Abstract
Dual-parameter flow cytometry, following bromodeoxyuridine (BrdUrd) incorporation and propidium iodide (PI) uptake into DNA, was used to study the effects of oestradiol and/or insulin on cell cycle kinetics of human breast cancer cells in vitro. After a lag-period of 6-12 h, an optimum in the percentage of S-phase cells was reached between 18 and 24 h after hormone administration. A 1 h pulse of oestradiol was as effective as the continuous presence of oestradiol in pushing the cells from quiescent growing cultures into the cell cycle. A 1 h pulse of insulin was less effective than continuous administration. The addition of doxorubicin resulted in an accumulation of the cells in the late S/G2M-phases. It is concluded that dual-parameter flow cytometry allows accurate assessment of the effects of hormones and chemotherapy on the cell cycle. Therefore this method is very suitable for studying the interaction of hormones and chemotherapy on cell growth.
Highlights
The cell cycle distribution of MCF-7 cells in culture was established by analysis of DNA distribution using pass filter and red (PI)-uptake and by dual-parameter flow cytometry
By analysis of DNA histograms obtained with PI-fluorescence only, no discrimination can be made between cells which are arrested in the S-phase and cells which are actively synthesising DNA
The PI-method is not appropriate to study accurately changes in cell cycle kinetics resulting from perturbation with cell cycle active cytotoxic agents
Summary
Cell cultureThe MCF-7 cell line was obtained from E.G. & G. Medium containing 5 tLg ml-1 phenol red, supplemented with 10% heat-inactivated (30 min at 56°C) fetal calf serum (FCS), 100 Uml-' penicillin, 100 igml-1 streptomycin, 50 pgml-' gentamycin and 10 sgml1' porcine insulin). Logarithmically growing cell cultures were trypsinised and seeded in T25-flasks at a density of 0.5 x 106 cells per flask, in experimental medium, i.e. RPMI-1640 medium, without phenol red and insulin, supplemented with antibiotics and 4.5% steroid hormone depleted FCS (obtained by treatment twice with 0.5% charcoal, 0.05% dextran T-70 (w/v) for 45 min at 50°C, and an intermediate 2 h incubation at 37°C with 2 U ml' of sulphatase). Experimental medium without additions (control), or supplemented with 0.03, 0.5 or 1.0 nM oestradiol (Merck, Darmstadt, FRG), 1.7 IlM porcine insulin (Organon BV, Oss, The Netherlands), or the combination of 1 nM oestradiol and 1.7IM insulin, was added to the cell cultures.
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