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Early functional apoptotic responses of thymocytes induced by Tri-n-butyltin
W Grundler1, P Dirscherl, W Beisker
1Flow Cytometry Group, GSF-National Research Center for Environment and Health, Neuherberg, Germany. Grundler@GSF.de
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 (Deltat = 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 microM), 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 microM), this [Ca2+]i increase was nearly transient during the observation time of 15 min. Higher TBT concentrations (0.5-10 microM), 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 [Ca(2+)]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.
Insights
This study introduces a flow cytometry technique to track early apoptosis markers in living cells. A steady-state intracellular calcium level, alongside changes in membrane permeability and cell volume, indicates an apoptotic cell fate within 15 minutes.
Area of Science:
- Cell Biology
- Apoptosis Research
- Flow Cytometry Applications
Background:
- Programmed cell death (apoptosis) is crucial in various scientific and clinical fields.
- Understanding early apoptotic events is key to deciphering temporal and causal relationships in cell death.
- Quantifying early functional events in apoptosis is essential for mechanistic insights.
Purpose of the Study:
- To establish a flow cytometric technique for quantifying time-dependent signals in living cells with high temporal resolution.
- To analyze the cellular response to apoptosis-stimulating agents over a 15-minute period.
- To correlate early fluorescence and scatter parameters with intracellular calcium, membrane permeability, and cell volume.
Main Methods:
- Developed a flow cytometry technique with 1-second temporal resolution (Δt = 1 s).
- Utilized a thermostatted sample tube holder for uninterrupted substance injection.
- Measured intracellular free Ca2+ concentration ([Ca2+]i) using Indo-1 fluorometry, membrane permeability via propidium iodide (PI) influx, and cell volume by forward scatter.
Main Results:
- Tri-n-butyltin (TBT) induced a rapid increase in [Ca2+]i in Jurkat T-cells (factor 1.2-6 within 2 min).
- Higher TBT concentrations (>0.5 μM) led to a steady-state Ca2+ signal (Ca-SST) in a subpopulation, correlating with increased PI fluorescence (approx. 4-fold).
- Apoptosis induction was associated with increased [Ca2+]i, altered membrane permeability, and decreased cell volume within 15 minutes.
Conclusions:
- The developed flow cytometry method allows for early detection of apoptosis-related events.
- A steady-state intracellular calcium level ([Ca2+]i), alongside increased membrane permeability and decreased cell volume, is a strong early indicator of apoptotic fate in Jurkat T-cells treated with TBT.
- This technique provides rapid, quantitative insights into the initial stages of apoptosis.