TWO-DIMENSIONAL GEL ELECTROPHORESIS(2-DE) ANALYSIS REVEALS DIFFERENTIALLY EXPRESSED PROTEIN PATTERNS IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) AFTER THYMOSIN Β4 (TB4) AND LENALIDOMIDE (LEN) TREATMENT
(Abstract release date: 05/19/16)
EHA Library. Bossio S. 06/09/16; 132596; E1047
Dr. Sabrina Bossio
Contributions
Contributions
Abstract
Abstract: E1047
Type: Eposter Presentation
Background
The proteomic approach is essential in the multi-disciplinary field of hematological research. Previously, using MALDI-TOF analysis we identified Tβ4, a G-actin sequestering protein involved in the regeneration of injured tissues and cell migration, as a downregulated protein in CLL patients, also confirmed by an independent GEP analysis comparing B-CLL cells and normal controls.
Aims
Here, using 2DE proteomic analysis we evaluated differential protein expression patterns after treatment with exogenous Tβ4 and/or Len, which has been shown to improve immune dysfunction in CLL by repairing actin polymerization and rapid signaling at the immunological synapse. We investigated whether purified B-CLL cells having high/low Tβ4 expression respond differently to migratory stimuli such as CXCL12/SDF1α.
Methods
Purified B-CLL lymphocytes were isolated from untreated Binet stage A CLL patients prospectively enrolled atdiagnosis (O-CLL1 clinicaltrial.gov identifier NCT00917540) and healthy controls. N=93 patients were evaluated for Tβ4 expression by Flow cytometry (FC, medianRFI=15.06, IQR:6.9–28.8).For migration assays, SDF1α (100nM) as chemoattractantwas used to determine the CLL migration index. B-CLL cells from two patient samples havinglow or highTβ4expression were pre-treated with Len (5mM) and then with Tβ4 (100nM)for 2DE analysis.
Results
CLL samples with the lowest Tβ4 expression (n=12) also had higher F-actin levels as evaluated by FC than normal controls (n=7) in a sample cohort (n=93). FC assessed Tβ4 expression in B-lymphocytes in CLL patients (n=93) vs controls (n=7). Tβ4RFIvalues showed Tβ4 expression was heterogeneous (Tβ4RFI=1.3–152.05;medianRFI=15.06) compared to controls (Tβ4RFI=3.4–70.9;medianRFI=38.3). Baseline F-actin expression is also influenced by different levels of Tβ4 expression: CLL CD19+CD5+ samples with Tβ4highalso had higher G-actin expression, with a lower F/G-ratio, and conversely Tβ4low samples, there wasa higher expression of F-actin, with a higher F/G-ratio (n=14), also confirmed by FC experiments. CD19+CD5+ CLL cells express high levels of functional CXCR4/CD184,which facilitates CLL cell migration beneath stromal cells. B-CLL strongly responded to the migratory stimulus SDF-1α. Furthermore, migration induced by SDF1α increased in CLL cells after Len (0.5 µM) treatment. Conversely, the CLL with Tβ4low was more responsive to Len inhibition, resulting in a reduction in migratory potential towards SDF1α. Purified B-CLL lysates (n=2) were analyzed by 2DE analysis. Protein profile analysis showed a number of protein spots ranging from 18 to 60kDa that were differentially expressed with respect to cells exhibiting Tβ4low/high expression. More importantly, treatment with Len or following Tβ4 replenishment also changed protein expression in function of the endogenous Tβ4
Conclusion
Our preliminary qualitative analysis suggests that the proteomic profile of CLL samples exhibiting higher or low Tβ4 levels also correspond to groups of proteins with a differential protein expression. Moreover, in the presence of Tβ4 or Len, or Tβ4/Len combination these proteins may respond with a strong inhibition or up-regulation of the expression of other proteins. Here,we show that 2DE evaluation of CLL profiles may be useful to identify changes in expression profiles of CLL proteins following treatment with different agents. These differences may translate into functional differences such as cell migration and cytoskeletal dynamics in the malignant clone.Special thanks to Celegene Corp and AIRC-CARICAL Regional Grant 16695.
Session topic: E-poster
Keyword(s): Actin, Chronic lymphocytic leukemia, Protein Expression
Type: Eposter Presentation
Background
The proteomic approach is essential in the multi-disciplinary field of hematological research. Previously, using MALDI-TOF analysis we identified Tβ4, a G-actin sequestering protein involved in the regeneration of injured tissues and cell migration, as a downregulated protein in CLL patients, also confirmed by an independent GEP analysis comparing B-CLL cells and normal controls.
Aims
Here, using 2DE proteomic analysis we evaluated differential protein expression patterns after treatment with exogenous Tβ4 and/or Len, which has been shown to improve immune dysfunction in CLL by repairing actin polymerization and rapid signaling at the immunological synapse. We investigated whether purified B-CLL cells having high/low Tβ4 expression respond differently to migratory stimuli such as CXCL12/SDF1α.
Methods
Purified B-CLL lymphocytes were isolated from untreated Binet stage A CLL patients prospectively enrolled atdiagnosis (O-CLL1 clinicaltrial.gov identifier NCT00917540) and healthy controls. N=93 patients were evaluated for Tβ4 expression by Flow cytometry (FC, medianRFI=15.06, IQR:6.9–28.8).For migration assays, SDF1α (100nM) as chemoattractantwas used to determine the CLL migration index. B-CLL cells from two patient samples havinglow or highTβ4expression were pre-treated with Len (5mM) and then with Tβ4 (100nM)for 2DE analysis.
Results
CLL samples with the lowest Tβ4 expression (n=12) also had higher F-actin levels as evaluated by FC than normal controls (n=7) in a sample cohort (n=93). FC assessed Tβ4 expression in B-lymphocytes in CLL patients (n=93) vs controls (n=7). Tβ4RFIvalues showed Tβ4 expression was heterogeneous (Tβ4RFI=1.3–152.05;medianRFI=15.06) compared to controls (Tβ4RFI=3.4–70.9;medianRFI=38.3). Baseline F-actin expression is also influenced by different levels of Tβ4 expression: CLL CD19+CD5+ samples with Tβ4highalso had higher G-actin expression, with a lower F/G-ratio, and conversely Tβ4low samples, there wasa higher expression of F-actin, with a higher F/G-ratio (n=14), also confirmed by FC experiments. CD19+CD5+ CLL cells express high levels of functional CXCR4/CD184,which facilitates CLL cell migration beneath stromal cells. B-CLL strongly responded to the migratory stimulus SDF-1α. Furthermore, migration induced by SDF1α increased in CLL cells after Len (0.5 µM) treatment. Conversely, the CLL with Tβ4low was more responsive to Len inhibition, resulting in a reduction in migratory potential towards SDF1α. Purified B-CLL lysates (n=2) were analyzed by 2DE analysis. Protein profile analysis showed a number of protein spots ranging from 18 to 60kDa that were differentially expressed with respect to cells exhibiting Tβ4low/high expression. More importantly, treatment with Len or following Tβ4 replenishment also changed protein expression in function of the endogenous Tβ4
Conclusion
Our preliminary qualitative analysis suggests that the proteomic profile of CLL samples exhibiting higher or low Tβ4 levels also correspond to groups of proteins with a differential protein expression. Moreover, in the presence of Tβ4 or Len, or Tβ4/Len combination these proteins may respond with a strong inhibition or up-regulation of the expression of other proteins. Here,we show that 2DE evaluation of CLL profiles may be useful to identify changes in expression profiles of CLL proteins following treatment with different agents. These differences may translate into functional differences such as cell migration and cytoskeletal dynamics in the malignant clone.Special thanks to Celegene Corp and AIRC-CARICAL Regional Grant 16695.
Session topic: E-poster
Keyword(s): Actin, Chronic lymphocytic leukemia, Protein Expression
Abstract: E1047
Type: Eposter Presentation
Background
The proteomic approach is essential in the multi-disciplinary field of hematological research. Previously, using MALDI-TOF analysis we identified Tβ4, a G-actin sequestering protein involved in the regeneration of injured tissues and cell migration, as a downregulated protein in CLL patients, also confirmed by an independent GEP analysis comparing B-CLL cells and normal controls.
Aims
Here, using 2DE proteomic analysis we evaluated differential protein expression patterns after treatment with exogenous Tβ4 and/or Len, which has been shown to improve immune dysfunction in CLL by repairing actin polymerization and rapid signaling at the immunological synapse. We investigated whether purified B-CLL cells having high/low Tβ4 expression respond differently to migratory stimuli such as CXCL12/SDF1α.
Methods
Purified B-CLL lymphocytes were isolated from untreated Binet stage A CLL patients prospectively enrolled atdiagnosis (O-CLL1 clinicaltrial.gov identifier NCT00917540) and healthy controls. N=93 patients were evaluated for Tβ4 expression by Flow cytometry (FC, medianRFI=15.06, IQR:6.9–28.8).For migration assays, SDF1α (100nM) as chemoattractantwas used to determine the CLL migration index. B-CLL cells from two patient samples havinglow or highTβ4expression were pre-treated with Len (5mM) and then with Tβ4 (100nM)for 2DE analysis.
Results
CLL samples with the lowest Tβ4 expression (n=12) also had higher F-actin levels as evaluated by FC than normal controls (n=7) in a sample cohort (n=93). FC assessed Tβ4 expression in B-lymphocytes in CLL patients (n=93) vs controls (n=7). Tβ4RFIvalues showed Tβ4 expression was heterogeneous (Tβ4RFI=1.3–152.05;medianRFI=15.06) compared to controls (Tβ4RFI=3.4–70.9;medianRFI=38.3). Baseline F-actin expression is also influenced by different levels of Tβ4 expression: CLL CD19+CD5+ samples with Tβ4highalso had higher G-actin expression, with a lower F/G-ratio, and conversely Tβ4low samples, there wasa higher expression of F-actin, with a higher F/G-ratio (n=14), also confirmed by FC experiments. CD19+CD5+ CLL cells express high levels of functional CXCR4/CD184,which facilitates CLL cell migration beneath stromal cells. B-CLL strongly responded to the migratory stimulus SDF-1α. Furthermore, migration induced by SDF1α increased in CLL cells after Len (0.5 µM) treatment. Conversely, the CLL with Tβ4low was more responsive to Len inhibition, resulting in a reduction in migratory potential towards SDF1α. Purified B-CLL lysates (n=2) were analyzed by 2DE analysis. Protein profile analysis showed a number of protein spots ranging from 18 to 60kDa that were differentially expressed with respect to cells exhibiting Tβ4low/high expression. More importantly, treatment with Len or following Tβ4 replenishment also changed protein expression in function of the endogenous Tβ4
Conclusion
Our preliminary qualitative analysis suggests that the proteomic profile of CLL samples exhibiting higher or low Tβ4 levels also correspond to groups of proteins with a differential protein expression. Moreover, in the presence of Tβ4 or Len, or Tβ4/Len combination these proteins may respond with a strong inhibition or up-regulation of the expression of other proteins. Here,we show that 2DE evaluation of CLL profiles may be useful to identify changes in expression profiles of CLL proteins following treatment with different agents. These differences may translate into functional differences such as cell migration and cytoskeletal dynamics in the malignant clone.Special thanks to Celegene Corp and AIRC-CARICAL Regional Grant 16695.
Session topic: E-poster
Keyword(s): Actin, Chronic lymphocytic leukemia, Protein Expression
Type: Eposter Presentation
Background
The proteomic approach is essential in the multi-disciplinary field of hematological research. Previously, using MALDI-TOF analysis we identified Tβ4, a G-actin sequestering protein involved in the regeneration of injured tissues and cell migration, as a downregulated protein in CLL patients, also confirmed by an independent GEP analysis comparing B-CLL cells and normal controls.
Aims
Here, using 2DE proteomic analysis we evaluated differential protein expression patterns after treatment with exogenous Tβ4 and/or Len, which has been shown to improve immune dysfunction in CLL by repairing actin polymerization and rapid signaling at the immunological synapse. We investigated whether purified B-CLL cells having high/low Tβ4 expression respond differently to migratory stimuli such as CXCL12/SDF1α.
Methods
Purified B-CLL lymphocytes were isolated from untreated Binet stage A CLL patients prospectively enrolled atdiagnosis (O-CLL1 clinicaltrial.gov identifier NCT00917540) and healthy controls. N=93 patients were evaluated for Tβ4 expression by Flow cytometry (FC, medianRFI=15.06, IQR:6.9–28.8).For migration assays, SDF1α (100nM) as chemoattractantwas used to determine the CLL migration index. B-CLL cells from two patient samples havinglow or highTβ4expression were pre-treated with Len (5mM) and then with Tβ4 (100nM)for 2DE analysis.
Results
CLL samples with the lowest Tβ4 expression (n=12) also had higher F-actin levels as evaluated by FC than normal controls (n=7) in a sample cohort (n=93). FC assessed Tβ4 expression in B-lymphocytes in CLL patients (n=93) vs controls (n=7). Tβ4RFIvalues showed Tβ4 expression was heterogeneous (Tβ4RFI=1.3–152.05;medianRFI=15.06) compared to controls (Tβ4RFI=3.4–70.9;medianRFI=38.3). Baseline F-actin expression is also influenced by different levels of Tβ4 expression: CLL CD19+CD5+ samples with Tβ4highalso had higher G-actin expression, with a lower F/G-ratio, and conversely Tβ4low samples, there wasa higher expression of F-actin, with a higher F/G-ratio (n=14), also confirmed by FC experiments. CD19+CD5+ CLL cells express high levels of functional CXCR4/CD184,which facilitates CLL cell migration beneath stromal cells. B-CLL strongly responded to the migratory stimulus SDF-1α. Furthermore, migration induced by SDF1α increased in CLL cells after Len (0.5 µM) treatment. Conversely, the CLL with Tβ4low was more responsive to Len inhibition, resulting in a reduction in migratory potential towards SDF1α. Purified B-CLL lysates (n=2) were analyzed by 2DE analysis. Protein profile analysis showed a number of protein spots ranging from 18 to 60kDa that were differentially expressed with respect to cells exhibiting Tβ4low/high expression. More importantly, treatment with Len or following Tβ4 replenishment also changed protein expression in function of the endogenous Tβ4
Conclusion
Our preliminary qualitative analysis suggests that the proteomic profile of CLL samples exhibiting higher or low Tβ4 levels also correspond to groups of proteins with a differential protein expression. Moreover, in the presence of Tβ4 or Len, or Tβ4/Len combination these proteins may respond with a strong inhibition or up-regulation of the expression of other proteins. Here,we show that 2DE evaluation of CLL profiles may be useful to identify changes in expression profiles of CLL proteins following treatment with different agents. These differences may translate into functional differences such as cell migration and cytoskeletal dynamics in the malignant clone.Special thanks to Celegene Corp and AIRC-CARICAL Regional Grant 16695.
Session topic: E-poster
Keyword(s): Actin, Chronic lymphocytic leukemia, Protein Expression
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