HOECHST-33342 - A DNA MARKER FOR STUDYING THE DYNAMICS OF CHROMATIN IN LIVING CELLS

The organization of eukaryotic chromatin is not static but changes as a function of cell status during proliferation, differentiation and tr ansformation. One major goal in cell biology is to study the chromatin organization without perturbing its function. A living cell is a dyna mic object and conventional biochemical analyses do not provide spatia l and temporal information. Fluorescence cytometry is suitable for str ucture-function analysis and can be considered, at the present time, a s an important means for understanding chromatin dynamics in living ce lls. An indirect, non invasive method for studying chromatin organizat ion, the distribution of condensed and decondensed domains, was develo pped using the fluorescent vital bisbenzimidazole probe Hoechst 33342 (Ho342). Ho342 is a non intercalating DNA specific fluorescent dye wit h a marked A-T preference. Non invasive staining conditions consisted of the combination of verapamil (VPL), a calcium channel blocker which acts in the same way as the membrane potential probe: DiO-C5-3 (Criss man et al, 1988) and Ho342. Special attention was paid to the kinetics of incorporation into cells, the effect of serum addition to the cult ure medium on the staining efficiency, the stoichiometry, the spectral characteristics of DNA-Ho342 complex, and the effect of the staining procedure on cell cycle progression and DNA synthesis. This fluorescen t dye was incorporated into living cells, and led to staining stoichio metry when using either 10 mu M Ho342 or 4 mu M in the presence of ver apamil 40 mu M, with similar kinetics of uptake. A plateau in fluoresc ence intensity of DNA-Ho342 complex was obtained following 60 min. upt ake, although stoichiometry was reached earlier (30 min.). Fluorescenc e intensity was artificially enhanced when the staining was performed in a medium containing bovine serum. The spectral characteristics of t he Ho342-DNA complex (Ho342 10 mu M and VPL-Ho342 4 mu M) were analyze d by confocal microspectrofluorometry which provides fluorescence spec tra from localized nuclear regions in a living cell. The results showe d that intra nuclear spectra did not vary as a function of time, of th e cell cycle phases, and of the intra nuclear position. In all cases, emission spectrum displayed a maximum at 450 nm and intensities measur ed at wavelengths below 400 nm or above 550 nm appeared to be negligib le compared to the maximum intensity. In the presence of VPL, the know n accumulation of cells in G2 induced by higher concentration of Ho342 was strongly decreased. In addition, the cells maintained their abili ty to incorporate bromodeoxyuridine. These experimental conditions usi ng the fluorescent probe Ho342 allowed the quantification of dynamic s tructural modifications of chromatin during cell cycle (fig. 1) (Paill asson et al, 1995).