CROSSTALK OF NOTCH1 AND NOTCH2 SIGNALING IN T-ALL CELL LINES
(Abstract release date: 05/19/16)
EHA Library. Okuhashi Y. 06/09/16; 134491; PB1591
Mr. Yuki Okuhashi
Contributions
Contributions
Abstract
Abstract: PB1591
Type: Publication Only
Background
Notch signaling is crucial for the growth of leukemia cells. We reported the effects of γ-secretase inhibitors (GSIs), which block Notch activation, on the growth of leukemia cells. GSIs suppressed the growth through induction of apoptosis in most of cell lines. Conversely, the growth of some cell lines were promoted by GSI treatment. For example, we previously reported GSI-XXI treatment suppressed the growth of KOPT-K1 cells while it promoted that of Jurkat cells, both of which are T-lymphoblastic leukemia (T-ALL) cells with constitutive Notch activation. However, the mechanisms of this phenomenon have not been fully clarified.
Aims
In this study, we investigated the mechanisms focusing on the crosstalk between NOTCH1 and NOTCH2 signaling in Jurkat cells.
Methods
Two T-ALL cell lines (KOPT-K1 and Jurkat) were used. The cells were transfected with siRNAs targeting NOTCH1 (siN1), NOTCH2 (siN2) or control siRNA by using the pipette tip chamber-based electroporation system. The effects of siN1 and siN2 on the expression levels of mRNA and protein of NOTCH-related molecules were examined by quantitative RT-PCR and immunoblotting, respectively. The effects of siN1 and siN2 on short-term growth were examined using a colorimetric WST-8 assay. To examine the effects of siRNA on morphological differentiation and apoptosis, cytospin preparations were prepared from harvested cells stained with Wright stain.
Results
Transfection with siN1 and siN2 selectively suppressed the expression of NOTCH1 and NOTCH2 mRNA and protein, respectively. NOTCH1 knockdown as well as NOTCH2 knockdown suppressed the growth and induced apoptosis of KOPT-K1 while they promoted the growth of Jurkat. In Jurkat, NOTCH1 knockdown increased the level of NOTCH2 protein. On the other hand, NOTCH2 knockdown increased the level of NOTCH1, cleaved NOTCH1 fragment (active form of NOTCH1), HES1 and MYC protein in Jurkat. In KOPT-K1, the expression of these protein was not significantly affected by siN1 and siN2. The knockdown NOTCH1 and NOTCH2 did not affect the expression of mTOR, Hedgehog and Wnt signal-related proteins in both cell lines.
Conclusion
Using siRNA-mediated knockdown experiments, we found that not only NOTCH1 knockdown but also NOTCH2 knockdown affected the growth of these NOTCH1-mutated, NOTCH2-unmutated T-ALL cell lines. The growth suppression by NOTCH2 knockdown did not involve the suppression of MYC expression, which was suppressed by NOTCH1 knockdown. We also found the crosstalk between NOTCH1 and NOTCH2 in Jurkat, which suggests that Jurkat might have reciprocally compensating system between two NOTCH. It might cause the resistance to GSIs. To our knowledge, this is the first report to show the interaction between NOTCH1 and NOTCH2 expression in T-ALL cell lines.
Session topic: E-poster
Keyword(s): Notch, SiRNA, T-ALL
Type: Publication Only
Background
Notch signaling is crucial for the growth of leukemia cells. We reported the effects of γ-secretase inhibitors (GSIs), which block Notch activation, on the growth of leukemia cells. GSIs suppressed the growth through induction of apoptosis in most of cell lines. Conversely, the growth of some cell lines were promoted by GSI treatment. For example, we previously reported GSI-XXI treatment suppressed the growth of KOPT-K1 cells while it promoted that of Jurkat cells, both of which are T-lymphoblastic leukemia (T-ALL) cells with constitutive Notch activation. However, the mechanisms of this phenomenon have not been fully clarified.
Aims
In this study, we investigated the mechanisms focusing on the crosstalk between NOTCH1 and NOTCH2 signaling in Jurkat cells.
Methods
Two T-ALL cell lines (KOPT-K1 and Jurkat) were used. The cells were transfected with siRNAs targeting NOTCH1 (siN1), NOTCH2 (siN2) or control siRNA by using the pipette tip chamber-based electroporation system. The effects of siN1 and siN2 on the expression levels of mRNA and protein of NOTCH-related molecules were examined by quantitative RT-PCR and immunoblotting, respectively. The effects of siN1 and siN2 on short-term growth were examined using a colorimetric WST-8 assay. To examine the effects of siRNA on morphological differentiation and apoptosis, cytospin preparations were prepared from harvested cells stained with Wright stain.
Results
Transfection with siN1 and siN2 selectively suppressed the expression of NOTCH1 and NOTCH2 mRNA and protein, respectively. NOTCH1 knockdown as well as NOTCH2 knockdown suppressed the growth and induced apoptosis of KOPT-K1 while they promoted the growth of Jurkat. In Jurkat, NOTCH1 knockdown increased the level of NOTCH2 protein. On the other hand, NOTCH2 knockdown increased the level of NOTCH1, cleaved NOTCH1 fragment (active form of NOTCH1), HES1 and MYC protein in Jurkat. In KOPT-K1, the expression of these protein was not significantly affected by siN1 and siN2. The knockdown NOTCH1 and NOTCH2 did not affect the expression of mTOR, Hedgehog and Wnt signal-related proteins in both cell lines.
Conclusion
Using siRNA-mediated knockdown experiments, we found that not only NOTCH1 knockdown but also NOTCH2 knockdown affected the growth of these NOTCH1-mutated, NOTCH2-unmutated T-ALL cell lines. The growth suppression by NOTCH2 knockdown did not involve the suppression of MYC expression, which was suppressed by NOTCH1 knockdown. We also found the crosstalk between NOTCH1 and NOTCH2 in Jurkat, which suggests that Jurkat might have reciprocally compensating system between two NOTCH. It might cause the resistance to GSIs. To our knowledge, this is the first report to show the interaction between NOTCH1 and NOTCH2 expression in T-ALL cell lines.
Session topic: E-poster
Keyword(s): Notch, SiRNA, T-ALL
Abstract: PB1591
Type: Publication Only
Background
Notch signaling is crucial for the growth of leukemia cells. We reported the effects of γ-secretase inhibitors (GSIs), which block Notch activation, on the growth of leukemia cells. GSIs suppressed the growth through induction of apoptosis in most of cell lines. Conversely, the growth of some cell lines were promoted by GSI treatment. For example, we previously reported GSI-XXI treatment suppressed the growth of KOPT-K1 cells while it promoted that of Jurkat cells, both of which are T-lymphoblastic leukemia (T-ALL) cells with constitutive Notch activation. However, the mechanisms of this phenomenon have not been fully clarified.
Aims
In this study, we investigated the mechanisms focusing on the crosstalk between NOTCH1 and NOTCH2 signaling in Jurkat cells.
Methods
Two T-ALL cell lines (KOPT-K1 and Jurkat) were used. The cells were transfected with siRNAs targeting NOTCH1 (siN1), NOTCH2 (siN2) or control siRNA by using the pipette tip chamber-based electroporation system. The effects of siN1 and siN2 on the expression levels of mRNA and protein of NOTCH-related molecules were examined by quantitative RT-PCR and immunoblotting, respectively. The effects of siN1 and siN2 on short-term growth were examined using a colorimetric WST-8 assay. To examine the effects of siRNA on morphological differentiation and apoptosis, cytospin preparations were prepared from harvested cells stained with Wright stain.
Results
Transfection with siN1 and siN2 selectively suppressed the expression of NOTCH1 and NOTCH2 mRNA and protein, respectively. NOTCH1 knockdown as well as NOTCH2 knockdown suppressed the growth and induced apoptosis of KOPT-K1 while they promoted the growth of Jurkat. In Jurkat, NOTCH1 knockdown increased the level of NOTCH2 protein. On the other hand, NOTCH2 knockdown increased the level of NOTCH1, cleaved NOTCH1 fragment (active form of NOTCH1), HES1 and MYC protein in Jurkat. In KOPT-K1, the expression of these protein was not significantly affected by siN1 and siN2. The knockdown NOTCH1 and NOTCH2 did not affect the expression of mTOR, Hedgehog and Wnt signal-related proteins in both cell lines.
Conclusion
Using siRNA-mediated knockdown experiments, we found that not only NOTCH1 knockdown but also NOTCH2 knockdown affected the growth of these NOTCH1-mutated, NOTCH2-unmutated T-ALL cell lines. The growth suppression by NOTCH2 knockdown did not involve the suppression of MYC expression, which was suppressed by NOTCH1 knockdown. We also found the crosstalk between NOTCH1 and NOTCH2 in Jurkat, which suggests that Jurkat might have reciprocally compensating system between two NOTCH. It might cause the resistance to GSIs. To our knowledge, this is the first report to show the interaction between NOTCH1 and NOTCH2 expression in T-ALL cell lines.
Session topic: E-poster
Keyword(s): Notch, SiRNA, T-ALL
Type: Publication Only
Background
Notch signaling is crucial for the growth of leukemia cells. We reported the effects of γ-secretase inhibitors (GSIs), which block Notch activation, on the growth of leukemia cells. GSIs suppressed the growth through induction of apoptosis in most of cell lines. Conversely, the growth of some cell lines were promoted by GSI treatment. For example, we previously reported GSI-XXI treatment suppressed the growth of KOPT-K1 cells while it promoted that of Jurkat cells, both of which are T-lymphoblastic leukemia (T-ALL) cells with constitutive Notch activation. However, the mechanisms of this phenomenon have not been fully clarified.
Aims
In this study, we investigated the mechanisms focusing on the crosstalk between NOTCH1 and NOTCH2 signaling in Jurkat cells.
Methods
Two T-ALL cell lines (KOPT-K1 and Jurkat) were used. The cells were transfected with siRNAs targeting NOTCH1 (siN1), NOTCH2 (siN2) or control siRNA by using the pipette tip chamber-based electroporation system. The effects of siN1 and siN2 on the expression levels of mRNA and protein of NOTCH-related molecules were examined by quantitative RT-PCR and immunoblotting, respectively. The effects of siN1 and siN2 on short-term growth were examined using a colorimetric WST-8 assay. To examine the effects of siRNA on morphological differentiation and apoptosis, cytospin preparations were prepared from harvested cells stained with Wright stain.
Results
Transfection with siN1 and siN2 selectively suppressed the expression of NOTCH1 and NOTCH2 mRNA and protein, respectively. NOTCH1 knockdown as well as NOTCH2 knockdown suppressed the growth and induced apoptosis of KOPT-K1 while they promoted the growth of Jurkat. In Jurkat, NOTCH1 knockdown increased the level of NOTCH2 protein. On the other hand, NOTCH2 knockdown increased the level of NOTCH1, cleaved NOTCH1 fragment (active form of NOTCH1), HES1 and MYC protein in Jurkat. In KOPT-K1, the expression of these protein was not significantly affected by siN1 and siN2. The knockdown NOTCH1 and NOTCH2 did not affect the expression of mTOR, Hedgehog and Wnt signal-related proteins in both cell lines.
Conclusion
Using siRNA-mediated knockdown experiments, we found that not only NOTCH1 knockdown but also NOTCH2 knockdown affected the growth of these NOTCH1-mutated, NOTCH2-unmutated T-ALL cell lines. The growth suppression by NOTCH2 knockdown did not involve the suppression of MYC expression, which was suppressed by NOTCH1 knockdown. We also found the crosstalk between NOTCH1 and NOTCH2 in Jurkat, which suggests that Jurkat might have reciprocally compensating system between two NOTCH. It might cause the resistance to GSIs. To our knowledge, this is the first report to show the interaction between NOTCH1 and NOTCH2 expression in T-ALL cell lines.
Session topic: E-poster
Keyword(s): Notch, SiRNA, T-ALL
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