THE EFFECT OF HYPOXIA ON NOTCH PATHWAY IN MULTIPLE MYELOMA NICHE
(Abstract release date: 05/19/16)
EHA Library. Garavelli S. 06/09/16; 134845; PB1945
Mrs. Silvia Garavelli
Contributions
Contributions
Abstract
Abstract: PB1945
Type: Publication Only
Background
Multiple myeloma (MM) is an incurable hematological tumor representing 13 % of all hematological malignancies and tumor cells accumulate in the bone marrow (BM) of patients.Several evidences demonstrate that Notch signaling mediates critical events in MM progression. The Notch pathway is highly conserved and consists of 4 receptors and two different ligand families (Delta and Serrate). Recently Notch receptors and ligands have been shown to be upregulated during MM progression and their signaling positively regulates cell proliferation, drug resistance and BM infiltration.Hypoxia is defined as a low oxygen condition and can induce angiogenesis; HIF-1α is the key mediator of hypoxic response and a transcription factor that can switch on different types of genes, which, in turn, regulate angiogenesis (VEGF) and proliferation of various stem cell populations. Recent reports indicate that HIF-1α may positively regulate Notch signaling and enhance the expression of Notch downstream genes. In MM, hypoxia can sustain stem cell-like population in MM and HIF-1α derived from bone marrow endothelial cells can have a role in promoting drug resistance in patients with MM.
Aims
The aim of this work is to evaluate if and how hypoxia can affect the activation of Notch signaling in MM microenvironment and the underlying mechanism.
Methods
OPM2, U266, H929 and RPMI8226 cell lines were cultured in complete RPMI-1640 medium. To induce an hypoxic-like condition, cells were treated with Cobalt Chloride (CoCl2) 100 μM for 24 h. To analyze the modulation of the expression of HIF-1α, the MM cells were treated with CoCl2 100 μM for 2h, 6h, 12h and 24h.We evaluated the effect of hypoxia on cells proliferation, apoptosis and cell cycle; Western Blot and qPCR were used to analyze how hypoxia can affect the activation of the Notch pathway.Cell proliferation: absolute cell counts were determined using the volumetric count tool of the BD FACSVerse™ System (BD Biosciences, USA).Apoptosis: MM cell lines were treated with CoCl2 100 μM for 24h and stained with Annexin V-FITC and Propidium Iodide, then processed using the BD FACSVerse™ System (BD Biosciences).Western Blot: 100 μg of proteins were loaded on a 8% polyacrilamide gel and transfer onto a nitrocellulose membrane. The membrane was incubated with the following antibodies: Actin (Santa Cruz Biotechnology), HIF-1α (Santa Cruz Biotechnology) and Notch2-cleaved (Abcam).Quantitative PCR reactions were carried out on a 7500 Fast Real-time PCR system (Applied Biosystems) using the MaximaTM SYBR Green/ROX qPCR Master Mix (ThermoScientific).
Results
The MM cell lines OPM2, U266, H929 were treated with CoCl2 100 μM for 24 to mimic hypoxic condition and we evaluated the effect on cell proliferation and apoptosis: hypoxia inhibits proliferation in all MM cell lines used (H929, U266 and OPM2) and induces apoptosis only in H929 cell lines.To analyze the molecular effects of hypoxia, we treated MM cell lines with CoCl2 100 μM for 2h, 6h, 12h and 24h and performed a Western Blot. HIF-1α is highly expressed in treated cells if compared to normal controls; the hypoxic condition induced the expression of pro-angiogenic factors i.e. VEGF and SDF-1α. Interestingly these have been reported to be controlled by Notch. Consistently, hypoxia induced the activation of Notch2 receptor.
Conclusion
Our results, although still preliminary, indicate that hypoxia can affect MM cell lines survival and proliferation and finally and that it is able to induce the activation of the oncogenic Notch pathway.
Session topic: E-poster
Keyword(s): Myeloma, Notch signaling
Type: Publication Only
Background
Multiple myeloma (MM) is an incurable hematological tumor representing 13 % of all hematological malignancies and tumor cells accumulate in the bone marrow (BM) of patients.Several evidences demonstrate that Notch signaling mediates critical events in MM progression. The Notch pathway is highly conserved and consists of 4 receptors and two different ligand families (Delta and Serrate). Recently Notch receptors and ligands have been shown to be upregulated during MM progression and their signaling positively regulates cell proliferation, drug resistance and BM infiltration.Hypoxia is defined as a low oxygen condition and can induce angiogenesis; HIF-1α is the key mediator of hypoxic response and a transcription factor that can switch on different types of genes, which, in turn, regulate angiogenesis (VEGF) and proliferation of various stem cell populations. Recent reports indicate that HIF-1α may positively regulate Notch signaling and enhance the expression of Notch downstream genes. In MM, hypoxia can sustain stem cell-like population in MM and HIF-1α derived from bone marrow endothelial cells can have a role in promoting drug resistance in patients with MM.
Aims
The aim of this work is to evaluate if and how hypoxia can affect the activation of Notch signaling in MM microenvironment and the underlying mechanism.
Methods
OPM2, U266, H929 and RPMI8226 cell lines were cultured in complete RPMI-1640 medium. To induce an hypoxic-like condition, cells were treated with Cobalt Chloride (CoCl2) 100 μM for 24 h. To analyze the modulation of the expression of HIF-1α, the MM cells were treated with CoCl2 100 μM for 2h, 6h, 12h and 24h.We evaluated the effect of hypoxia on cells proliferation, apoptosis and cell cycle; Western Blot and qPCR were used to analyze how hypoxia can affect the activation of the Notch pathway.Cell proliferation: absolute cell counts were determined using the volumetric count tool of the BD FACSVerse™ System (BD Biosciences, USA).Apoptosis: MM cell lines were treated with CoCl2 100 μM for 24h and stained with Annexin V-FITC and Propidium Iodide, then processed using the BD FACSVerse™ System (BD Biosciences).Western Blot: 100 μg of proteins were loaded on a 8% polyacrilamide gel and transfer onto a nitrocellulose membrane. The membrane was incubated with the following antibodies: Actin (Santa Cruz Biotechnology), HIF-1α (Santa Cruz Biotechnology) and Notch2-cleaved (Abcam).Quantitative PCR reactions were carried out on a 7500 Fast Real-time PCR system (Applied Biosystems) using the MaximaTM SYBR Green/ROX qPCR Master Mix (ThermoScientific).
Results
The MM cell lines OPM2, U266, H929 were treated with CoCl2 100 μM for 24 to mimic hypoxic condition and we evaluated the effect on cell proliferation and apoptosis: hypoxia inhibits proliferation in all MM cell lines used (H929, U266 and OPM2) and induces apoptosis only in H929 cell lines.To analyze the molecular effects of hypoxia, we treated MM cell lines with CoCl2 100 μM for 2h, 6h, 12h and 24h and performed a Western Blot. HIF-1α is highly expressed in treated cells if compared to normal controls; the hypoxic condition induced the expression of pro-angiogenic factors i.e. VEGF and SDF-1α. Interestingly these have been reported to be controlled by Notch. Consistently, hypoxia induced the activation of Notch2 receptor.
Conclusion
Our results, although still preliminary, indicate that hypoxia can affect MM cell lines survival and proliferation and finally and that it is able to induce the activation of the oncogenic Notch pathway.
Session topic: E-poster
Keyword(s): Myeloma, Notch signaling
Abstract: PB1945
Type: Publication Only
Background
Multiple myeloma (MM) is an incurable hematological tumor representing 13 % of all hematological malignancies and tumor cells accumulate in the bone marrow (BM) of patients.Several evidences demonstrate that Notch signaling mediates critical events in MM progression. The Notch pathway is highly conserved and consists of 4 receptors and two different ligand families (Delta and Serrate). Recently Notch receptors and ligands have been shown to be upregulated during MM progression and their signaling positively regulates cell proliferation, drug resistance and BM infiltration.Hypoxia is defined as a low oxygen condition and can induce angiogenesis; HIF-1α is the key mediator of hypoxic response and a transcription factor that can switch on different types of genes, which, in turn, regulate angiogenesis (VEGF) and proliferation of various stem cell populations. Recent reports indicate that HIF-1α may positively regulate Notch signaling and enhance the expression of Notch downstream genes. In MM, hypoxia can sustain stem cell-like population in MM and HIF-1α derived from bone marrow endothelial cells can have a role in promoting drug resistance in patients with MM.
Aims
The aim of this work is to evaluate if and how hypoxia can affect the activation of Notch signaling in MM microenvironment and the underlying mechanism.
Methods
OPM2, U266, H929 and RPMI8226 cell lines were cultured in complete RPMI-1640 medium. To induce an hypoxic-like condition, cells were treated with Cobalt Chloride (CoCl2) 100 μM for 24 h. To analyze the modulation of the expression of HIF-1α, the MM cells were treated with CoCl2 100 μM for 2h, 6h, 12h and 24h.We evaluated the effect of hypoxia on cells proliferation, apoptosis and cell cycle; Western Blot and qPCR were used to analyze how hypoxia can affect the activation of the Notch pathway.Cell proliferation: absolute cell counts were determined using the volumetric count tool of the BD FACSVerse™ System (BD Biosciences, USA).Apoptosis: MM cell lines were treated with CoCl2 100 μM for 24h and stained with Annexin V-FITC and Propidium Iodide, then processed using the BD FACSVerse™ System (BD Biosciences).Western Blot: 100 μg of proteins were loaded on a 8% polyacrilamide gel and transfer onto a nitrocellulose membrane. The membrane was incubated with the following antibodies: Actin (Santa Cruz Biotechnology), HIF-1α (Santa Cruz Biotechnology) and Notch2-cleaved (Abcam).Quantitative PCR reactions were carried out on a 7500 Fast Real-time PCR system (Applied Biosystems) using the MaximaTM SYBR Green/ROX qPCR Master Mix (ThermoScientific).
Results
The MM cell lines OPM2, U266, H929 were treated with CoCl2 100 μM for 24 to mimic hypoxic condition and we evaluated the effect on cell proliferation and apoptosis: hypoxia inhibits proliferation in all MM cell lines used (H929, U266 and OPM2) and induces apoptosis only in H929 cell lines.To analyze the molecular effects of hypoxia, we treated MM cell lines with CoCl2 100 μM for 2h, 6h, 12h and 24h and performed a Western Blot. HIF-1α is highly expressed in treated cells if compared to normal controls; the hypoxic condition induced the expression of pro-angiogenic factors i.e. VEGF and SDF-1α. Interestingly these have been reported to be controlled by Notch. Consistently, hypoxia induced the activation of Notch2 receptor.
Conclusion
Our results, although still preliminary, indicate that hypoxia can affect MM cell lines survival and proliferation and finally and that it is able to induce the activation of the oncogenic Notch pathway.
Session topic: E-poster
Keyword(s): Myeloma, Notch signaling
Type: Publication Only
Background
Multiple myeloma (MM) is an incurable hematological tumor representing 13 % of all hematological malignancies and tumor cells accumulate in the bone marrow (BM) of patients.Several evidences demonstrate that Notch signaling mediates critical events in MM progression. The Notch pathway is highly conserved and consists of 4 receptors and two different ligand families (Delta and Serrate). Recently Notch receptors and ligands have been shown to be upregulated during MM progression and their signaling positively regulates cell proliferation, drug resistance and BM infiltration.Hypoxia is defined as a low oxygen condition and can induce angiogenesis; HIF-1α is the key mediator of hypoxic response and a transcription factor that can switch on different types of genes, which, in turn, regulate angiogenesis (VEGF) and proliferation of various stem cell populations. Recent reports indicate that HIF-1α may positively regulate Notch signaling and enhance the expression of Notch downstream genes. In MM, hypoxia can sustain stem cell-like population in MM and HIF-1α derived from bone marrow endothelial cells can have a role in promoting drug resistance in patients with MM.
Aims
The aim of this work is to evaluate if and how hypoxia can affect the activation of Notch signaling in MM microenvironment and the underlying mechanism.
Methods
OPM2, U266, H929 and RPMI8226 cell lines were cultured in complete RPMI-1640 medium. To induce an hypoxic-like condition, cells were treated with Cobalt Chloride (CoCl2) 100 μM for 24 h. To analyze the modulation of the expression of HIF-1α, the MM cells were treated with CoCl2 100 μM for 2h, 6h, 12h and 24h.We evaluated the effect of hypoxia on cells proliferation, apoptosis and cell cycle; Western Blot and qPCR were used to analyze how hypoxia can affect the activation of the Notch pathway.Cell proliferation: absolute cell counts were determined using the volumetric count tool of the BD FACSVerse™ System (BD Biosciences, USA).Apoptosis: MM cell lines were treated with CoCl2 100 μM for 24h and stained with Annexin V-FITC and Propidium Iodide, then processed using the BD FACSVerse™ System (BD Biosciences).Western Blot: 100 μg of proteins were loaded on a 8% polyacrilamide gel and transfer onto a nitrocellulose membrane. The membrane was incubated with the following antibodies: Actin (Santa Cruz Biotechnology), HIF-1α (Santa Cruz Biotechnology) and Notch2-cleaved (Abcam).Quantitative PCR reactions were carried out on a 7500 Fast Real-time PCR system (Applied Biosystems) using the MaximaTM SYBR Green/ROX qPCR Master Mix (ThermoScientific).
Results
The MM cell lines OPM2, U266, H929 were treated with CoCl2 100 μM for 24 to mimic hypoxic condition and we evaluated the effect on cell proliferation and apoptosis: hypoxia inhibits proliferation in all MM cell lines used (H929, U266 and OPM2) and induces apoptosis only in H929 cell lines.To analyze the molecular effects of hypoxia, we treated MM cell lines with CoCl2 100 μM for 2h, 6h, 12h and 24h and performed a Western Blot. HIF-1α is highly expressed in treated cells if compared to normal controls; the hypoxic condition induced the expression of pro-angiogenic factors i.e. VEGF and SDF-1α. Interestingly these have been reported to be controlled by Notch. Consistently, hypoxia induced the activation of Notch2 receptor.
Conclusion
Our results, although still preliminary, indicate that hypoxia can affect MM cell lines survival and proliferation and finally and that it is able to induce the activation of the oncogenic Notch pathway.
Session topic: E-poster
Keyword(s): Myeloma, Notch signaling
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