Melanoma cells exhibit strong intracellular TASK-3-specific immunopositivity in both tissue sections and cell culture

Abstract

Amplification of the kcnk9 gene and overexpression of the encoded channel protein (TASK-3) seems to be involved in carcinogenesis. In the present work, TASK-3 expression of melanoma cells has been studied. For the investigation of TASK-3-specific immunolabelling, a monoclonal antibody has been developed and applied along with two, commercially available polyclonal antibodies targeting different epitopes of the channel protein. Both primary and metastatic melanoma cells proved to be TASK-3 positive, showing prominent intracellular TASK-3-specific labelling; mostly concentrating around or in the proximity of the nuclei. The immunoreaction was associated with the nuclear envelope, and with the processes of the cells and it was also present in the cell surface membrane. Specificity of the immunolabelling was confirmed by Western blot and transfection experiments. As TASK-3 immunopositivity of benign melanocytes could also be demonstrated, the presence or absence of TASK-3 channels cannot differentiate between malignant and non-malignant melanocytic tumours.

Molecular Background of Leak K+Currents: Two-Pore Domain Potassium Channels

“…Similarly, TASK-3 immunoreactivity was diffusely distributed in the cytoplasm with some perinuclear condensation in gastric carcinoma cells, and the level of expression did not differ significantly from the normal gastric mucosa (177). TASK-3 was also detected predominantly in the perinuclear region of malignant melanoma cells and normal melanocytes (276). Interestingly, in the control experiment, TASK-3 immunolabeling showed clear plasma membrane localization in adrenal glomerulosa cells (276).…”

“…TASK-3 was also detected predominantly in the perinuclear region of malignant melanoma cells and normal melanocytes (276). Interestingly, in the control experiment, TASK-3 immunolabeling showed clear plasma membrane localization in adrenal glomerulosa cells (276). Closer analysis localized TASK-3 immunopositivity to the mitochondria both in malignantly transformed (melanoma) cells and normal keratinocytes (293).…”

Mitochondrial expression of the two-pore domain TASK-3 channels in malignantly transformed and non-malignant human cells

“…Amplification of the kcnk9 gene (encoding the TASK-3 protein) has been noted in a number of malignant tumours, accompanied by a 5-to 100-fold overexpression of the channel protein in a significant portion (44%) of the investigated breast and lung cancers [24]. TASK-3 expression was also reported in malignant stomach and large intestinal tumours [19], and in a recent study, very strong TASK-3 expression has been noted in malignant melanoma cells both in vitro and in vivo [26]. Immunocytochemical and immunohistochemical studies pointed out that the TASK-3 expression was present mostly intracellularly, with relatively insignificant cell surface labelling, suggesting that there might be cell organelles with significant TASK-3 expression.…”

“…The immunohistochemical methods were similar to those reported earlier [26]. All experiments on human tissues were conducted with the authorization of the Ethical Committee of the University of Debrecen as well as with the written consent of the patients.…”

“…Blocking was performed with PBS containing 1% bovine serum albumin for 30 min at room temperature followed by the application of the primary antibody (4°C, overnight). When immunolabelling was performed on whole cells, a monoclonal antibody (1:200-1:2,000) was employed (KO514, Clone KCNK75, Sigma) whose specificity was confirmed elsewhere [26]. The cells were rinsed in PBS (three times) on the following day, incubated with the secondary antibody [fluorescein isothiocyanate (FITC)-conjugated horse anti-mouse antibody; 1:200; Vector Laboratories, Burlingame, CA, USA] for 1 h at room temperature followed by three rinses with PBS.…”