The depletion of cellular iron can lead to the inhibition of ribonucleotide reductase, preventing new DNA synthesis and hence inhibiting cell proliferation. Electron paramagnetic resonance (EPR) spectroscopy has been used to examine simultaneously for the first time the relationship between chelation of intracellular iron and the rate of removal and regeneration of the tyrosyl radical of ribonucleotide reductase within intact human leukemia K562 cells. The different physiochemical characteristics of relatively hydrophobic low molecular weight bidentate hydroxypyridinone chelators and the higher molecular weight hexadentate ferrioxamine have been exploited to elucidate these interactions further. The base-line concentration of EPR-detectable mononuclear nonheme iron complexes was 3.15 ؎ 1.05 M, rising on incubation with chelators more rapidly with hydroxypyridinones than with desferrioxamine. Hydroxypyridinones also removed the tyrosyl radical more rapidly, apparently as a consequence of depletion of the intracellular iron pools necessary to regenerate the active enzyme and compatible with their reportedly greater cell toxicity. The radical decay rate is consistent with previous models, suggesting that iron is spontaneously removed from mammalian ribonucleotide reductase. Upon removal of extracellular chelator the regeneration of the tyrosyl radical was significantly faster for hydroxypyridinones than for desferrioxamine, consistent with their differential effects on cell cycle synchronization.
The interactions of iron chelators with intracellular iron pools have been examined by measuring the subcellular distribution of radiolabelled desferrioxamine (DFO) and the orally active hydroxypyridinone (HPO) chelator 1,2-diethyl-3-hydroxypyridin-4-one (CP94), as well as the ability of these chelators to modify the subcellular distribution of 59Fe delivered by the receptor mediated endocytosis of transferrin. K562 cells were pulsed with 59Fe transferrin and challenged with DFO or CP94 (100 microM IBE) for 20 or 240 min and then subjected to subcellular fractionation. At 20 min there was a significant decrease (P < 0.05) in both lysosomal/particulate 59Fe (75% of control) and cytosolic 59Fe ferritin (50% of control) in cells incubated with CP94, unlike cells treated with DFO where no decrease was observed. By 240 min, in addition to the above, 59Fe accumulation was significantly decreased in the nuclear, mitochondrial, and low molecular weight cytosolic fractions with CP94 (P < 0.05). With DFO a significant decrease in 59Fe in only the lysosomal/particulate and cytosolic ferritin compartments was observed at 240 min (P < 0.05). At this time, however, there was a significant accumulation of both cytosolic low molecular weight 59Fe and cytosolic DFO. The relatively rapid decrease of 59Fe within intracellular compartments seen with CP94 compared to DFO was paralleled by a significantly higher accumulation of CP94 than DFO in nuclear, lysosomal/particulate and low molecular weight cytosolic compartments at 20 min (P < 0.05). These results suggest that transferrin derived endosomal iron may be chelated by HPOs, unlike DFO, due to their faster uptake into these organelles. The more rapid access of HPOs than DFO to certain intracellular iron pools may explain the greater possibility of HPOs to inhibit proliferation of cells in vivo.
We describe a protocol for the synchronisation of normal and tumour cells grown in suspension cultures using 3-hydroxypyridin-4-one iron chelators. These compounds inhibit ribonucleotide reductase, one of the rate limiting enzymes in DNA synthesis, and so block the cell cycle in late G1 phase. After removal of the chelator or repletion of cellular iron, cells progress through the cycle and remain synchronised for at least one full cell cycle. Cell viability is unaffected for at least 72 hours post-incubation and chelator treatment has no effect on RNA and protein synthesis. This method of synchronisation has been successful with all cell lines tested including normal and leukaemic human cell lines.
The relationship between the oral efficacy and the acute toxicity of hydroxypyridin-4-one iron chelators has been investigated to clarify structure-function relationships of these compounds in vivo and to identify compounds with the maximum therapeutic safety margin. By comparing 59Fe excretion following oral or intraperitoneal administration of increasing doses of each chelator to iron-overloaded mice, the most effective compounds have been identified. These have partition coefficients (Kpart) above 0.3 in the iron-free form with a trend of increasing oral efficacy with increasing Kpart values (r = .6). However, this is achieved at a cost of increasing acute toxicity, as shown by a linear correlation between 59Fe excretion increase per unit dose and 1/LD50 (r = .83). A sharp increase in the LD50 values is observed for compounds with Kpart values above 1.0, suggesting that such compounds are unlikely to possess a sufficient therapeutic safety margin. Below a Kpart of 1.0, acute toxicity is relatively independent of lipid solubility. All the compounds are less toxic by the oral route than by the intraperitoneal route, although iron excretion is not significantly different by these two routes. At least five compounds (CP51, CP94, CP93, CP96, and CP21) are more effective orally than the same dose of intraperitoneal desferrioxamine (DFO) (P less than or equal to .02) or orally administered L1(CP20) (P less than or equal to .02).
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