Reactive oxygen species, but not mitochondrial membrane potential, is associated with radiation-induced apoptosis of AHH-1 human lymphoblastoid cells.

The aim of the study was to investigate the sensitivity of AHH-1 human lymphoblastoid cells to radiation and its relevance to intracellular events, specifically alteration in cellular energy-producing systems. AHH-1 human lymphoblastoid cells were irradiated with 6 Gy of gamma radiation, and then were collected at the indicated time points. Parallel studies were conducted to assess the effects of radiation on the cell proliferation and apoptotic index. Mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) production were monitored. A marked decrease of cell viability was observed as early as 12 h postirradiation and fraction of apoptotic cells was highest at 24 h. Intracellular ROS generation measured with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) appeared to be highest as early as 30 min postirradiation and resumed to normal level at 6 h. Unexpectedly, the fluorescence intensity of Rhodamine 123 for measuring MMP did not change during the first 3h after radiation and exhibited an aberrant increase at 6 h. The results suggest that AHH-1 cells are sensitive to radiation-induced apoptosis and ROS generation is an early phase in the apoptosis process. Moreover, the results might cast doubts on those studies using Rhodamine 123 which hypothesized that the fall in MMP is one of the early events of apoptosis.

Increase of CD4(+)CD25(+) T cells in Smad3(-/-) mice.

AIM: To investigate the changes of lymphocyte subpopulations, especially CD4(+)CD25(+) T regulatory cells in Smad3(-/-) mice. METHODS: Hematological changes and changes of lymphocyte subpopulations were detected in Smad3(-/-) mice using cell counter and flow cytometry, respectively, and compared to their littermate controls. RESULTS: The numbers of neutrophils and lymphocytes in peripheral blood were significantly increased in Smad3(-/-) mice compared to littermate controls. CD19(+) expressing cells in blood and spleen, and CD8(+) T cells in thymus were all markedly decreased in Smad3(-/-) mice. More important, Smad3(-/-) mice had an increased population of CD4(+)CD25(+) T cells in peripheral lymphoid tissues, including thymus, spleen, and lymph nodes. CONCLUSION: These observations suggest that the changes of lymphocyte subpopulations might play a role in susceptibility to inflammation of Smad3(-/-) mice.

[Relationship between apoptosis of mouse thymic lymphocytes and expressions of bax, bcl-2 and bcl-XL after gamma-ray radiation with lethal dose].

AIM: To observe the relationship between apoptosis of mouse thymic lymphocytes and the expressions of bax, bcl-2 and bcl-X(L) after gamma-ray radiation with lethal dose and provide the basis for treatment of acute severe radiation sickness. METHODS: 250 cleaning-grade C57 mice were randomly divided into 6 groups and treated with 0, 6, 9, 12, 15 and 20 Gy of whole body single gamma-irradiation, respectivety. The mice were killed by dislocation and then the thymus and peripheral blood samples were taken at 1-28 days and 3-12 months after irradiation. The lymphocytic apoptosis was analyzed by TUNEL. The expressions of bax, bcl-2 and bcl-X(L) were detected by in situ hybridization and immunohistochemical staining. RESULTS: (1)Mouse peripheral leucocytes showed a transient elevation at 6 h after radiation and then decreased rapidly. The leucocyte's number reduced to minimal level at day 7 after 6 Gy gamma-irradiation and returned to basic normal value until one month after radiation. (2)24 h after irradiation the apoptotic rate of thymic lymphocytes increased swiftly, and apoptotic rate was positively correlated with radiation dose in the range of 6-12 Gy. There was no such a correlation after >/=15 Gy irradiation. (3)24 h after 6 Gy irradiation the apoptotic rate of thymic lymphocytes reached the maximal level. Afterwards it began to decrease, but still higher than that of control group even after 12 months. (4)3 h after 6 Gy radiation the expression of Bax protein in thymic lymphocytes increased immediately, and reached the highest value at 24 h. On the other hand, the expressions of Bcl-2 and Bcl-X(L) proteins reduced evidently at 3 h after radiation, reaching the lowest level at 24 h. Analysis of bax and bcl-2 mRNAs was concordant with the protein expression results. CONCLUSION: After 6-12 Gy gamma-ray irradiation, the apoptotic rate of thymic lymphocytes is positively correlated with the radiation doses, suggesting that apoptosis is a major way of thymic lymphocyte death after

[Apoptosis and modulatory mechanism of human AHH-1 T lymphocytes induced by gamma-irradiation].

AIM: To explore the apoptotic characteristics and modulatory mechanism of human AHH-1 T lymphoblast induced by ionizing radiation and provide an experimental basis for preventing and treating immune damage in acute radiation sickness. METHODS: TdT-mediated dUTP nick end labeling (TUNEL) and May-Grunwald Giemsa (MGG) staining, MTT colorimetry and immunohistochemical staining were used to detect the T lymphocyte apoptosis, cell survival and the expressions of Bax, Bcl-XL and caspase-3 proteins. RESULTS: (1) Over a definite dose range, apoptotic rate of AHH-1T lymphocytes increased with the elevation of radiation doses, while the lymphocytic survival rate decreased sharply, both showing a good dose-effect relationship. (2) The expression of apoptosis-accelerating protein Bax enhanced obviously with the increase of radiation doses, while apoptosis-inhibiting protein Bcl-XL trended to persistent decline. (3)The activity of activated apoptosis-executing protein caspase-3 heightened significantly with increased radiation doses, which revealed a better corresponding relationship to apoptotic rate. CONCLUSION: A great number of apoptotic AHH-1 T cells may be one of major accounts for the lymphocyte rapid reduction and depressed organism immune function induced by irradiation. Bax, Bcl-XL and caspase-3 proteins play an important role in the regulation of radiation-induced T cell apoptosis.