G0-G1 cell cycle phase transition as revealed by fluorescence resonance energy transfer: analysis of human fibroblast chromatin

Abstract

In the present study, microspectrofluorometry and digital imaging procedures were used to investigate by fluorescence Resonance Energy Transfer (FRET) analysis the changes of chromatin organization during the transition from G0 quiescent state to G1 phase. G0-G1 transition is a key event in cell cycle progress depending on the activation of specific genes and the concomitant silencing of others, which both entail spatial chromatin rearrangement. Normal human fibroblasts arrested in G0-phase by culture in low-serum contanining medium and stimulated to re-enter G1 by serum addition were used as cell model. To investigate the occurrence and timing of these supramolecular chromatin changes, we estimated the relative FRET efficiency in single cells after double-staining with two DNA-specific dyes which non-covalently bind to double-helical DNA. Hoechst 33258 and propidium iodide were used as a donor-acceptor dye pair since they exhibit particularly favourable spectral characteristics, that allow the calculation procedure to be semplified. The results of FRET analysis were compared to those of the immunocytochemical labelling of two nuclear proteins (i.e., Ki-67 and statin) whose expression is an established marker of potentially proliferating G1 cells or resting G0 cells, respectively. FRET efficiency was lower in G0 than in G1 fibroblasts: this is likely due to a higher chromatin packaging in quiescent cells which especially hinders the intercalation of propidium iodide molecules, thus making the interaction with the donor molecules less favourable, in terms of relative distance and spatial orientation. FRET efficiency significantly increased shortly (1h) after serum stimulation of quiescent fibroblasts, thus indicating that chromatin is rearranged in parallel with activation of cycle-related gene; it is worth noting that these signs largely preceded the occurrence of immunopositivity for Ki-67, which was detectable only 24h after serum stimulation. FRET-based analyses, which already proved to be suitable for studying the overall chromatin organization in differentiated cells, may now be envisaged as a powerful tool for detecting, in single cells, more subtle changes linked to the activation of early cycle-related genes.