TRANSIENT OVEREXPRESSION OF HUMAN H-FERRITIN AND L-FERRITIN CHAINS INCOS CELLS

The understanding of the in vitro mechanisms of ferritin iron incorpor ation has greatly increased in recent years with the studies of recomb inant and mutant ferritins. However, little is known about how this pr otein functions in vivo, mainly because of the lack of cellular models in which ferritin expression can be modulated independently from iron . To this aim, primate fibroblastoid COS-7 cells were transiently tran sfected with cDNAs for human ferritin H- and L-chains under simian vir us 40 promoter and analysed within 66 h. Ferritin accumulation reached levels 300-500-fold higher than background, with about 40% of the cel ls being transfected. Thus ferritin concentration in individual cells was increased up to 1000-fold over controls with no evident signs of t oxicity. The exogenous ferritin subunits were correctly assembled into homopolymers, but did not affect either the size or the subunit compo sition of the endogenous heteropolymeric fraction of ferritin, which r emained essentially unchanged in the transfected and non-transfected c ells. After 18 h of incubation with [Fe-59]ferric-nitrilotriacetate, c ellular iron incorporation was similar in the transfected and non-tran sfected cells and most of the protein-bound radioactivity was associat ed with ferritin heteropolymers, while H-and L-homopolymers remained i ron-free. Cell co-transfection with cDNAs for H-and L-chains produced ferritin heteropolymers that also did not increase cellular iron incor poration. It is concluded that transient transfection of COS cells ind uces a high level of expression of ferritin subunits that do not co-as semble with the endogenous ferritins and have no evident activity in i ron incorporation/metabolism.