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

Background: Programmed cell death, also termed apoptosis, is the main focus of interest in a variety of scientific and clinical areas. For a better un derstanding of the mechanisms of apoptosis, from the onset of the cellular death program to the late stages of apoptosis or apoptotic necrosis, very e arly functional events have to be quantified because they might be involved in temporal and causal relationships between apoptosis-related key process es. Methods: We have established a flow cytometric technique to quantify time-d ependent signals simultaneously with high temporal resolution (Delta = 1 s) in living cells. With this technique, the response of cells to apoptosis-s timulating agents can be analyzed over 15 min. For this purpose, a thermost atted sample tube holder for repeatable interruption-free injection of subs tances into the cell suspension was developed. Early detectable fluorescenc e and scatter parameters were related to intracellular free Ca2+ concentrat ion, [Ca2+](i) (Indo-1 fluorometry), membrane permeability (propidium iodid e [PI] influx), and cell volume (forward scatter). Results: A T-cell line (Jurkat) sen ed as a model system. Apoptosis R as in duced by the biozid Tri-n-butyltin (TBT). Dependent on the TBT concentratio n (0.3-10 muM), the mean free [Ca2+](i) increased by a factor of 1.2-6 duri ng a short time interval of just 2 min. Especially after low TBT concentrat ions (<0.5 muM), this [Ca2+](i) increase was nearly transient during the ob servation time of 15 min. Higher TBT concentrations (0.5-10 muM), 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 o bserved. The analysis of the simultaneously registered PI signals of the Ca -SST cells showed a shift to increasing PI fluorescence (by a factor of abo ut 4) with increasing Ca2+ concentrations. In Ca-TR cells, the PI fluoresce nce 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 measur ed during the same early time period. Conclusions: The simultaneously analyzed parameters (i.e., [Ca2+](i), membr ane permeability, and cell volume) suggested that, in our model system of J urkat T-cells treated with TBT, an apoptotic cell fate was indicated very e arly (within 15 min) by the steady-state [Ca2+], level. Cytometry 44:45-56, 2001. (C) 2001 Wiley-Liss, Inc.