Tracking the cell cycle origins for escape from topotecan action by breast cancer cells.

The anticancer agent topotecan is considered to be S-phase specific. This implies that cancer cells that are not actively replicating DNA could resist the effects of the drug. The cycle specificity of topotecan action was investigated in MCF-7 cells, using time-lapse microscopy to link the initial cell cycle position during acute exposures to topotecan with the antiproliferative consequences for individual cells. The bioactive dose range (0.5-10 microM) for 1-h topotecan exposures was defined by rapid drug delivery and topoisomerase I trapping. Topotecan caused pan-cycle induction and activation of p53. Lineage analysis of the time-lapse sequences identified cells initially in S-phase and G2, and defined the time to mitosis for cells originating from G2, S-phase and G1. Topotecan prevented all mitoses from S-phase cells and G1 cells (half-maximal effects at 0.14 microM and 0.96 microM, respectively). No dose of topotecan completely prevented mitosis among G2 cells, and at saturating doses of topotecan about half the cells of G2 origin continued dividing (the half-maximal effects was at 0.31 microM). Overall, topotecan differentially targeted G1-, S- and G2-phase cells, but many G2 cells were resistant to topotecan, presenting a clear route for cell cycle-mediated drug resistance.

The effects of wild-type and mutant HFE expression upon cellular iron uptake in transfected human embryonic kidney cells.

In order to measure the effects of HFE (haemochromatosis) upon iron uptake, stable expression of wild-type and C282Y, H63D and S65C mutant HFE cDNA was established in HEK 293 cells. Control cells were transfected with empty vector. Expression of HFE mRNA and protein was detected in the cell lines transfected with HFE cDNA, but not in the control cell line. The ferritin concentration in wild-type cells cultured in 40 microM ferric ammonium citrate was 69% of that in control cells and 81% of that in C282Y cells. The ferritin concentration in H63D cells was intermediate between wild-type and C282Y and the ferritin concentration in S65C cells was similar to wild-type cells. Uptake of transferrin-iron in wild-type, C282Y and control cells was measured over 45 min. The Hill coefficients for transferrin-iron uptake were similar. The V(max) for transferrin-iron uptake in wild-type cells was 59.5% of control cells and 69.5% of C282Y cells. Estimates of K(m) were 232 nM for wild-type cells, 338 nM for C282Y cells and 570 nM for controls. Transferrin receptor levels were lowered, but not significantly, in the HFE transfected cells. The results show that HFE reduces transferrin-iron uptake, probably as an uncompetitive inhibitor.

Factor V Leiden and the common haemochromatosis mutation HFE C282Y: is there an association in familial venous thromboembolic disease?

The involvement in venous thrombosis of the two most common mutations of the hereditary haemochromatosis gene (HFE C282Y and HFE H63D) was investigated in 239 patients with objectively proven venous thrombosis. Neither mutation showed an increased prevalence in the cohort (HFE C282Y: 13.0% (95% CI 9.3-17.8) patients, 16.2% (95% CI 14.3-18.2) controls; HFE H63D: 28.3% (95% CI 22.9-34.3) patients, 28.1% (95% CI 25.8-30.6) controls. Neither mutation was increased in patients with factor V Leiden (FVL) compared to those without. However, HFE C282Y was increased among patients who had both FVL and a family history of thrombosis (7/20), compared with those with FVL and no family history (1/22) (relative risk 7.97, 95% CI 1.5-43.1, P = 0.016).