Intracellular Iron-Binding Macromolecules in HeLa Cells

Abstract

The concentration of the iron-chelating agent, desferrioxamine (Desferal), that just inhibits iron entry into HeLa cells is also the concentration that inhibits DNA synthesis. As a first step in clarification of the mechanism whereby iron may partake in DNA synthesis, we have partially characterized several of the intracellular iron-binding sites. Most cytoplasmic iron appears to be bound to a polysaccharide containing glucose that sediments at about 32 S. Nucleolar iron is bound to a single protein, the mobility of which is independent of the concentration of sodium dodecyl sulfate in an acrylamide gel. In contrast the pattern and mobility of nuclear iron, other than nucleolar, is heterogeneous and markedly affected by the concentration of sodium dodecyl sulfate. The evidence suggests that nuclear iron is bound to protein through one or more intermediate(s).Addition of the iron-chelating agent, desferrioxamine (Desferal, Ciba), to HeLa cells selectively prevents entry of 55Fe (as citrate) and reversibly inhibits DNA synthesis, with little effect on RNA or protein synthesis (1). In the present paper we note that the concentration of desferrioxamine that just inhibits entry of iron into cells is precisely that concentration that also causes cessation of DNA synthesis, implying that perpetual addition or replacement of iron is critical for cell division. Thus it is of interest to characterize the intracellular iron-binding sites in HeLa cells since one or more of them may function in the regulation of DNA synthesis. Granick (2) has suggested that ferritin is the principal iron-binding site in organs such as spleen and liver, while Wacker and Vallee (3) have given evidence indicating that most tissue iron is bound to RNA. Eichorn (4) showed that Fe++ stabilized hydrogen bonds in solutions of calf-thymus DNA and felt that the metal probably complexed with P04-3 groups. We here present data suggesting that, in contrast to the findings of Wacker and Vallee, most cytoplasmic iron is bound to polysaccharide(s); nuclear iron, on the other hand, appears to be bound to several proteins, possibly by way of an intermediate. Although nucleoplasmic iron has a heterogeneous distribution on acrylamide gels, nucleolar iron is bound to a single protein. Thus, while most body iron is undoubtedly bound to certain longrecognized repositories such as heme, transferrin, ferritin, and various enzymes, e.g., cytochrome oxidase (5), it now appears that a significant fraction has a previously unsuspected distribution and the macromolecules involved probably have functions yet to be elucidated. MATERIALS AND METHODSCells. HeLa cells, subline S3, maintained in Eagle's suspension culture medium (6) supplemented with 7% fetal-calf serum were used throughout.Effects of Desferrioxamine Concentration on Macromolecular Synthesis. Aliquots containing 50 ml of logarithmically growing HeLa cells (2 X 105 cells per ml) in suspension culture were treated with desferrioxamine at concentrations ranging from 0-1 mg/ml. Uptake of ['4C]thymidine, [1...

Mucopolysaccharides associated with nuclei of cultured mammalian cells.

“…of rat liver nuclei by heparin. Another possible function of acidic polysaccharides in regulating DNA synthesis is indicated by the studies of Robbins et al (27). Kinoshita has presented evidence (28) for a heparin-like polysaccharide which is released within the cytoplasm during cleavage of sea Proc.…”

A cytochemical study of the embryo sac in Lilium longiflorum. I. Changes in the distribution of iron during the embryo sac development.

“…Although all nuclei or chromosomes and nucleoli in the embryo sac have no Prussian blue deposits, it may not be possible to conclude that they contain no iron or iron compounds. Because Robbins andPederson (1970, 1972) have reported on the existence of iron in the nuclei and nucleoli of the HeLa cells, and moreover, because there is a possibility that masked iron is negative against the Prussian blue reaction. …”

Nuclear incorporation of iron during the eukaryotic cell cycle

“…The following year it was shown that human neuronal cells also contain nucleolar iron (Sukhorukovaa et al, 2013), although no explanation was provided. It should be noted that the presence of nucleolar iron in HeLa cells was reported some decades ago (Robbins et al, 1972). In the case of HeLa cells, it was clear that iron bound to proteins in the nucleolus and it was suggested that the organelle could be a Fe repository for iron-dependent DNA synthesis proteins.…”