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.

Assessment of the usefulness of the murine cytotoxic T cell line CTLL-2 for immunotoxicity screening by transcriptomics

“…ER stress also induces leakage of calcium from the ER leading to activation of NFB, calcineurin and NFAT that induces oxidative stress and T cell activation in primary thymocytes and Jurkat cells and ultimately leads to apoptosis as well (Katika et al, 2012b;van Kol et al, 2011). There are strong indications that TBTO acts directly on the endoplasmic reticulum inducing a raise of intracellular Ca 2+ release from the ER leading to the same calcium induced responses as mentioned above for DON (Aw et al, 1990;Bissonnette et al, 2010;Chow et al, 1992;Grundler et al, 2001;Kass and Orrenius, 1999;Katika et al, 2012b;Lane et al, 2009).…”

Quantification of temporary and permanent subpopulations of bull sperm by an optimized SYBR‐14/propidium iodide assay

“…These demands are fulfilled by the adequate use of Hamilton microliter syringes (needle length 120 mm) and a modified sample tube head developed by us (14).…”

“…This setup allowed an interruption-free data registration, whereas the time delay between stimulation and registration of its response remains smaller than 3 s due to a short transport time from tube to nozzle enabled by an experimentally standardized stirring and boost procedure (14). To run quantitative kinetic experiments with living cells temperature regulation and stabilization are basic requirements to be achieved for the sample tube solution within small temperature limits (Յ 0.1°C) over long periods of time.…”

“…To run quantitative kinetic experiments with living cells temperature regulation and stabilization are basic requirements to be achieved for the sample tube solution within small temperature limits (Յ 0.1°C) over long periods of time. The thermostatted sample tube holder (stabilizing the temperature at 37 Ϯ 0.1°C) is well suited for this prerequisites (14,15). In practice, two of these holders were used: one for the actual measurements, the other one for prestaining or prewarming of the next sample tube solution.…”

Immunotoxicogenomics : gene expression profiling as a tool to study immunotoxicity

“…Inhibition of lymphocyte proliferation by TBT has been observed in vivo and in vitro [110,112,[132][133][134]. Induction of apoptosis by has been ascribed to a rise in the intracellular calcium concentration, possibly mediating release of cytochrome c and activation of caspases [126,127,[135][136][137][138][139][140][141][142][143][144][145][146][147][148][149][150]. The latter effect has been suggested to cause cytoskeletal disruption [151,152] and calcium may activate protein kinases and phosphatases and phospholipases [141,153].…”