimulation was completely reversed by the antiestrogen tamoxifen. In addition, male nude mice transplanted with IIB-MEL-J tumor were treated with FLU when tumors were palpable. FLU was effective in diminishing tumor growth and increasing survival rate of the animals. As a conclusion, the presence of functional androgen receptors in these cells has been demonstrated by growth inhibition in vitro and in vivo with antiandrogens, and their atypical nature is suggested by binding cross-reactivity and competition studies.