Limits...
Bad can act as a key regulator of T cell apoptosis and T cell development.

Mok CL, Gil-Gómez G, Williams O, Coles M, Taga S, Tolaini M, Norton T, Kioussis D, Brady HJ - J. Exp. Med. (1999)

Bottom Line: Here, we show that Bad expression levels are greatly increased in thymocytes during apoptosis.The numbers of T cells are greatly depleted and the processes of T cell development and selection are perturbed.These data suggest that Bad can act as a key regulator of T cell apoptosis and that this is a consequence of its upregulation after exposure to death stimuli.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Immunology, MRC National Institute for Medical Research, London NW7 1AA, United Kingdom.

ABSTRACT
Bad is a distant relative of Bcl-2 and acts to promote cell death. Here, we show that Bad expression levels are greatly increased in thymocytes during apoptosis. We generated bad transgenic mice to study the action of upregulated Bad expression on T cell apoptosis. The T cells from these mice are highly sensitive to apoptotic stimuli, including anti-CD95. The numbers of T cells are greatly depleted and the processes of T cell development and selection are perturbed. We show that the proapoptotic function of Bad in primary T cells is regulated by Akt kinase and that Bad overexpression enhances both cell cycle progression and interleukin 2 production after T cell activation. These data suggest that Bad can act as a key regulator of T cell apoptosis and that this is a consequence of its upregulation after exposure to death stimuli.

Show MeSH

Related in: MedlinePlus

Akt kinase activity in bad thymocytes is constitutively higher  and is inhibited by wortmannin. Total cell lysates were prepared from  thymocytes of bad transgenic mice and control littermates which had been  cultured for up to 4 h. Akt was immunoprecipitated and an in vitro kinase  assay was carried out using histone H2B as substrate. (A) The constitutive  (0 h) level of kinase activity and the activity versus time of the nontransgenic control is shown in the left panel, and that of the bad transgenic  mice in the right panel. (B) Akt kinase activity of bad transgenic thymocytes is shown in the absence (left) or presence (right) of 2 μM wortmannin over 4 h.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2192908&req=5

Figure 8: Akt kinase activity in bad thymocytes is constitutively higher and is inhibited by wortmannin. Total cell lysates were prepared from thymocytes of bad transgenic mice and control littermates which had been cultured for up to 4 h. Akt was immunoprecipitated and an in vitro kinase assay was carried out using histone H2B as substrate. (A) The constitutive (0 h) level of kinase activity and the activity versus time of the nontransgenic control is shown in the left panel, and that of the bad transgenic mice in the right panel. (B) Akt kinase activity of bad transgenic thymocytes is shown in the absence (left) or presence (right) of 2 μM wortmannin over 4 h.

Mentions: To confirm that the effect of the PI-3-K inhibitors was on Akt activation, we directly measured the Akt kinase activity in thymocytes from bad transgenic mice in the presence or absence of the inhibitors. Akt kinase activity was assayed by in vitro kinase assay after Akt immunoprecipitation using histone H2B as substrate (23). Akt kinase activity was measured in thymocytes from bad transgenic and nontransgenic mice after in vitro culture. The constitutive level of Akt kinase activity (0 h) is much higher in bad transgenic mice than in nontransgenic mice (Fig. 8 A). This high level of Akt kinase activity falls as the level of apoptosis increases with time, whereas the level of Akt kinase activity in the wild type remains negligible. In the presence of wortmannin the rate of decrease of Akt activity is greater in the bad transgenic thymocytes than without wortmannin (Fig. 8 B). For example, as determined by densitometry, the level of Akt kinase activity is almost halved in bad transgenic thymocytes after 2–3 h in culture in the presence of wortmannin. Thus, the effect of PI-3-K inhibitors in accelerating Bad-induced apoptosis appears to be mediated via the reduction of Akt kinase activity. However, we were surprised to discover that bad transgenic thymocytes have a significantly elevated level of constitutive Akt kinase activity. This suggests that, although Bad is biochemically downstream of Akt, elevated levels of Bad can influence the level of Akt activity, possibly by a paracrine/autocrine feedback mechanism.


Bad can act as a key regulator of T cell apoptosis and T cell development.

Mok CL, Gil-Gómez G, Williams O, Coles M, Taga S, Tolaini M, Norton T, Kioussis D, Brady HJ - J. Exp. Med. (1999)

Akt kinase activity in bad thymocytes is constitutively higher  and is inhibited by wortmannin. Total cell lysates were prepared from  thymocytes of bad transgenic mice and control littermates which had been  cultured for up to 4 h. Akt was immunoprecipitated and an in vitro kinase  assay was carried out using histone H2B as substrate. (A) The constitutive  (0 h) level of kinase activity and the activity versus time of the nontransgenic control is shown in the left panel, and that of the bad transgenic  mice in the right panel. (B) Akt kinase activity of bad transgenic thymocytes is shown in the absence (left) or presence (right) of 2 μM wortmannin over 4 h.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2192908&req=5

Figure 8: Akt kinase activity in bad thymocytes is constitutively higher and is inhibited by wortmannin. Total cell lysates were prepared from thymocytes of bad transgenic mice and control littermates which had been cultured for up to 4 h. Akt was immunoprecipitated and an in vitro kinase assay was carried out using histone H2B as substrate. (A) The constitutive (0 h) level of kinase activity and the activity versus time of the nontransgenic control is shown in the left panel, and that of the bad transgenic mice in the right panel. (B) Akt kinase activity of bad transgenic thymocytes is shown in the absence (left) or presence (right) of 2 μM wortmannin over 4 h.
Mentions: To confirm that the effect of the PI-3-K inhibitors was on Akt activation, we directly measured the Akt kinase activity in thymocytes from bad transgenic mice in the presence or absence of the inhibitors. Akt kinase activity was assayed by in vitro kinase assay after Akt immunoprecipitation using histone H2B as substrate (23). Akt kinase activity was measured in thymocytes from bad transgenic and nontransgenic mice after in vitro culture. The constitutive level of Akt kinase activity (0 h) is much higher in bad transgenic mice than in nontransgenic mice (Fig. 8 A). This high level of Akt kinase activity falls as the level of apoptosis increases with time, whereas the level of Akt kinase activity in the wild type remains negligible. In the presence of wortmannin the rate of decrease of Akt activity is greater in the bad transgenic thymocytes than without wortmannin (Fig. 8 B). For example, as determined by densitometry, the level of Akt kinase activity is almost halved in bad transgenic thymocytes after 2–3 h in culture in the presence of wortmannin. Thus, the effect of PI-3-K inhibitors in accelerating Bad-induced apoptosis appears to be mediated via the reduction of Akt kinase activity. However, we were surprised to discover that bad transgenic thymocytes have a significantly elevated level of constitutive Akt kinase activity. This suggests that, although Bad is biochemically downstream of Akt, elevated levels of Bad can influence the level of Akt activity, possibly by a paracrine/autocrine feedback mechanism.

Bottom Line: Here, we show that Bad expression levels are greatly increased in thymocytes during apoptosis.The numbers of T cells are greatly depleted and the processes of T cell development and selection are perturbed.These data suggest that Bad can act as a key regulator of T cell apoptosis and that this is a consequence of its upregulation after exposure to death stimuli.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Immunology, MRC National Institute for Medical Research, London NW7 1AA, United Kingdom.

ABSTRACT
Bad is a distant relative of Bcl-2 and acts to promote cell death. Here, we show that Bad expression levels are greatly increased in thymocytes during apoptosis. We generated bad transgenic mice to study the action of upregulated Bad expression on T cell apoptosis. The T cells from these mice are highly sensitive to apoptotic stimuli, including anti-CD95. The numbers of T cells are greatly depleted and the processes of T cell development and selection are perturbed. We show that the proapoptotic function of Bad in primary T cells is regulated by Akt kinase and that Bad overexpression enhances both cell cycle progression and interleukin 2 production after T cell activation. These data suggest that Bad can act as a key regulator of T cell apoptosis and that this is a consequence of its upregulation after exposure to death stimuli.

Show MeSH
Related in: MedlinePlus