Preparation of iron-deficient tissue culture medium by deferoxamine-Sepharose treatment and application to the differential actions of apotransferrin and diferric transferrin

Abstract

We have shown that triiodothyronine-dependent GH 1 rat pituitary cell growth in serum-free defined culture required apotransferrin (apoTf) (D. A. Sirbasku, et al., Biochemistry 30, 295–304, 7466–7477, 1991). These studies were done in “low-Fe” medium without Fe(III) Fe(II) salts. Nonetheless, significant concentrations of iron may have been contributed by other components, making this medium unsuitable for study of the differential effects of apoTf and diferric transferrin (2Fe·Tf). Measuring residual iron in culture medium has been troublesome because the most sensitive method (i.e., atomic absorption) detected levels only in excess of 10 ng/ml and did not distinguish between the forms of iron present. To estimate the Fe(III) available to bind to apoTf, we developed a more sensitive and specific method. Urea-polyacrylamide gel electrophoresis (PAGE) separates apoTf, the two monoferric transferrins, and 2Fe·Tf. [ 125I]apoTf was incubated with medium, or components, and the formation of [ 125I]-2Fe·Tf was monitored by urea-PAGE/autoradiography. By this method, the concentration of Fe(III) in low-Fe medium was estimated at 8.4 to 20 ng/ml and the sources were identified. We next sought to remove the Fe(III). Standard chelators were ineffective or cytotoxic. In contrast, an affinity method with deferoxamine-Sepharose depleted ≥90% of the Fe(III). In this medium, apoTf and 2Fe·Tf showed differential effects with GH 1 cells and with MCF-7, MTW9 PL2 , and MDCK cells. With the methods described here, the effects of apoTf and 2Fe·Tf on growth can be studied separately.