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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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Only DR4-, but not DR5-mediated, apoptosis requires mitochondrial amplification in HCT15 cells. (A) Overexpression of mitochondrial-localised Bcl-2. HCT15 cells were stably transfected with mitochondrial-localised Bcl-2 (Bcl-2-ActA: Bcl-2) expressing plasmid or empty vector (EV). Overexpression of Blcl-2-ActA was confirmed using western blotting. (B) Bcl-2 ActA protects HCT15 cells from DR4-, but not from DR5- or rhTRAIL-induced apoptosis. Cells were treated with 10 nM of crosslinked or uncrosslinked agonistic DR4 and DR5 antibodies or 50 ng ml–1 of rhTRAIL for 12 h and apoptosis was measured using Annexin V staining (DR4, DR5, rhTRAIL labels). Results are presented as means±s.e.m. of three independent experiments. Asterisks (*) designate significant difference (P<0.05) between the indicated sample pairs.
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fig4: Only DR4-, but not DR5-mediated, apoptosis requires mitochondrial amplification in HCT15 cells. (A) Overexpression of mitochondrial-localised Bcl-2. HCT15 cells were stably transfected with mitochondrial-localised Bcl-2 (Bcl-2-ActA: Bcl-2) expressing plasmid or empty vector (EV). Overexpression of Blcl-2-ActA was confirmed using western blotting. (B) Bcl-2 ActA protects HCT15 cells from DR4-, but not from DR5- or rhTRAIL-induced apoptosis. Cells were treated with 10 nM of crosslinked or uncrosslinked agonistic DR4 and DR5 antibodies or 50 ng ml–1 of rhTRAIL for 12 h and apoptosis was measured using Annexin V staining (DR4, DR5, rhTRAIL labels). Results are presented as means±s.e.m. of three independent experiments. Asterisks (*) designate significant difference (P<0.05) between the indicated sample pairs.

Mentions: As overexpression of DN-Egr-1 affected only the DR5-mediated but not the DR4-mediated apoptotic pathway, we wanted to determine whether DR5 and DR4 signal apoptosis through the same pathway in HCT15 cells. As Egr-1 has been reported to regulate the expression of Bcl-2 proteins (Huang et al, 1998b; Ahmed, 2004), first the requirement for mitochondrial amplification for DR4- and DR5-mediated apoptosis was assessed. To this end, stable, mitochondrial-targeted Bcl-2 overexpressing HCT15 cells were generated (mass pool of stable transfectants of Bcl-2-ActA overexpressing cells; Figure 4A) and treated with agonistic DR4 and DR5 antibodies (10 nM) or rhTRAIL (50 ng ml–1). Cells were treated for 12 h to allow all cells affected to undergo apoptosis. Bcl-2 overexpression reduced the level of apoptosis induced by DR4, but not by DR5 or rhTRAIL (Figure 4B), indicating that in HCT15 cells the DR4-induced apoptotic pathway requires mitochondrial amplification, whereas the DR5-induced pathway does not. The effect of DN-Egr-1 on the expression of key mitochondrial proteins was nonetheless examined. Western blot analysis showed that overexpression of DN-EGR-1 did not alter the expression levels of Bax, Bcl-2, Bcl-XL, Mcl-1 or XIAP (Supplementary Figure 2B).


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)

Only DR4-, but not DR5-mediated, apoptosis requires mitochondrial amplification in HCT15 cells. (A) Overexpression of mitochondrial-localised Bcl-2. HCT15 cells were stably transfected with mitochondrial-localised Bcl-2 (Bcl-2-ActA: Bcl-2) expressing plasmid or empty vector (EV). Overexpression of Blcl-2-ActA was confirmed using western blotting. (B) Bcl-2 ActA protects HCT15 cells from DR4-, but not from DR5- or rhTRAIL-induced apoptosis. Cells were treated with 10 nM of crosslinked or uncrosslinked agonistic DR4 and DR5 antibodies or 50 ng ml–1 of rhTRAIL for 12 h and apoptosis was measured using Annexin V staining (DR4, DR5, rhTRAIL labels). Results are presented as means±s.e.m. of three independent experiments. Asterisks (*) designate significant difference (P<0.05) between the indicated sample pairs.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2837577&req=5

fig4: Only DR4-, but not DR5-mediated, apoptosis requires mitochondrial amplification in HCT15 cells. (A) Overexpression of mitochondrial-localised Bcl-2. HCT15 cells were stably transfected with mitochondrial-localised Bcl-2 (Bcl-2-ActA: Bcl-2) expressing plasmid or empty vector (EV). Overexpression of Blcl-2-ActA was confirmed using western blotting. (B) Bcl-2 ActA protects HCT15 cells from DR4-, but not from DR5- or rhTRAIL-induced apoptosis. Cells were treated with 10 nM of crosslinked or uncrosslinked agonistic DR4 and DR5 antibodies or 50 ng ml–1 of rhTRAIL for 12 h and apoptosis was measured using Annexin V staining (DR4, DR5, rhTRAIL labels). Results are presented as means±s.e.m. of three independent experiments. Asterisks (*) designate significant difference (P<0.05) between the indicated sample pairs.
Mentions: As overexpression of DN-Egr-1 affected only the DR5-mediated but not the DR4-mediated apoptotic pathway, we wanted to determine whether DR5 and DR4 signal apoptosis through the same pathway in HCT15 cells. As Egr-1 has been reported to regulate the expression of Bcl-2 proteins (Huang et al, 1998b; Ahmed, 2004), first the requirement for mitochondrial amplification for DR4- and DR5-mediated apoptosis was assessed. To this end, stable, mitochondrial-targeted Bcl-2 overexpressing HCT15 cells were generated (mass pool of stable transfectants of Bcl-2-ActA overexpressing cells; Figure 4A) and treated with agonistic DR4 and DR5 antibodies (10 nM) or rhTRAIL (50 ng ml–1). Cells were treated for 12 h to allow all cells affected to undergo apoptosis. Bcl-2 overexpression reduced the level of apoptosis induced by DR4, but not by DR5 or rhTRAIL (Figure 4B), indicating that in HCT15 cells the DR4-induced apoptotic pathway requires mitochondrial amplification, whereas the DR5-induced pathway does not. The effect of DN-Egr-1 on the expression of key mitochondrial proteins was nonetheless examined. Western blot analysis showed that overexpression of DN-EGR-1 did not alter the expression levels of Bax, Bcl-2, Bcl-XL, Mcl-1 or XIAP (Supplementary Figure 2B).

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