|Briske Anderson, Mary
Submitted to: Journal of Federation of American Societies for Experimental Biology
Publication Type: Abstract Only
Publication Acceptance Date: 12/10/1999
Publication Date: 3/15/2000
Citation: Reeves, P.G., DeMars, L.C., Briske-Anderson, M.J. 2000. Physiologic concentrations of media zinc alter the relative abundance of ATP7A MRNA and protein in caco-2 cells [abstract]. The Federation of American Societies for Experimental Biology Journal. 14:A228.
Technical Abstract: Previous studies have shown that modest elevations of zinc (Zn) in the growth media of Caco-2 cells depressed the rate of copper (Cu) efflux to the basolateral side of the cells. The present study determined if this effect is related to a down-regulation of the copper transporter ATP7A. Cells were seeded onto Falcon membranes with high pore density and maintained in DME medium supplemented with 10% FBS, nonessential amino acids, glucose, and glutamine. This medium normally contains 5 uM Zn and 0.5 uM Cu. From d 15 to 21 after seeding, cells were exposed to 5, 25 or 50 uM Zn. At d 21, some of the cells were harvested and mRNA was isolated with oligo (dT)-cellulose. ATP7A mRNA was detected by Northern blotting by using a 1876 bp cDNA probe containing 3 of the metal binding domains and 4 transmembrane regions. Other cells were prepared for the determination of ATP7A protein by Western blotting using an antibody prepared with the peptide, ydpklqtpktlqeaiddm, relatively specific for ATP7A. The results showed that ATP7A mRNA was reduced in cells treated for 7 d with 50 uM but not 25 uM media Zn when compared to cells treated with 5 uM Zn. Further, Western blotting showed that three major proteins cross reacted with the antibody, with molecular masses of approximately 55, 100, and 130 kDa. The intensities of the heavier bands were visibly reduced as the media zinc concentration increased in excess of 5 uM, but the intensity of band 55 was not changed. Although other laboratories have shown that short- term treatment of certain cell types does not affect ATP7A, this study shows that treatment of up to 7 d with 50 uM Zn affects both ATP7A mRNA and protein in Caco-2 cells. (cDNA probe and antibody were supplied by the Laboratory of Dr. J. Gitschier, UCSF).