Zhi-Wei Lai

Identification of Stage-Specific Gene Modulation during Early Thymocyte Development by Whole-Genome Profiling Analysis after Aryl Hydrocarbon Receptor Activation

The aryl hydrocarbon receptor (AHR) is a basic helix-loop-helix transcription factor, implicated as an important modulator of the immune system and of early thymocyte development. We have shown previously that AHR activation by the environmental contaminant and potent AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads to a significant decline in the percentage of S-phase cells in the CD3−CD4−CD8− triple-negative stage (TN) 3 and TN4 T-cell committed thymocytes 9 to 12 h after exposure. In the more immature TN1- or TN2-stage cells, no effect on cell cycle was observed. To identify early molecular targets, which could provide insight into how the AHR acts as a modulator of thymocyte development and cell cycle regulation, we performed gene-profiling experiments using RNA isolated from four intrathymic progenitor populations in which the AHR was activated for 6 or 12 h. This microarray analysis of AHR activation identified 108 distinct gene probes that were significantly modulated in the TN1–4 thymocyte progenitor stages. Although most of the genes identified have specific AHR recognition sequences, only seven genes were altered exclusively in the two T-cell committed stages of early thymocyte development (TN3 and TN4) in which the decline of S-phase cells is seen. Moreover, all seven of these genes were reduced in expression, and five of the seven are associated with cell cycle regulatory processes. These seven genes are novel targets for modulation by the TCDD-activated AHR and may be involved in the observed cell-cycle arrest and suppression of early thymocyte development.

Prenatal Exposure to Cigarette Smoke Alters Later-Life Antitumor Cytotoxic T-Lymphocyte (CTL) Activity Via Possible Changes in T-Regulatory Cells

Epidemiological studies suggest that maternal smoking increases the incidence in the progeny of certain childhood cancers. Our previous study in mice demonstrated the feasibility of such an association by demonstrating that prenatal exposure to cigarette smoke (CS) elevated the incidence of transplanted tumors and reduced cytotoxic T-lymphocyte (CTL) activity in juvenile male offspring. The current study extends these findings by investigating the relationship between CS-induced CTL suppression and effects on regulators of effector T-cell activity, such as T-regulatory (Treg; CD4+CD25+Foxp3+) cells and transforming growth factor (TGF)-β. Results here demonstrate that in utero exposure to CS, at a maternal particle concentration of 15 mg/m3 (4 h/d, 5 d/wk), significantly reduced ex vivo CTL activity of whole splenocytes (and isolated CD8+ cells) against tumor cells both before and after injection of prenatally exposed mice with EL4 lymphoma cells. In contrast, prenatal CS exposure significantly increased levels of thymic Treg cells in a time-dependent manner following tumor cell injection. In vitro production of TGF-β by splenocytes recovered from prenatally exposed, tumor-bearing mice was also altered. Neither prenatal CS exposure nor subsequent administration of EL4 cells exerted any marked effects on lymphoid organ weights, cellularity, or histologic profiles. Given that Treg cells and TGF-β suppress effector T-cell activities, these findings suggest possible immune mechanisms by which early exposure to CS reduces CTL tumoricidal activity during tumor cell development. Data suggest that children of smoking mothers may be less able to mount an appropriate adaptive immune response to tumors, thus increasing their risk for some cancers later in life.

Bcl2-Independent Chromatin Cleavage is a Very Early Event during Induction of Apoptosis in Mouse Thymocytes after Treatment with Either Dexamethasone or Ionizing Radiation

Abstract Hahn, P. J., Lai, Z-W., Nevaldine, B., Schiff, N., Fiore, N. C. and Silverstone, A. E. Bcl2-Independent Chromatin Cleavage is a Very Early Event during Induction of Apoptosis in Mouse Thymocytes after Treatment with Either Dexamethasone or Ionizing Radiation. Radiat. Res. 160, 559–567 (2003). We have quantified the emergence of early chromatin breaks during the signal transduction phase of apoptosis in mouse thymocytes after treatment with either ionizing radiation or dexamethasone. Dexamethasone at 1 μM can induce significant levels of DNA breaks (equivalent to the amount induced directly by 7.5 Gy ionizing radiation) within 0.5 h of treatment. The execution phase of apoptosis was not observed until 4–6 h after the same treatment. The presence of the Bcl2 transgene under the control of the p56lck promoter almost completely inhibited apoptosis up to 24 h after treatment, but it had virtually no effect on the early chromatin cleavage occurring in the first 6 h. Ionizing radiation induced chromatin cleavage both directly by damaging DNA and indirectly with kinetics similar to the induction of chromatin cleavage by dexamethasone. The presence of the Bcl2 transgene had no effect on the direct or indirect radiation-induced cleavage in the first 6 h, but after the first 6 h, the Bcl2 gene inhibited further radiation-induced chromatin cleavage. These results suggest that endonucleases are activated within minutes of treatment with either dexamethasone or ionizing radiation as part of the very early signal transduction phase of apoptosis, and prior to the irreversible commitment to cell death.