EXPRESSION OF MENKES DISEASE GENE IN MAMMARY-CARCINOMA CELLS

Two P-type ATPases, MNK and WND were recently shown to be defective in the human disorders of copper transport, Menkes disease and Wilson di sease respectively. These proteins are important in copper homeostasis but their full physiological function has not been established. This study uses the human breast carcinoma line, PMC42, to investigate copp er transport in the mammary gland. Northern blot analysis indicated th at both MNK and WND mRNA are expressed in these cells. Western blot an alysis with an MNK-specific antibody demonstrated a band of approx. 17 8 kDa, close to the expected size of 163 kDa. Treatment of PMC42 cells with lactational hormones (oestrogen and progesterone for 3 days foll owed by dexamethasone, insulin and prolactin for a further 3 days) did not produce an obvious increase in MNK expression as measured by Nort hern and Western blots. By using indirect immunofluorescence with the MNK antibody, the intracellular distribution of MNK was found to be pr edominantly perinuclear, consistent with Golgi localization. Punctate staining was also seen in a smaller proportion of cells, suggesting th at some MNK is associated with endosomes. Treatment of PMC42 cells wit h lactational hormones increased the intensity of the perinuclear and punctate fluorescence. Exposure of cells to 100 mM copper resulted in the dispersion of the fluorescence towards the periphery of the cell. The results suggest a role for MNK in the secretion of copper into mil k and that PMC42 cells are a valuable model for investigating the deta iled cellular function of MNK and WND.