A role for bcl-2 in notch1-dependent transcription in thymic lymphoma cells

Abstract

Notch1 is a transcription factor important for T-cell development. Notch1 is active in double negative (DN) thymocytes, while being depressed in double positive (DP) thymocytes. Synchronously, the expression of Bcl-2 becomes downregulated during the transition from DN to DP thymocytes. We previously observed that overexpression of an intracellular active Notch1 (ICN) in Bcl-2-positive 2B4 T cells leads to the transcription of Notch1-regulated genes. However, these genes were not induced in Bcl-2-negative DP PD1.6 thymic lymphoma cells overexpressing ICN. Here we show that, when Bcl-2 is simultaneously introduced into these cells, Notch-regulated genes are transcribed. Only in the presence of both Bcl-2 and ICN, PD1.6 thymic lymphoma cells become resistant to glucocorticoid (GC)-induced apoptosis. Our data suggest that Bcl-2 plays a role in modulating Notch1 function in T cells.

Figure 1

(a) Induction of Deltex1 and Hes1 expression in PD1.6 cells overexpressing both ICN-Notch1 and Bcl-2. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of the indicated cells. GAPDH was used as loading control. (b) Western blot analysis of Bcl-2 and Notch1 expression in PD1.6, PD1.6Bcl-2, PD1.6Notch1, and PD1.6Bcl-2Notch1 cells. α-Tubulin was used as loading control.

Figure 2

PD1.6 cells overexpressing both ICN-Notch1 and Bcl-2 become resistant to dexamethasone (Dex)-induced apoptosis. The cells were exposed to 100 nM Dex for the indicated time periods, followed by analysis of propidium iodide (PI) positive cells on flow cytometry. Untreated cells were used as control. *P < 0.04  **P < 0.01.

Figure 3

PD1.6Bcl-2Notch1 cells show reduced GR expression. Western blot analysis of untreated or Dex-treated (100 nM, 2 hrs) cells. α-Tubulin was used as loading control.