Early functional apoptotic responses of thymocytes induced by Tri‐n‐butyltin

Abstract

Background Programmed cell death, also termed apoptosis, is the main focus of interest in a variety of scientific and clinical areas. For a better understanding of the mechanisms of apoptosis, from the onset of the cellular death program to the late stages of apoptosis or apoptotic necrosis, very early functional events have to be quantified because they might be involved in temporal and causal relationships between apoptosis-related key processes. Methods We have established a flow cytometric technique to quantify time-dependent signals simultaneously with high temporal resolution (Δt = 1 s) in living cells. With this technique, the response of cells to apoptosis-stimulating agents can be analyzed over 15 min. For this purpose, a thermostatted sample tube holder for repeatable interruption-free injection of substances into the cell suspension was developed. Early detectable fluorescence and scatter parameters were related to intracellular free Ca2+ concentration, [Ca2+]i (Indo-1 fluorometry), membrane permeability (propidium iodide [PI] influx), and cell volume (forward scatter). Results A T-cell line (Jurkat) served as a model system. Apoptosis was induced by the biozid Tri-n-butyltin (TBT). Dependent on the TBT concentration (0.3–10 μM), the mean free [Ca2+]i increased by a factor of 1.2–6 during a short time interval of just 2 min. Especially after low TBT concentrations (<0.5 μM), this [Ca2+]i increase was nearly transient during the observation time of 15 min. Higher TBT concentrations (0.5–10 μM), however, induced a transient increase of [Ca2+]i (Ca-TR) only in a fraction of the cells; in another subpopulation, a steady-state Ca2+ signal (Ca-SST) was observed. The analysis of the simultaneously registered PI signals of the Ca-SST cells showed a shift to increasing PI fluorescence (by a factor of about 4) with increasing Ca2+ concentrations. In Ca-TR cells, the PI fluorescence remained nearly unchanged. These apoptosis-related changes (increase in [Ca2+]i and membrane permeability) could be confirmed by the additional observation of a TBT concentration-dependent decrease in cell volume measured during the same early time period. Conclusions The simultaneously analyzed parameters (i.e., [Ca2+]i, membrane permeability, and cell volume) suggested that, in our model system of Jurkat T-cells treated with TBT, an apoptotic cell fate was indicated very early (within 15 min) by the steady-state [Ca2+]i level. Cytometry 44:45–56, 2001. © 2001 Wiley-Liss, Inc.