Limits...
Bcl-2-mediated drug resistance: inhibition of apoptosis by blocking nuclear factor of activated T lymphocytes (NFAT)-induced Fas ligand transcription.

Srivastava RK, Sasaki CY, Hardwick JM, Longo DL - J. Exp. Med. (1999)

Bottom Line: However, the mechanism of the antiapoptotic effects is unclear.The effects of Bcl-2 can be overcome, at least partially, through phosphorylation of Bcl-2.Phosphorylated Bcl-2 cannot bind calcineurin, and NFAT activation, FasL expression, and apoptosis can occur after Bcl-2 phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunology, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224-6825, USA. rakeshs@vax.grc.nia.nih.gov

ABSTRACT
Bcl-2 inhibits apoptosis induced by a variety of stimuli, including chemotherapy drugs and glucocorticoids. It is generally accepted that Bcl-2 exerts its antiapoptotic effects mainly by dimerizing with proapoptotic members of the Bcl-2 family such as Bax and Bad. However, the mechanism of the antiapoptotic effects is unclear. Paclitaxel and other drugs that disturb microtubule dynamics kill cells in a Fas/Fas ligand (FasL)-dependent manner; antibody to FasL inhibits paclitaxel-induced apoptosis. We have found that Bcl-2 overexpression leads to the prevention of chemotherapy (paclitaxel)-induced expression of FasL and blocks paclitaxel-induced apoptosis. The mechanism of this effect is that Bcl-2 prevents the nuclear translocation of NFAT (nuclear factor of activated T lymphocytes, a transcription factor activated by microtubule damage) by binding and sequestering calcineurin, a calcium-dependent phosphatase that must dephosphorylate NFAT to move to the nucleus. Without NFAT nuclear translocation, the FasL gene is not transcribed. Thus, it appears that paclitaxel and other drugs that disturb microtubule function kill cells at least in part through the induction of FasL. Furthermore, Bcl-2 antagonizes drug-induced apoptosis by inhibiting calcineurin activation, blocking NFAT nuclear translocation, and preventing FasL expression. The effects of Bcl-2 can be overcome, at least partially, through phosphorylation of Bcl-2. Phosphorylated Bcl-2 cannot bind calcineurin, and NFAT activation, FasL expression, and apoptosis can occur after Bcl-2 phosphorylation.

Show MeSH

Related in: MedlinePlus

Bcl-2 inhibits paclitaxel-induced apoptosis. (A) Jurkat cells were stably transfected with either pSSFV-neo or pSFFV-Bcl-2 plasmid. (B) MDA-MB-231 cells were stably transfected with either pSSFV-neo or pSFFV-Bcl-2 plasmid. (C) Jurkat cells (JT/Neo and JT/Bcl-2) were treated with paclitaxel (0.001, 0.01, and 0.1 μM) with or without anti-FasL neutralizing antibody (NOK-2; 1 μg/ml) for 48 h. Cells were stained with DAPI and visualized under fluorescence microscopy. Cells with fragmented nuclei or condensed chromatin were counted as apoptotic. Data (mean ± SE of quadruplicate determinations) represent one of three separate experiments that gave similar results. (D) MDA/MB/231 (MDA/Neo and MDA/Bcl-2) were treated with paclitaxel (0.001, 0.01, and 0.1 μM) with or without anti-FasL neutralizing antibody (NOK-2; 1 μg/ml) for 48 h. Cells were stained with DAPI and visualized under fluorescence microscopy. Cells with fragmented and condensed chromatin were counted as apoptotic. Data (mean ± SE of quadruplicate determinations) represent one of three separate experiments that gave similar results. (E) JT/Neo and JT/Bcl-2 cells were treated with paclitaxel (0.001, 0.01, and 0.1 μM) with or without anti-Fas blocking antibody (1 μg/ml) for 48 h. Apoptotic nuclei were counted as described for D. (F) JT/Neo and JT/mut CD95 cells were treated with paclitaxel (0.001, 0.01, and 0.1 μM) for 48 h. Apoptotic nuclei were counted as described for D.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2195578&req=5

Figure 1: Bcl-2 inhibits paclitaxel-induced apoptosis. (A) Jurkat cells were stably transfected with either pSSFV-neo or pSFFV-Bcl-2 plasmid. (B) MDA-MB-231 cells were stably transfected with either pSSFV-neo or pSFFV-Bcl-2 plasmid. (C) Jurkat cells (JT/Neo and JT/Bcl-2) were treated with paclitaxel (0.001, 0.01, and 0.1 μM) with or without anti-FasL neutralizing antibody (NOK-2; 1 μg/ml) for 48 h. Cells were stained with DAPI and visualized under fluorescence microscopy. Cells with fragmented nuclei or condensed chromatin were counted as apoptotic. Data (mean ± SE of quadruplicate determinations) represent one of three separate experiments that gave similar results. (D) MDA/MB/231 (MDA/Neo and MDA/Bcl-2) were treated with paclitaxel (0.001, 0.01, and 0.1 μM) with or without anti-FasL neutralizing antibody (NOK-2; 1 μg/ml) for 48 h. Cells were stained with DAPI and visualized under fluorescence microscopy. Cells with fragmented and condensed chromatin were counted as apoptotic. Data (mean ± SE of quadruplicate determinations) represent one of three separate experiments that gave similar results. (E) JT/Neo and JT/Bcl-2 cells were treated with paclitaxel (0.001, 0.01, and 0.1 μM) with or without anti-Fas blocking antibody (1 μg/ml) for 48 h. Apoptotic nuclei were counted as described for D. (F) JT/Neo and JT/mut CD95 cells were treated with paclitaxel (0.001, 0.01, and 0.1 μM) for 48 h. Apoptotic nuclei were counted as described for D.

Mentions: FasL induction has been demonstrated in activation-induced cell death in T cells 4748495051 and in the death of other cell types induced by anticancer drugs 52, gamma irradiation 53, and UV light 54. We investigated the possibility of the involvement of the FasL/Fas pathway in paclitaxel-induced apoptosis. Jurkat cells or MDA-MB-231 cells were stably transfected with either pSSFV-Neo or pSSFV-Bcl-2 expression vector to assess the protective effects of Bcl-2 on paclitaxel-induced apoptosis (Fig. 1a and Fig. b). Treatment of cells with paclitaxel resulted in induction of apoptosis in a dose-dependent manner, and overexpression of Bcl-2 inhibited paclitaxel-induced apoptosis in Jurkat cells (Fig. 1 C). The paclitaxel dose–response curve suggests a 10-fold increase in resistance in cells overexpressing Bcl-2. Neutralization of FasL by treatment of cells with anti-FasL antibody (NOK-2) significantly inhibited paclitaxel-induced apoptosis in both JT/Neo and JT/Bcl-2 cells. Indeed, very little tumor cell death could be documented in Bcl-2–overexpressing Jurkat cells exposed to anti-FasL antibody. To examine the role of Bcl-2 in paclitaxel-induced apoptosis, we used MDA-MB-231 breast cancer cells, which do not express endogenous Bcl-2 (Fig. 1 B). Overexpression of Bcl-2 in MDA cells inhibited paclitaxel-induced apoptosis by greater than two logs (Fig. 1 D). Neutralization of FasL by anti-FasL antibody (NOK-2) significantly inhibited paclitaxel-induced apoptosis in MDA/Neo but had little effect in MDA/Bcl-2 cells. Incubation of cells with Fas- blocking antibody inhibited paclitaxel-induced apoptosis in JT/Neo and JT/Bcl-2 cells. Similarly, overexpression of mutant CD95/Fas (mutant receptors lacking intracellular cytoplasmic domains) inhibited paclitaxel-induced apoptosis (Fig. 1 F). Taken together, these data demonstrate that (a) paclitaxel-induced apoptosis can be inhibited by Bcl-2 and (b) the FasL/Fas pathway, at least in part, mediates paclitaxel-induced apoptosis.


Bcl-2-mediated drug resistance: inhibition of apoptosis by blocking nuclear factor of activated T lymphocytes (NFAT)-induced Fas ligand transcription.

Srivastava RK, Sasaki CY, Hardwick JM, Longo DL - J. Exp. Med. (1999)

Bcl-2 inhibits paclitaxel-induced apoptosis. (A) Jurkat cells were stably transfected with either pSSFV-neo or pSFFV-Bcl-2 plasmid. (B) MDA-MB-231 cells were stably transfected with either pSSFV-neo or pSFFV-Bcl-2 plasmid. (C) Jurkat cells (JT/Neo and JT/Bcl-2) were treated with paclitaxel (0.001, 0.01, and 0.1 μM) with or without anti-FasL neutralizing antibody (NOK-2; 1 μg/ml) for 48 h. Cells were stained with DAPI and visualized under fluorescence microscopy. Cells with fragmented nuclei or condensed chromatin were counted as apoptotic. Data (mean ± SE of quadruplicate determinations) represent one of three separate experiments that gave similar results. (D) MDA/MB/231 (MDA/Neo and MDA/Bcl-2) were treated with paclitaxel (0.001, 0.01, and 0.1 μM) with or without anti-FasL neutralizing antibody (NOK-2; 1 μg/ml) for 48 h. Cells were stained with DAPI and visualized under fluorescence microscopy. Cells with fragmented and condensed chromatin were counted as apoptotic. Data (mean ± SE of quadruplicate determinations) represent one of three separate experiments that gave similar results. (E) JT/Neo and JT/Bcl-2 cells were treated with paclitaxel (0.001, 0.01, and 0.1 μM) with or without anti-Fas blocking antibody (1 μg/ml) for 48 h. Apoptotic nuclei were counted as described for D. (F) JT/Neo and JT/mut CD95 cells were treated with paclitaxel (0.001, 0.01, and 0.1 μM) for 48 h. Apoptotic nuclei were counted as described for D.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195578&req=5

Figure 1: Bcl-2 inhibits paclitaxel-induced apoptosis. (A) Jurkat cells were stably transfected with either pSSFV-neo or pSFFV-Bcl-2 plasmid. (B) MDA-MB-231 cells were stably transfected with either pSSFV-neo or pSFFV-Bcl-2 plasmid. (C) Jurkat cells (JT/Neo and JT/Bcl-2) were treated with paclitaxel (0.001, 0.01, and 0.1 μM) with or without anti-FasL neutralizing antibody (NOK-2; 1 μg/ml) for 48 h. Cells were stained with DAPI and visualized under fluorescence microscopy. Cells with fragmented nuclei or condensed chromatin were counted as apoptotic. Data (mean ± SE of quadruplicate determinations) represent one of three separate experiments that gave similar results. (D) MDA/MB/231 (MDA/Neo and MDA/Bcl-2) were treated with paclitaxel (0.001, 0.01, and 0.1 μM) with or without anti-FasL neutralizing antibody (NOK-2; 1 μg/ml) for 48 h. Cells were stained with DAPI and visualized under fluorescence microscopy. Cells with fragmented and condensed chromatin were counted as apoptotic. Data (mean ± SE of quadruplicate determinations) represent one of three separate experiments that gave similar results. (E) JT/Neo and JT/Bcl-2 cells were treated with paclitaxel (0.001, 0.01, and 0.1 μM) with or without anti-Fas blocking antibody (1 μg/ml) for 48 h. Apoptotic nuclei were counted as described for D. (F) JT/Neo and JT/mut CD95 cells were treated with paclitaxel (0.001, 0.01, and 0.1 μM) for 48 h. Apoptotic nuclei were counted as described for D.
Mentions: FasL induction has been demonstrated in activation-induced cell death in T cells 4748495051 and in the death of other cell types induced by anticancer drugs 52, gamma irradiation 53, and UV light 54. We investigated the possibility of the involvement of the FasL/Fas pathway in paclitaxel-induced apoptosis. Jurkat cells or MDA-MB-231 cells were stably transfected with either pSSFV-Neo or pSSFV-Bcl-2 expression vector to assess the protective effects of Bcl-2 on paclitaxel-induced apoptosis (Fig. 1a and Fig. b). Treatment of cells with paclitaxel resulted in induction of apoptosis in a dose-dependent manner, and overexpression of Bcl-2 inhibited paclitaxel-induced apoptosis in Jurkat cells (Fig. 1 C). The paclitaxel dose–response curve suggests a 10-fold increase in resistance in cells overexpressing Bcl-2. Neutralization of FasL by treatment of cells with anti-FasL antibody (NOK-2) significantly inhibited paclitaxel-induced apoptosis in both JT/Neo and JT/Bcl-2 cells. Indeed, very little tumor cell death could be documented in Bcl-2–overexpressing Jurkat cells exposed to anti-FasL antibody. To examine the role of Bcl-2 in paclitaxel-induced apoptosis, we used MDA-MB-231 breast cancer cells, which do not express endogenous Bcl-2 (Fig. 1 B). Overexpression of Bcl-2 in MDA cells inhibited paclitaxel-induced apoptosis by greater than two logs (Fig. 1 D). Neutralization of FasL by anti-FasL antibody (NOK-2) significantly inhibited paclitaxel-induced apoptosis in MDA/Neo but had little effect in MDA/Bcl-2 cells. Incubation of cells with Fas- blocking antibody inhibited paclitaxel-induced apoptosis in JT/Neo and JT/Bcl-2 cells. Similarly, overexpression of mutant CD95/Fas (mutant receptors lacking intracellular cytoplasmic domains) inhibited paclitaxel-induced apoptosis (Fig. 1 F). Taken together, these data demonstrate that (a) paclitaxel-induced apoptosis can be inhibited by Bcl-2 and (b) the FasL/Fas pathway, at least in part, mediates paclitaxel-induced apoptosis.

Bottom Line: However, the mechanism of the antiapoptotic effects is unclear.The effects of Bcl-2 can be overcome, at least partially, through phosphorylation of Bcl-2.Phosphorylated Bcl-2 cannot bind calcineurin, and NFAT activation, FasL expression, and apoptosis can occur after Bcl-2 phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunology, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224-6825, USA. rakeshs@vax.grc.nia.nih.gov

ABSTRACT
Bcl-2 inhibits apoptosis induced by a variety of stimuli, including chemotherapy drugs and glucocorticoids. It is generally accepted that Bcl-2 exerts its antiapoptotic effects mainly by dimerizing with proapoptotic members of the Bcl-2 family such as Bax and Bad. However, the mechanism of the antiapoptotic effects is unclear. Paclitaxel and other drugs that disturb microtubule dynamics kill cells in a Fas/Fas ligand (FasL)-dependent manner; antibody to FasL inhibits paclitaxel-induced apoptosis. We have found that Bcl-2 overexpression leads to the prevention of chemotherapy (paclitaxel)-induced expression of FasL and blocks paclitaxel-induced apoptosis. The mechanism of this effect is that Bcl-2 prevents the nuclear translocation of NFAT (nuclear factor of activated T lymphocytes, a transcription factor activated by microtubule damage) by binding and sequestering calcineurin, a calcium-dependent phosphatase that must dephosphorylate NFAT to move to the nucleus. Without NFAT nuclear translocation, the FasL gene is not transcribed. Thus, it appears that paclitaxel and other drugs that disturb microtubule function kill cells at least in part through the induction of FasL. Furthermore, Bcl-2 antagonizes drug-induced apoptosis by inhibiting calcineurin activation, blocking NFAT nuclear translocation, and preventing FasL expression. The effects of Bcl-2 can be overcome, at least partially, through phosphorylation of Bcl-2. Phosphorylated Bcl-2 cannot bind calcineurin, and NFAT activation, FasL expression, and apoptosis can occur after Bcl-2 phosphorylation.

Show MeSH
Related in: MedlinePlus