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Early growth response-1 is a regulator of DR5-induced apoptosis in colon cancer cells.

Mahalingam D, Natoni A, Keane M, Samali A, Szegezdi E - Br. J. Cancer (2010)

Bottom Line: Our results also show that DR4 mediates a type II, mitochondrion-dependent apoptotic pathway, whereas DR5 induces a mitochondrion-independent, type I apoptosis in HCT15 colon carcinoma cells.Egr-1 drives c-FLIP expression and the short splice variant of c-FLIP (c-FLIP(S)) specifically inhibits DR5 activation.Selective knockdown of c-FLIP(S) sensitises cells to DR5-induced but not DR4-induced apoptosis and Egr-1 exerts an effect as an inhibitor of the DR5-induced apoptotic pathway, possibly by regulating the expression of c-FLIP(S).

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and National Centre of Biomedical Engineering Science, National University of Ireland, Galway, Ireland.

ABSTRACT

Background: Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) induces tumour cell apoptosis by binding to death receptor 4 (DR4) and DR5. DR4 and DR5 activation however can also induce inflammatory and pro-survival signalling. It is not known how these different cellular responses are regulated and what the individual role of DR4 vs DR5 is in these processes.

Methods: DNA microarray study was carried out to identify genes differentially expressed after DR4 and DR5 activation. RT-PCR and western blotting was used to examine the expression of early growth response gene-1 (Egr-1) and the proteins of the TRAIL signalling pathway. The function of Egr-1 was studied by siRNA-mediated knockdown and overexpression of a dominant-negative version of Egr-1.

Results: We show that the immediate early gene, Egr-1, regulates TRAIL sensitivity. Egr-1 is constitutively expressed in colon cancer cells and further induced upon activation of DR4 or DR5. Our results also show that DR4 mediates a type II, mitochondrion-dependent apoptotic pathway, whereas DR5 induces a mitochondrion-independent, type I apoptosis in HCT15 colon carcinoma cells. Egr-1 drives c-FLIP expression and the short splice variant of c-FLIP (c-FLIP(S)) specifically inhibits DR5 activation.

Conclusion: Selective knockdown of c-FLIP(S) sensitises cells to DR5-induced but not DR4-induced apoptosis and Egr-1 exerts an effect as an inhibitor of the DR5-induced apoptotic pathway, possibly by regulating the expression of c-FLIP(S).

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Inhibition or knockdown of Egr-1 potentiates rhTRAIL- and DR5-induced apoptosis. (A) Effect of dominant-negative Egr-1 (DN-Egr-1) expression on TRAIL-, DR4- and DR5-induced apoptosis in HCT15 cells. HCT15 cells were transiently transfected with EBGN-Egr-1 (DN) or empty vector (EV). Cell lysates of parental cells (C), EV- and DN-Egr-1-transfected cells were analysed for overexpression of DN-Egr-1 at 24 h after transfection using western blotting (inlet). HCT15 cells transfected with pEBGN-EGR-1 (DN-Egr-1) were treated at 24 h after transfection with either 10 nM of crosslinked agonistic DR4/DR5 antibody or 50 ng ml–1 rhTRAIL for 5 h and apoptosis was assessed using Annexin V staining (DR4, DR5 and rhTRAIL). (B) Knockdown of Egr-1 sensitises HCT15 cells to TRAIL and DR5-induced apoptosis. HCT15 cells were transiently transfected with a Smartpool siRNA mix against Egr-1 (Egr-1) or scrambled siRNA (control siRNA, C). Knockdown of Egr-1 was confirmed 24 h after transfection using Western blotting (inlet). Cells were treated as described in point (A) and induction of apoptosis was measured with Annexin V staining. Results are presented as means±s.e.m. of at least three independent experiments; *Significant difference with P<0.05.
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fig3: Inhibition or knockdown of Egr-1 potentiates rhTRAIL- and DR5-induced apoptosis. (A) Effect of dominant-negative Egr-1 (DN-Egr-1) expression on TRAIL-, DR4- and DR5-induced apoptosis in HCT15 cells. HCT15 cells were transiently transfected with EBGN-Egr-1 (DN) or empty vector (EV). Cell lysates of parental cells (C), EV- and DN-Egr-1-transfected cells were analysed for overexpression of DN-Egr-1 at 24 h after transfection using western blotting (inlet). HCT15 cells transfected with pEBGN-EGR-1 (DN-Egr-1) were treated at 24 h after transfection with either 10 nM of crosslinked agonistic DR4/DR5 antibody or 50 ng ml–1 rhTRAIL for 5 h and apoptosis was assessed using Annexin V staining (DR4, DR5 and rhTRAIL). (B) Knockdown of Egr-1 sensitises HCT15 cells to TRAIL and DR5-induced apoptosis. HCT15 cells were transiently transfected with a Smartpool siRNA mix against Egr-1 (Egr-1) or scrambled siRNA (control siRNA, C). Knockdown of Egr-1 was confirmed 24 h after transfection using Western blotting (inlet). Cells were treated as described in point (A) and induction of apoptosis was measured with Annexin V staining. Results are presented as means±s.e.m. of at least three independent experiments; *Significant difference with P<0.05.

Mentions: To determine whether Egr-1 has any role in TRAIL-induced apoptosis, HCT15 cells were transiently transfected with a plasmid expressing dominant-negative Egr-1 (EBGN-Egr-1) that contains only the DNA-binding domain of Egr-1 fused to GST (Al-Sarraj et al, 2005). Overexpression of dominant-negative Egr-1 protein (DN-Egr-1) was confirmed by western blot analysis using Egr-1 antibody (inlet, Figure 3A). On the blot, the lower (approximately 56 kDa) band represents the truncated, DN-Egr-1. To inhibit Egr-1 activity, 2.5 μg of DN-Egr-1 plasmid was transfected into the cells, as this amount was found to fully block Egr-1 transcriptional activity for at least 48 h after transfection (Supplementary Figure 2A). After 5 h treatment with 10 nM agonistic DR5 antibody or rhTRAIL, HCT15 cells overexpressing DN-Egr-1 suffered significantly more apoptosis than untransfected cells or cells transfected with the empty vector (Figure 3A). Interestingly, no enhancement in apoptosis was observed in cells treated with agonistic DR4 antibody (Figure 3A). Knockdown of Egr-1 with siRNA (Smartpool, Dharmacon) also increased the sensitivity of HCT15 cells to DR5 activation, but not to DR4 activation (Figure 3B).


Early growth response-1 is a regulator of DR5-induced apoptosis in colon cancer cells.

Mahalingam D, Natoni A, Keane M, Samali A, Szegezdi E - Br. J. Cancer (2010)

Inhibition or knockdown of Egr-1 potentiates rhTRAIL- and DR5-induced apoptosis. (A) Effect of dominant-negative Egr-1 (DN-Egr-1) expression on TRAIL-, DR4- and DR5-induced apoptosis in HCT15 cells. HCT15 cells were transiently transfected with EBGN-Egr-1 (DN) or empty vector (EV). Cell lysates of parental cells (C), EV- and DN-Egr-1-transfected cells were analysed for overexpression of DN-Egr-1 at 24 h after transfection using western blotting (inlet). HCT15 cells transfected with pEBGN-EGR-1 (DN-Egr-1) were treated at 24 h after transfection with either 10 nM of crosslinked agonistic DR4/DR5 antibody or 50 ng ml–1 rhTRAIL for 5 h and apoptosis was assessed using Annexin V staining (DR4, DR5 and rhTRAIL). (B) Knockdown of Egr-1 sensitises HCT15 cells to TRAIL and DR5-induced apoptosis. HCT15 cells were transiently transfected with a Smartpool siRNA mix against Egr-1 (Egr-1) or scrambled siRNA (control siRNA, C). Knockdown of Egr-1 was confirmed 24 h after transfection using Western blotting (inlet). Cells were treated as described in point (A) and induction of apoptosis was measured with Annexin V staining. Results are presented as means±s.e.m. of at least three independent experiments; *Significant difference with P<0.05.
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fig3: Inhibition or knockdown of Egr-1 potentiates rhTRAIL- and DR5-induced apoptosis. (A) Effect of dominant-negative Egr-1 (DN-Egr-1) expression on TRAIL-, DR4- and DR5-induced apoptosis in HCT15 cells. HCT15 cells were transiently transfected with EBGN-Egr-1 (DN) or empty vector (EV). Cell lysates of parental cells (C), EV- and DN-Egr-1-transfected cells were analysed for overexpression of DN-Egr-1 at 24 h after transfection using western blotting (inlet). HCT15 cells transfected with pEBGN-EGR-1 (DN-Egr-1) were treated at 24 h after transfection with either 10 nM of crosslinked agonistic DR4/DR5 antibody or 50 ng ml–1 rhTRAIL for 5 h and apoptosis was assessed using Annexin V staining (DR4, DR5 and rhTRAIL). (B) Knockdown of Egr-1 sensitises HCT15 cells to TRAIL and DR5-induced apoptosis. HCT15 cells were transiently transfected with a Smartpool siRNA mix against Egr-1 (Egr-1) or scrambled siRNA (control siRNA, C). Knockdown of Egr-1 was confirmed 24 h after transfection using Western blotting (inlet). Cells were treated as described in point (A) and induction of apoptosis was measured with Annexin V staining. Results are presented as means±s.e.m. of at least three independent experiments; *Significant difference with P<0.05.
Mentions: To determine whether Egr-1 has any role in TRAIL-induced apoptosis, HCT15 cells were transiently transfected with a plasmid expressing dominant-negative Egr-1 (EBGN-Egr-1) that contains only the DNA-binding domain of Egr-1 fused to GST (Al-Sarraj et al, 2005). Overexpression of dominant-negative Egr-1 protein (DN-Egr-1) was confirmed by western blot analysis using Egr-1 antibody (inlet, Figure 3A). On the blot, the lower (approximately 56 kDa) band represents the truncated, DN-Egr-1. To inhibit Egr-1 activity, 2.5 μg of DN-Egr-1 plasmid was transfected into the cells, as this amount was found to fully block Egr-1 transcriptional activity for at least 48 h after transfection (Supplementary Figure 2A). After 5 h treatment with 10 nM agonistic DR5 antibody or rhTRAIL, HCT15 cells overexpressing DN-Egr-1 suffered significantly more apoptosis than untransfected cells or cells transfected with the empty vector (Figure 3A). Interestingly, no enhancement in apoptosis was observed in cells treated with agonistic DR4 antibody (Figure 3A). Knockdown of Egr-1 with siRNA (Smartpool, Dharmacon) also increased the sensitivity of HCT15 cells to DR5 activation, but not to DR4 activation (Figure 3B).

Bottom Line: Our results also show that DR4 mediates a type II, mitochondrion-dependent apoptotic pathway, whereas DR5 induces a mitochondrion-independent, type I apoptosis in HCT15 colon carcinoma cells.Egr-1 drives c-FLIP expression and the short splice variant of c-FLIP (c-FLIP(S)) specifically inhibits DR5 activation.Selective knockdown of c-FLIP(S) sensitises cells to DR5-induced but not DR4-induced apoptosis and Egr-1 exerts an effect as an inhibitor of the DR5-induced apoptotic pathway, possibly by regulating the expression of c-FLIP(S).

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and National Centre of Biomedical Engineering Science, National University of Ireland, Galway, Ireland.

ABSTRACT

Background: Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) induces tumour cell apoptosis by binding to death receptor 4 (DR4) and DR5. DR4 and DR5 activation however can also induce inflammatory and pro-survival signalling. It is not known how these different cellular responses are regulated and what the individual role of DR4 vs DR5 is in these processes.

Methods: DNA microarray study was carried out to identify genes differentially expressed after DR4 and DR5 activation. RT-PCR and western blotting was used to examine the expression of early growth response gene-1 (Egr-1) and the proteins of the TRAIL signalling pathway. The function of Egr-1 was studied by siRNA-mediated knockdown and overexpression of a dominant-negative version of Egr-1.

Results: We show that the immediate early gene, Egr-1, regulates TRAIL sensitivity. Egr-1 is constitutively expressed in colon cancer cells and further induced upon activation of DR4 or DR5. Our results also show that DR4 mediates a type II, mitochondrion-dependent apoptotic pathway, whereas DR5 induces a mitochondrion-independent, type I apoptosis in HCT15 colon carcinoma cells. Egr-1 drives c-FLIP expression and the short splice variant of c-FLIP (c-FLIP(S)) specifically inhibits DR5 activation.

Conclusion: Selective knockdown of c-FLIP(S) sensitises cells to DR5-induced but not DR4-induced apoptosis and Egr-1 exerts an effect as an inhibitor of the DR5-induced apoptotic pathway, possibly by regulating the expression of c-FLIP(S).

Show MeSH
Related in: MedlinePlus