Coincident expression of Menkes gene with copper efflux in human placental cells

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Abstract
BeWo cells, a human choriocarcinoma cell line, have a high-affinity system for transporting copper ions into the cell (Km = 0.21 microM) but are sluggish in releasing copper back into the medium from preloaded cells. The slow efflux rate has recently been shown to correlate with a failure of BeWo cells to express the Menkes transcript [Y. Qian, E. Tiffany-Castiglioni, and E. D. Harris. Am. J. Physiol. 271 (Cell Physiol. 40). In press]. We have now determined that only when BeWo cells were grown on plastic surfaces such as petri dishes or flasks did they display negligible release and enhanced retention of 67Cu. Reverse transcriptase-polymerase chain reaction with the use of primers selective for the Menkes gene failed to show any evidence of a Menkes transcript in cells cultured on plastic surfaces. In contrast, cells grown on porous filters previously shown to allow apical and basolateral surfaces to develop did display the transcript and showed significant copper release with normal retention. Release of copper from filter-grown cells was blocked with p-chloromercuribenzoate, thus confirming sulfhydryl group involvement. Absorption of the 67Cu, either as a free ion or bound to ceruloplasmin, was unaffected by the different culture conditions. The data link the Menkes gene product with the ability of cells to release copper ions. They also suggest that the expression of the Menkes gene may be regulated by the development of polarized cell membranes.