Limits...
Differential roles of Hath1, MUC2 and P27Kip1 in relation with gamma-secretase inhibition in human colonic carcinomas: a translational study.

Souazé F, Bou-Hanna C, Kandel C, Leclair F, Devallière J, Charreau B, Bézieau S, Mosnier JF, Laboisse CL - PLoS ONE (2013)

Bottom Line: Hath1, a bHLH transcription factor negatively regulated by the γ-secretase-dependent Notch pathway, is required for intestinal secretory cell differentiation.Human colon carcinoma cell lines that differentiate along a mucin secreting (MUC2/MUC5AC) and/or enterocytic (DPPIV) lineages were maintained on inserts with or without a γ-secretase inhibitor (DBZ).In cultured primary human colon carcinomas, Hath1 was up-regulated in 7 out of 10 tumors upon DBZ treatment.

View Article: PubMed Central - PubMed

Affiliation: EA Biometadys, Université de Nantes, Nantes, France. frederique.souaze@inserm.fr

ABSTRACT
Hath1, a bHLH transcription factor negatively regulated by the γ-secretase-dependent Notch pathway, is required for intestinal secretory cell differentiation. Our aim was fourfold: 1) determine whether Hath1 is able to alter the phenotype of colon cancer cells that are committed to a differentiated phenotype, 2) determine whether the Hath1-dependent alteration of differentiation is coupled to a restriction of anchorage-dependent growth, 3) decipher the respective roles of three putative tumor suppressor genes Hath1, MUC2 and P27kip1 in this coupling and, 4) examine how our findings translate to primary tumors. Human colon carcinoma cell lines that differentiate along a mucin secreting (MUC2/MUC5AC) and/or enterocytic (DPPIV) lineages were maintained on inserts with or without a γ-secretase inhibitor (DBZ). Then the cells were detached and their ability to survive/proliferate in the absence of substratum was assessed. γ-secretase inhibition led to a Hath1-mediated preferential induction of MUC2 over MUC5AC, without DPPIV modification, in association with a decrease in anchorage-independent growth. While P27kip1 silencing relieved the cells from the Hath1-induced decrease of anchorage-independent growth, MUC2 silencing did not modify this parameter. Hath1 ectopic expression in the Hath1 negative enterocytic Caco2 cells led to a decreased anchorage-independent growth in a P27kip1-independent manner. In cultured primary human colon carcinomas, Hath1 was up-regulated in 7 out of 10 tumors upon DBZ treatment. Parallel MUC2 up-regulation occurred in 4 (4/7) and P27kip1 in only 2 (2/7) tumors. Interestingly, the response patterns of primary tumors to DBZ fitted with the hierarchical model of divergent signalling derived from our findings on cell lines.

Show MeSH

Related in: MedlinePlus

Effect of γ-secretase inhibition on the percentage of MUC2 positive cells and the expression of differentiation-associated genes.A- Percentage of MUC2 positive cells in control and DBZ-treated cells maintained on filters was determined by immunohistochemistry as described in material and methods. Mean ± SEM of 3 filters after counting at least 200 cells/filter (***, p<0.001 DBZ vs control). B- RT-PCR detection of Hath1, MUC2, MUC5AC, DPPIV mRNA in HT29-Cl.27H filter cultures treated with DBZ for 21 days. The results of real-time quantitative PCR are expressed relative to the expression level of control cultures after normalization to actin gene expression. Mean relative expression to the control DMSO; Mean ± SEM of 4 experiments; ***, P<0.001; **, P<0.01 (DBZ vs control). C- RT-PCR detection of Hath1 and MUC2 mRNA in DBZ-treated HT29-Cl.27H cells infected by AdN2ICD Adenovirus (30 or 40 moi/cell); Mean ± SEM of 3 experiments; *, P<0.05. D- RT-PCR detection of MUC2, MUC5AC and DPP-IV mRNA in polyclonal populations of Caco2 cells stably transfected by Hath1 expression vector. (For comparison purpose the Cts for MUC2 PCR are: Caco2-cmv Ct = 32; Caco2-Hath1 Ct = 26.5 and Ht29-Cl.27H Ct = 26). Insert: c-myc tag was detected by immunoblot on polyclonal populations of Caco2 cells stably transfected by empty vector or Hath1 expression vector.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3569436&req=5

pone-0055904-g002: Effect of γ-secretase inhibition on the percentage of MUC2 positive cells and the expression of differentiation-associated genes.A- Percentage of MUC2 positive cells in control and DBZ-treated cells maintained on filters was determined by immunohistochemistry as described in material and methods. Mean ± SEM of 3 filters after counting at least 200 cells/filter (***, p<0.001 DBZ vs control). B- RT-PCR detection of Hath1, MUC2, MUC5AC, DPPIV mRNA in HT29-Cl.27H filter cultures treated with DBZ for 21 days. The results of real-time quantitative PCR are expressed relative to the expression level of control cultures after normalization to actin gene expression. Mean relative expression to the control DMSO; Mean ± SEM of 4 experiments; ***, P<0.001; **, P<0.01 (DBZ vs control). C- RT-PCR detection of Hath1 and MUC2 mRNA in DBZ-treated HT29-Cl.27H cells infected by AdN2ICD Adenovirus (30 or 40 moi/cell); Mean ± SEM of 3 experiments; *, P<0.05. D- RT-PCR detection of MUC2, MUC5AC and DPP-IV mRNA in polyclonal populations of Caco2 cells stably transfected by Hath1 expression vector. (For comparison purpose the Cts for MUC2 PCR are: Caco2-cmv Ct = 32; Caco2-Hath1 Ct = 26.5 and Ht29-Cl.27H Ct = 26). Insert: c-myc tag was detected by immunoblot on polyclonal populations of Caco2 cells stably transfected by empty vector or Hath1 expression vector.

Mentions: HT29-Cl.27H cells maintained for 21 days on porous inserts formed polarized monolayers (Figure 1A, lane 1). Alcian blue, a stain for acidic mucin that is normally restricted to the intestinal goblet cells, stained some epithelial cells (Figure 1A, lane 2). Accordingly, immunohistochemistry showed that only few mucin-secreting cells expressed MUC2. The staining was restricted to the perinuclear region corresponding to the cis-Golgi since the antibody was specific for the peptidic epitope of human MUC2 (Figure 1A, lane 3). Numerous cells were stained by a mix of monoclonal antibodies specific for both the peptidic and carbohydrate chains of MUC5AC mucins that are normally expressed by the gastric mucosa (Figure 1A, lane 4). In addition, enterocytic cells were present, with an apical brush border stained with anti-DPPIV antibody (Figure 1A, lane 5). A 21-day treatment with DBZ of filter-grown cells was not cytotoxic as shown on HES staining (Figure 1A, lane 1). Interestingly, DBZ treatment led to a dramatic increase in alcian blue positive goblet cells (Figure 1A, lane 2). This was paralleled by a 6 to 9 fold increase in MUC2 positive epithelial cells (Figure 1A, lane 3 and Figure 2A). Finally, the staining patterns of MUC5AC and DPPIV did not change (Figure 1, lanes 4–5). This finding suggested that γ-secretase inhibition had no effect on the enterocytic differentiation.


Differential roles of Hath1, MUC2 and P27Kip1 in relation with gamma-secretase inhibition in human colonic carcinomas: a translational study.

Souazé F, Bou-Hanna C, Kandel C, Leclair F, Devallière J, Charreau B, Bézieau S, Mosnier JF, Laboisse CL - PLoS ONE (2013)

Effect of γ-secretase inhibition on the percentage of MUC2 positive cells and the expression of differentiation-associated genes.A- Percentage of MUC2 positive cells in control and DBZ-treated cells maintained on filters was determined by immunohistochemistry as described in material and methods. Mean ± SEM of 3 filters after counting at least 200 cells/filter (***, p<0.001 DBZ vs control). B- RT-PCR detection of Hath1, MUC2, MUC5AC, DPPIV mRNA in HT29-Cl.27H filter cultures treated with DBZ for 21 days. The results of real-time quantitative PCR are expressed relative to the expression level of control cultures after normalization to actin gene expression. Mean relative expression to the control DMSO; Mean ± SEM of 4 experiments; ***, P<0.001; **, P<0.01 (DBZ vs control). C- RT-PCR detection of Hath1 and MUC2 mRNA in DBZ-treated HT29-Cl.27H cells infected by AdN2ICD Adenovirus (30 or 40 moi/cell); Mean ± SEM of 3 experiments; *, P<0.05. D- RT-PCR detection of MUC2, MUC5AC and DPP-IV mRNA in polyclonal populations of Caco2 cells stably transfected by Hath1 expression vector. (For comparison purpose the Cts for MUC2 PCR are: Caco2-cmv Ct = 32; Caco2-Hath1 Ct = 26.5 and Ht29-Cl.27H Ct = 26). Insert: c-myc tag was detected by immunoblot on polyclonal populations of Caco2 cells stably transfected by empty vector or Hath1 expression vector.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3569436&req=5

pone-0055904-g002: Effect of γ-secretase inhibition on the percentage of MUC2 positive cells and the expression of differentiation-associated genes.A- Percentage of MUC2 positive cells in control and DBZ-treated cells maintained on filters was determined by immunohistochemistry as described in material and methods. Mean ± SEM of 3 filters after counting at least 200 cells/filter (***, p<0.001 DBZ vs control). B- RT-PCR detection of Hath1, MUC2, MUC5AC, DPPIV mRNA in HT29-Cl.27H filter cultures treated with DBZ for 21 days. The results of real-time quantitative PCR are expressed relative to the expression level of control cultures after normalization to actin gene expression. Mean relative expression to the control DMSO; Mean ± SEM of 4 experiments; ***, P<0.001; **, P<0.01 (DBZ vs control). C- RT-PCR detection of Hath1 and MUC2 mRNA in DBZ-treated HT29-Cl.27H cells infected by AdN2ICD Adenovirus (30 or 40 moi/cell); Mean ± SEM of 3 experiments; *, P<0.05. D- RT-PCR detection of MUC2, MUC5AC and DPP-IV mRNA in polyclonal populations of Caco2 cells stably transfected by Hath1 expression vector. (For comparison purpose the Cts for MUC2 PCR are: Caco2-cmv Ct = 32; Caco2-Hath1 Ct = 26.5 and Ht29-Cl.27H Ct = 26). Insert: c-myc tag was detected by immunoblot on polyclonal populations of Caco2 cells stably transfected by empty vector or Hath1 expression vector.
Mentions: HT29-Cl.27H cells maintained for 21 days on porous inserts formed polarized monolayers (Figure 1A, lane 1). Alcian blue, a stain for acidic mucin that is normally restricted to the intestinal goblet cells, stained some epithelial cells (Figure 1A, lane 2). Accordingly, immunohistochemistry showed that only few mucin-secreting cells expressed MUC2. The staining was restricted to the perinuclear region corresponding to the cis-Golgi since the antibody was specific for the peptidic epitope of human MUC2 (Figure 1A, lane 3). Numerous cells were stained by a mix of monoclonal antibodies specific for both the peptidic and carbohydrate chains of MUC5AC mucins that are normally expressed by the gastric mucosa (Figure 1A, lane 4). In addition, enterocytic cells were present, with an apical brush border stained with anti-DPPIV antibody (Figure 1A, lane 5). A 21-day treatment with DBZ of filter-grown cells was not cytotoxic as shown on HES staining (Figure 1A, lane 1). Interestingly, DBZ treatment led to a dramatic increase in alcian blue positive goblet cells (Figure 1A, lane 2). This was paralleled by a 6 to 9 fold increase in MUC2 positive epithelial cells (Figure 1A, lane 3 and Figure 2A). Finally, the staining patterns of MUC5AC and DPPIV did not change (Figure 1, lanes 4–5). This finding suggested that γ-secretase inhibition had no effect on the enterocytic differentiation.

Bottom Line: Hath1, a bHLH transcription factor negatively regulated by the γ-secretase-dependent Notch pathway, is required for intestinal secretory cell differentiation.Human colon carcinoma cell lines that differentiate along a mucin secreting (MUC2/MUC5AC) and/or enterocytic (DPPIV) lineages were maintained on inserts with or without a γ-secretase inhibitor (DBZ).In cultured primary human colon carcinomas, Hath1 was up-regulated in 7 out of 10 tumors upon DBZ treatment.

View Article: PubMed Central - PubMed

Affiliation: EA Biometadys, Université de Nantes, Nantes, France. frederique.souaze@inserm.fr

ABSTRACT
Hath1, a bHLH transcription factor negatively regulated by the γ-secretase-dependent Notch pathway, is required for intestinal secretory cell differentiation. Our aim was fourfold: 1) determine whether Hath1 is able to alter the phenotype of colon cancer cells that are committed to a differentiated phenotype, 2) determine whether the Hath1-dependent alteration of differentiation is coupled to a restriction of anchorage-dependent growth, 3) decipher the respective roles of three putative tumor suppressor genes Hath1, MUC2 and P27kip1 in this coupling and, 4) examine how our findings translate to primary tumors. Human colon carcinoma cell lines that differentiate along a mucin secreting (MUC2/MUC5AC) and/or enterocytic (DPPIV) lineages were maintained on inserts with or without a γ-secretase inhibitor (DBZ). Then the cells were detached and their ability to survive/proliferate in the absence of substratum was assessed. γ-secretase inhibition led to a Hath1-mediated preferential induction of MUC2 over MUC5AC, without DPPIV modification, in association with a decrease in anchorage-independent growth. While P27kip1 silencing relieved the cells from the Hath1-induced decrease of anchorage-independent growth, MUC2 silencing did not modify this parameter. Hath1 ectopic expression in the Hath1 negative enterocytic Caco2 cells led to a decreased anchorage-independent growth in a P27kip1-independent manner. In cultured primary human colon carcinomas, Hath1 was up-regulated in 7 out of 10 tumors upon DBZ treatment. Parallel MUC2 up-regulation occurred in 4 (4/7) and P27kip1 in only 2 (2/7) tumors. Interestingly, the response patterns of primary tumors to DBZ fitted with the hierarchical model of divergent signalling derived from our findings on cell lines.

Show MeSH
Related in: MedlinePlus