Dex-induced Apoptosis Research Articles

Degradation of constitutive transcription factors during apoptosis in rat thymocytes.

Dexamethasone (Dex) accelerates the rate of apoptosis in thymocytes by a process thought to require gene expression. Among the genes implicated in the regulation of this phenomenon are the immediate early genes such as c-fos and c-jun, whose expression is modulated by a complement of preexisting transcription factors. We have analyzed the DNA-binding activity of these constitutive transcription factors during Dex-induced apoptosis in thymocytes to assess their functionality. We observed a progressive loss of the DNA-binding proteins in parallel with the appearance of the characteristic morphological and biochemical features of apoptosis. At the same time we have found a general increase in the nuclear proteolytic activity concomitant with a significant loss of the nuclear nonhistone chromosomal proteins. Indeed, cotreatment of thymocytes with the nonspecific serine protease inhibitor phenylmethylsulphonyl fluoride was able to partially protect the stability of the DNA-binding proteins and alter the expression of the c-fos and c-jun genes but did not inhibit apoptosis. Our results suggest that the action of a protease(s) is responsible for the degradation of constitutive transcription factors during Dex-induced apoptosis, rendering the death pathway irreversible.

Dexamethasone and interleukins modulate apoptosis of murine thymocytes and peripheral T-lymphocytes

Glucocorticoid hormones (GCH) induce apoptotic cell death in immature thymocytes through an active process, characterized by extensive DNA fragmentation into oligonucleosomal subunits. This requires macromolecular synthesis and is inhibited by interleukins subunits. This requires macromolecular analyse the possible effect of GCH on more differentiated lymphocytes, i.e. peripheral (from lymph nodes and spleen) T-lymphocytes. The results show that in vitro dexamethasone (DEX) induces DNA fragmentation and cell death not only in thymocytes but also in mature T cells. We also tested the possible role of interleukins (ILs) in the modulation of apoptotic cell death. We show that DEXinduced apoptosis is inhibited by IL-2 and IL-4 and that the IL-4 induced inhibition correlates with induction of c - jun (a component of AP-1 transcription factor). Furthermore high doses of IL-2 are able to induce apoptosis in both thymocytes and peripheral T cells. These data indicate that both thymocytes and peripheral T cells undergo apoptosis in response to appropriate stimuli and suggest that GCH and ILs interact in regulating T-lymphocytes apoptotic death.

Tributyltin and Dexamethasone Induce Apoptosis in Rat Thymocytes by Mutually Antagonistic Mechanisms

The nuclei of apoptotic thymocytes can be identified by flow cytometry as a subpopulation exhibiting reduced DNA content. We observed that rat thymocyte cultures exposed to 1.0-2.5 μM tri-n-butyltin methoxide (TBT) exhibited a rapid time- and concentration-dependent induction of apoptosis, with > 85% of cells exhibiting reduced DNA content within 1 hr after exposure to 2.0-2.5 μM TBT. In contrast, exposure to 1.0 μM dexamethasone phosphate (DEX) resulted in a gradual time-dependent increase to ∼45% induction of apoptosis by 6 hr versus ∼15% spontaneous induction in controls. However, simultaneous exposure to TBT and DEX resulted in a decreased response: although TBT concentrations between 0.1 and 0.5 μM did not induce apoptosis, they reduced the ability of DEX to initiate apoptosis; while at TBT concentrations ≥ 1.0 μM, simultaneous exposure to DEX substantially decreased the extent of TBT-induced apoptosis and cytotoxicity. Furthermore, while treatment with the protein synthesis inhibitor cycloheximide or the protein kinase C inhibitor H-7 completely blocked DEX-induced apoptosis, neither significantly reduced induction of apoptosis by TBT. Taken together, the toxicant-specific differences in the timing and extent of apoptotic induction and the dissimilar responses to CHX and H-7 suggest that TBT and DEX initiate endonuclease-mediated apoptotic cell death through different mechanisms. Moreover, the ability of each agent to retard the action of the other suggests that these mechanisms are directly or indirectly antagonistic.

Heat shock induces apoptosis in mouse thymocytes and protects them from glucocorticoid-induced cell death

Thymocyte death is a complex phenomenon under the control of different signals and stimuli. We evaluated the effect of elevated temperature (heat shock, HS) on mouse thymocyte apoptosis. Incubation of thymocytes at 43 °C for 20 min induced DNA fragmentation and cell death, but it was also able to decrease the apoptosis induced by dexamethasone (DEX), TPA or Ca 2+ ionophore. The anti-apoptotic effect was correlated with induction of heat shock proteins (HSPs) and abolished by protein synthesis inhibition. On the other hand, HS-induced unlike DEX-induced apoptosis was not inhibited by protein synthesis and mRNA transcription inhibitors, the PKC inhibitors H-7 and staurosporine, or interleukin-4 (IL-4), but only by Zn 2+. These results suggest that HS interferes in thymocyte death by either inducing or inhibiting thymocyte apoptosis and that the induction process mechanisms are different from those of GCH.

A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry

Corticosteroids, calcium ionophores and anti-CD3 monoclonal antibodies kill mouse thymocytes incubated in vitro. Cell death is preceded by extensive DNA fragmentation into oligonucleosomal subunits. This type of cell death (apoptosis), which physiologically occurs in the intrathymic process of immune cell selection, is usually evaluated by either electrophoretic or colorimetric methods which measure DNA fragmentation in the nuclear extracts. These techniques are unable to determine the percentage of apopotic nuclei or recognize the apoptotic cells in a heterogeneous cell population. We have developed a flow cytometric method for measuring the percentage of apoptotic nuclei after propidium iodide staining in hypotonic buffer and have compared it with the classical colorimetric and electrophoretic techniques using dexamethasone (DEX)-treated mouse thymocytes. Apoptotic nuclei appeared as a broad hypodiploid DNA peak which was easily discriminable from the narrow peak of thymocytes with normal (diploid) DNA content in the red fluorescence channels. When the DEX-induced apoptosis was inhibited by either low-temperature (4°C) incubation or cycloheximide treatment, no hypodiploid DNA peak appeared. Similarly, thymocyte death induced by sodium azide, a substance with cell-killing activity through non-apoptotic mechanisms, did not result in any variation in the normal DNA peak. The flow cytometric data showed an excellent correlation with the results obtained with both electrophoretic and colorimetric methods. This new rapid, simple and reproducible method should prove useful for assessing apoptosis of specific cell populations in heterogeneous tissues such as bone marrow, thymus and lymph nodes.