Single‐cell analysis of the relationship among transferrin receptors, proliferation, and cell cycle phase in K562 cells

Abstract

Multiparameter single-cell analysis by flow cytometry was used to distinguish between size-related changes in K562 cell transferrin receptor (TfR) expression and changes in membrane receptor density throughout the cell cycle and over time in culture. Lightscatter pulse-width time-of-flight, a direct and readily calibrated measure of cell diameter, was used to calculate receptor density as the average number of receptors per unit cell surface area. Cell surface TfRs were unimodally distributed over the cell population and were present throughout the cell cycle. The number of receptors increased as cells progressed through the cell cycle, but cell cycle phase was also correlated with cell volume. However, when size heterogeneity was factored out by reanalysis of listmode data, there was a clear cell-cycle effect: among cells of the same size, both the number of receptors per cell and the receptor density increased from G1 to S to G2IM. TfEt expression was also followed over time in culture after dilution into fresh medium. A decrease in growth rate after four days was preceded by one to two days by a decrease in both number of TfRs per cell and mean receptor density, indicating that decreased TfR expression represented true "down-regulation" and not just decreased cell size or an increase in the proportion of smaller G1 cells. This type of analysis is generally applicable for resolving the effects of cell size heterogeneity and cell cycle on membrane protein distribution and for other studies of ligand-receptor interaction.

Doublet discrimination in DNA cell‐cycle analysis

“…Although a G 2 + M cell has twice the volume of a G 0/1 cell, diameter only increases by ≈26% (based on the formula relating the volume V of a sphere to its diameter D, i.e., V = (3.14/6)D 3 , since if V G2 = 2V G1 , D G2 = (2) 1/3 D G1 = 1.26D G1 ), by contrast, the combined diameter of a G 0/1 doublet is at least twice that of a single G 0/1 event, providing that it aligns in the direction of flow. In the case of uninucleate cells, the diameter of a G 2 nucleus compared to a G 0/1 nucleus correlates very well with that predicted by this theory (16).…”

Cell cycle analysis by flow cytometry: Principles and applications

“…DNA content has been studied in combination with numerous cellular components, such as membrane antigens [27,104], nucleolar or nuclear antigens [35], cytoplasmic antigens [167] hormone receptors [131], surface proteins [77] or oncogene products [61,90]. It allows to follow cellular constituent evolution during the cycle, to identify some phase specific components, such as proliferating cell nuclear antigen (PCNA) for S phase [32], or to detect proliferation markers, like Ki67 [108].…”

Anti-HMG-CoA Reductase, Anti-diabetic, Anti-urease, Anti-tyrosinase and Anti-leukemia Cancer Potentials of Panicolin as a Natural Compound:<i>In vitro</i> and <i>in silico</i> Study

“…The interactions of panicolin with the highly expressed surface receptors in the studied cell lines were estimated by molecular docking. Platelet-activating factor receptor (PDB ID: 5ZKP) was selected for HL-60 26) , CD13 (PDB ID: 4FYT) was selected for THP-1 27) , transferrin receptor (PDB ID: 6OKD) was chosen for K562 28) , and CD44 (PDB ID: 4PZ3) was chosen for Molt-4 29) . The chemical activities of panicolin were determined against these proteins.…”