Transferrin receptor expression during exponential and plateau phase growth of human tumour cells in culture

Abstract

Transferrin receptor expression by the human tumour cell lines CCRF-CEM leukaemia and PMC-22B melanoma was studied, measuring the specific binding of fluorescein isothiocyanate (FITC)-labelled transferrin using a fluorescence-activated cell sorter. By measuring the fluorescence of cells stained at subsaturating concentrations of conjugate it was possible to calculate the average numbers of receptors per cell and the binding affinity by Scatchard analysis. These values (1.9 X 10(5) binding sites/cell, KA 1.2 X 10(9) M-1 for CCRF-CEM during exponential growth and 6.9 X 10(4) binding sites/cell, KA 1.4 X 10(-9) M-1 for PMC-22B) are in close agreement with previously published data obtained using radiolabelled transferrin. The present method, however, allowed the transferrin receptor expression of individual cells within a population to be measured and thus it has been possible to test the hypothesis that transferrin receptor is a marker for cycling cells. Frequency-distribution histograms of transferrin receptor showed a wide range of values for both cell lines during exponential growth. When the extreme ranges were sorted and the cells examined for cellular DNA content it was found that those with the highest transferrin receptor expression were enriched with cells in S, G2, and M phases of the cell cycle, whereas those with low transferrin receptor expression were mainly in G1. However, two-parameter-correlated dot plots of transferrin receptor expression versus DNA content showed there was considerable overlap between the ranges of receptor expression for the different cell cycle compartments. Using a stathmokinetic method we have measured the proportion of quiescent cells in fed plateau phase cultures. Transferrin receptor expression was downgraded under these growth conditions but, contrary to expectation, the decline affected the population uniformly, without the emergence of a distinct, transferrin receptor-negative subpopulation corresponding to the increasing proportion of quiescent cells. Thus, although transferrin receptor expression bears some relation to cell cycle phase and reflects the proliferative activity of populations of cells, it is incapable of identifying individual cells which are out of cycle.

Transferrin receptor on peripheral blood lymphocytes in iron deficiency anaemia

“…Several in vitro studies have documented close relationships among T lymphocytes, transferrin receptor, iron and cell proliferation. In in vitro lymphocyte cultures, blockade of TfR or elimination of iron from the culture medium was shown to result in decreased proliferation (Teatle, 1990;Hedley et al, 1985;Musgrove et al, 1984). Although in a previous report of a few patients with mild iron deficiency anaemia the number of CD71 þ peripheral blood lymphocytes was found to be normal (Pattanapanyasat, 1990), our study indicated that severe iron deficiency resulted in significantly lower numbers of CD71 þ lymphocytes in peripheral blood.…”

“…Removing Tf or iron from the medium causes reduced cell growth and eventual cell death. These observations indicate that in mononuclear cells TfR expression is associated with cell activation, growth control and cell proliferation (Cook et al, 1993;May & Cuatrecasas, 1985;Teatle, 1990;Hedley et al, 1985;Musgrove et al, 1984;Shannon et al, 1986).…”

Role of transferrin, Fe, and transferrin receptors in myeloid leukemia cell growth. Studies with an antitransferrin receptor monoclonal antibody.

“…Although FeNTA apparently overcame 42/6 inhibition in some settings, these observations did not explain failure to inhibit some ANLL and granulocyte/macrophage progenitor colony-forming cells (13). Recent studies suggest varying levels of TfR display by dividing T lymphoblasts (27 In two studies, FCS HL60 cells were grown with 1.25% DMSO, and day 5 cell growth was 76% of that of FCS controls.…”

Flow cytometric quantitation of nucleoside transporter sites on human leukemic cells

“…For example, a flow cytometric study of the binding of fluoresceinated epidermal growth factor to A431 epidermoid carcinoma cells has yielded information on the affinity of ligand-receptor interaction as well as the density of receptor sites (4). Flow cytometry has also been applied to equilibrium binding analysis of fluorphore-labeled transferrin, lectins, steroids and insulin to various cells (11,12,16). Shohami et al (15) previously reported the synthesis of a dansyl derivative of 6-thioguanosine which binds to and inhibits the es nucleoside transporter and was used in a spectrofluorometric study of human erythrocytes.…”