Apoptosis Of Human Thymocytes Research Articles

Apoptosis in human thymocytes after treatment with glucocorticoids

Treatment of unfractionated human thymocytes in culture with the synthetic glucocorticoid dexamethasone induced cell death, as measured by trypan blue exclusion, after several hours of incubation. In purified subsets of human cortical and medullary thymocytes dexamethasone caused cell lysis with similar kinetics in both populations; 50% of thymocytes were killed after 20-24 h of incubation with the steroid. The mechanism of dexamethasone-induced cell death seems to correspond to apoptosis since degradation of DNA into oligonucleosome-sized fragments could be observed in the cultures treated with the steroid. A certain degree of DNA fragmentation and cell death could also be observed in control cultures of thymocytes. In contrast, peripheral T lymphocytes were resistant to the cytolytic effect of glucocorticoid hormone. The killing of human thymocytes by dexamethasone was inhibited by cycloheximide, suggesting that this cell death program requires a fully operating protein synthesis machinery and perhaps the induction of new proteins.

Involvement of cAMP in CD3 T cell receptor complex‐ and CD2‐mediated apoptosis of human thymocytes

During intrathymic T cell development, elimination of autoreactive T cell clones by programmed cell death (PCD or apoptosis) is an essential mechanism for self tolerance. The precise intracellular second messengers that lead to this process remain to be determined. In the present work, we show that treatment of freshly isolated thymocytes with an antagonist of the cAMP pathway, the Rp-cAMP, significantly decreases spontaneous death by apoptosis of human thymocytes in vitro. Addition of Rp-cAMP also rescues thymocytes from activation-induced apoptosis following the ligation of surface CD3/T cell receptor complex or CD2 antigens. A cAMP analog, the dibutyryl(Dibut)-cAMP increases PCD of human thymocytes in a dose-dependent manner. Growth and rescue from PCD of thymocytes in the presence of interleukin (IL)-2 or IL-4 are also enhanced by Rp-cAMP and inhibited by Dibut-cAMP. Finally, we detect substantial levels of intracellular cAMP in freshly isolated thymocytes. This study reveals the involvement of cAMP as a second messenger during the apoptosis of normal human thymocytes.

Pyrrolidine dithiocarbamate, a potent inhibitor of nuclear factor κB (NF-κB) activation, prevents apoptosis in human promyelocytic leukemia HL-60 cells and thymocytes

We examined the effect of pyrrolidine dithiocarbamate (PDTC), which potently blocks the activation of nuclear factor κB (NF-κB), on the induction of apoptosis by a variety of agents. Treatment of a human promyelocytic leukemia cell line, HL-60, with 10 μg/mL etoposide or 2 μM 1-β- d-arabinofuranosylcytosine induced NF-κB activation within 1 hr and subsequently caused apoptosis within 3–4 hr. The simultaneous addition of 50–500 μM PDTC with these agents blocked NF-κB activation and completely abrogated both morphologically apoptotic changes and internucleosomal DNA fragmentation for up to 6 hr. However, PDTC failed to inhibit the endonuclease activity contained in the whole cell lysates. The inhibitory effect of PDTC was also observed in etoposide- and dexamethasone-induced apoptosis in human thymocytes at a concentration of 1–10 μM. Since PDTC has both antioxidant and metal-ion chelating activities, we tested the effects of N- acetyl- l-cysteine (NAC) (antioxidant) or o-phenanthroline (OP) (metal-ion chelator) on the induction of apoptosis. Pretreatment of HL-60 cells or thymocytes with 100–500 μM OP for 2 hr, but not 10–60 mM NAC, suppressed subsequent occurrence of apoptosis induced by etoposide. These results suggest that the activation of NF-κB plays an important role in the apoptotic process of human hematopoietic cells.

A rapid method for measuring apoptosis and dual-color immunofluorescence by single laser flow cytometry

A sensitive method for quantification of cells undergoing apoptisis that permits the simultaneous measurement of dual-color cell surface immunofluorescence is presented. Unfixed cells are stained with 7-amino-actinomycin D (7-AAD) for discrimination of live from early apoptotic cells and from cells which have lost membrane integrity (late apoptotic or necrotic, dead cells). Owing to its spectral characteristics 7-AAD can be combined with fluorescein-isothiocyanate (FITC) and phycoerythrin (PE) cell surface staining. After staining, the samples can be treated with paraformaldehyde (PF) solution to eliminate the risk for exposure of laboratory personnel to biohazardous agents and to preserve the cells through fixation for later analysis on the flow cytometer. The value of the method is shown on the measurement of apoptosis in human thymocytes and in human peripheral blood mononuclear cells (PBMC) exposed to various inducers of active cell death. The method is validated by fluorescent activated cell sorting in combination with morphologic examination of the sorted cells. The technique we are presenting is particularly valuable in a clinical setting because it allows rapid multiparameter analysis of apoptosis in combination with cell surface phenotype on biohazardous samples with single laser instrumentation.

Effects of phorbol esters on radiation - induced apoptosis in human thymocytes

Effects of phorbol esters on radiation - induced apoptosis in human thymocytes

Genistein Induces Apoptosis in Immature Human Thymocytes by Inhibiting Topoisomerase-II

The toxicity of genistein, an inhibitor of tyrosine kinases and topoisomerase-II, on human thymocytes was investigated. Genistein induced marked chromatin fragmentation indicative of apoptosis in human thymocyte cultures. Genistein-induced thymocyte apoptosis is unlikely due to an inhibition of basal tyrosine kinase activity, since another tyrosine kinase inhibitor, herbimycin A, does not induce thymocyte apoptosis, whereas other topoisomerase-II inhibitors do. The thymocyte subpopulation most sensitive to genistein-induced apoptosis exhibited a CD3−CD4+CD8+ phenotype. This subpopulation of thymocytes is also sensitive to glucocorticoid-induced apoptosis; however, differences between genistein- and glucocorticoid-induced apoptosis were noted. In particular, unlike glucocorticoid-induced apoptosis, genistein-induced apoptosis does not involve changes in [Ca2+]i and cannot be blocked by activation of protein kinase C.