CALCIUM INFLUX IS SHAPED BY THE MUTATIONAL STATUS IN CIRCULATING CD34+ CELLS OF PATIENTS WITH MYELOFIBROSIS
(Abstract release date: 05/21/15)
EHA Library. Rosti V. 06/14/15; 103163; S818
Disclosure(s): Fondazione IRCCS Policlinico San MatteoCentro per lo Studio e la Cura della Mielofibrosi - L.S.R. Area Biotecnologie
Dr. Vittorio Rosti
Contributions
Contributions
Abstract
Abstract: S818
Type: Oral Presentation
Presentation during EHA20: From 14.06.2015 08:45 to 14.06.2015 09:00
Location: Room Strauss 2
Background
Mutations of the JAK2, CALR, and MPL are detectable in the hematopoietic cells of about 90% of patients with myelofibrosis. Whereas JAK2 and MPL mutations are represented by point mutations resulting in a gain of function of the gene, CALR mutations are mainly represented by either a 52-bp deletion or a 5 bp insertion, resulting in a novel C-terminal peptide sequence. This domain regulates the Ca2+-binding activity of the protein and the two mutations are predicted to modify the Ca2+ storage capacity in the endoplasmic reticulum (ER). The ER-Ca2+ content of a cell depends on the balance between the so-called Store Operated Calcium Entry (SOCE) and the ER-dependent Ca2+ release and, in turn, it regulates many important cell functions, such as proliferation, apoptosis and gene expression. In physiologic conditions, most of the ER-bound Ca2+ is stored in the smooth-ER (s-ER). Little is known about the effects of the mutations on intracellular Ca2+ homeostasis in CD34+ cells of patients with myelofibrosis.
Aims
We have investigated the intracellular Ca2+ release and SOCE in circulating CD34+ cells of patients with MF with either the V617FJAK2 mutation (n=7) or a mutation of the CALR gene (n=7), and of healthy subjects (HS, n=4). We have also studied the ultrastructural extension of the s-ER of CD34+ cells of the same subjects.
Methods
After loading CD34+ cells with 4 mcM fura-2 acetoxymethyl ester, depletion of intracellular Ca2+ stores was induced by adding 10 mcM cyclopiazonic acid (CPA) to a 0 Ca2+ bathing medium. Ca2+ was then added to the extracellular solution eliciting a rise in [Ca2+]i due to Ca2+ influx through open store-operated Ca2+ channels. The amplitude of the peak Ca2+ response to CPA, was measured as the difference between the ratio at the peak and the mean ratio of 1 min baseline before the peak. SOCE amplitude was measured as the difference between the peak and the mean ratio of 1 min baseline recorded before the readdition of Ca2+. Extension of sER was assessed by electron microscopy (EM) followed by analysis by ImageJ software. Data are expressed as mean +/- SD.
Results
Resting levels of Ca2+ of CALR-mutated CD34+ cells were not statistically different from those of JAK2-mutated cells, but higher compared to those of HS. CALR-mutated CD34+ cells released more Ca2+ from ER stores compared to JAK2-mutated cells (60.6 nM ± 30.9 vs 26.7 nM ± 24.9, respectively, P=0.038) and compared to HS (27.6 ± 19.8 nM, P=0.041 compared to CALR-mutated cells). SOCE of CALR-mutated CD34+ cells (318.5 nM ± 206.1) was slightly increased compared to JAK2-mutated patients (242.8 ± 146.8) and lower than SOCE of CD34+ cells of HS (104.3 nM ± 66.5), without reaching a statistical significance. At EM evaluation, the extension of vescicles of the sER was higher in CALR-mutated CD34+ cells compared to JAK2-mutated cells (0.82 vs 0.24 mcm, respectively, P=0.0001) and to HS-cells (0.101 mcm, P=0.0007).
Summary
Our results show that CALR mutation in CD34+ cells of patients with PMF is associated with a rearrangement of intracellular Ca++ balance, with a significant difference in Ca++ release compared to that observed both in JAK2-mutated- and HS-CD34+ cells. In keeping with the higher Ca2+ release, we observed an increased extension of s-ER. Considering the pivotal role of Ca2+ in regulating many cell functions, including proliferation and differentiation, these results seem to suggest that CALR mutation could be implicated in the myeloproliferative phenotype that characterizes PMF patients carrying CALR mutations by interfering with the signaling role of Ca2+.
Keyword(s): Calcium, CD34, Mutation, Myelofibrosis
Session topic: Novel insights into the mechanisms involved in MPNs
Type: Oral Presentation
Presentation during EHA20: From 14.06.2015 08:45 to 14.06.2015 09:00
Location: Room Strauss 2
Background
Mutations of the JAK2, CALR, and MPL are detectable in the hematopoietic cells of about 90% of patients with myelofibrosis. Whereas JAK2 and MPL mutations are represented by point mutations resulting in a gain of function of the gene, CALR mutations are mainly represented by either a 52-bp deletion or a 5 bp insertion, resulting in a novel C-terminal peptide sequence. This domain regulates the Ca2+-binding activity of the protein and the two mutations are predicted to modify the Ca2+ storage capacity in the endoplasmic reticulum (ER). The ER-Ca2+ content of a cell depends on the balance between the so-called Store Operated Calcium Entry (SOCE) and the ER-dependent Ca2+ release and, in turn, it regulates many important cell functions, such as proliferation, apoptosis and gene expression. In physiologic conditions, most of the ER-bound Ca2+ is stored in the smooth-ER (s-ER). Little is known about the effects of the mutations on intracellular Ca2+ homeostasis in CD34+ cells of patients with myelofibrosis.
Aims
We have investigated the intracellular Ca2+ release and SOCE in circulating CD34+ cells of patients with MF with either the V617FJAK2 mutation (n=7) or a mutation of the CALR gene (n=7), and of healthy subjects (HS, n=4). We have also studied the ultrastructural extension of the s-ER of CD34+ cells of the same subjects.
Methods
After loading CD34+ cells with 4 mcM fura-2 acetoxymethyl ester, depletion of intracellular Ca2+ stores was induced by adding 10 mcM cyclopiazonic acid (CPA) to a 0 Ca2+ bathing medium. Ca2+ was then added to the extracellular solution eliciting a rise in [Ca2+]i due to Ca2+ influx through open store-operated Ca2+ channels. The amplitude of the peak Ca2+ response to CPA, was measured as the difference between the ratio at the peak and the mean ratio of 1 min baseline before the peak. SOCE amplitude was measured as the difference between the peak and the mean ratio of 1 min baseline recorded before the readdition of Ca2+. Extension of sER was assessed by electron microscopy (EM) followed by analysis by ImageJ software. Data are expressed as mean +/- SD.
Results
Resting levels of Ca2+ of CALR-mutated CD34+ cells were not statistically different from those of JAK2-mutated cells, but higher compared to those of HS. CALR-mutated CD34+ cells released more Ca2+ from ER stores compared to JAK2-mutated cells (60.6 nM ± 30.9 vs 26.7 nM ± 24.9, respectively, P=0.038) and compared to HS (27.6 ± 19.8 nM, P=0.041 compared to CALR-mutated cells). SOCE of CALR-mutated CD34+ cells (318.5 nM ± 206.1) was slightly increased compared to JAK2-mutated patients (242.8 ± 146.8) and lower than SOCE of CD34+ cells of HS (104.3 nM ± 66.5), without reaching a statistical significance. At EM evaluation, the extension of vescicles of the sER was higher in CALR-mutated CD34+ cells compared to JAK2-mutated cells (0.82 vs 0.24 mcm, respectively, P=0.0001) and to HS-cells (0.101 mcm, P=0.0007).
Summary
Our results show that CALR mutation in CD34+ cells of patients with PMF is associated with a rearrangement of intracellular Ca++ balance, with a significant difference in Ca++ release compared to that observed both in JAK2-mutated- and HS-CD34+ cells. In keeping with the higher Ca2+ release, we observed an increased extension of s-ER. Considering the pivotal role of Ca2+ in regulating many cell functions, including proliferation and differentiation, these results seem to suggest that CALR mutation could be implicated in the myeloproliferative phenotype that characterizes PMF patients carrying CALR mutations by interfering with the signaling role of Ca2+.
Keyword(s): Calcium, CD34, Mutation, Myelofibrosis
Session topic: Novel insights into the mechanisms involved in MPNs
Abstract: S818
Type: Oral Presentation
Presentation during EHA20: From 14.06.2015 08:45 to 14.06.2015 09:00
Location: Room Strauss 2
Background
Mutations of the JAK2, CALR, and MPL are detectable in the hematopoietic cells of about 90% of patients with myelofibrosis. Whereas JAK2 and MPL mutations are represented by point mutations resulting in a gain of function of the gene, CALR mutations are mainly represented by either a 52-bp deletion or a 5 bp insertion, resulting in a novel C-terminal peptide sequence. This domain regulates the Ca2+-binding activity of the protein and the two mutations are predicted to modify the Ca2+ storage capacity in the endoplasmic reticulum (ER). The ER-Ca2+ content of a cell depends on the balance between the so-called Store Operated Calcium Entry (SOCE) and the ER-dependent Ca2+ release and, in turn, it regulates many important cell functions, such as proliferation, apoptosis and gene expression. In physiologic conditions, most of the ER-bound Ca2+ is stored in the smooth-ER (s-ER). Little is known about the effects of the mutations on intracellular Ca2+ homeostasis in CD34+ cells of patients with myelofibrosis.
Aims
We have investigated the intracellular Ca2+ release and SOCE in circulating CD34+ cells of patients with MF with either the V617FJAK2 mutation (n=7) or a mutation of the CALR gene (n=7), and of healthy subjects (HS, n=4). We have also studied the ultrastructural extension of the s-ER of CD34+ cells of the same subjects.
Methods
After loading CD34+ cells with 4 mcM fura-2 acetoxymethyl ester, depletion of intracellular Ca2+ stores was induced by adding 10 mcM cyclopiazonic acid (CPA) to a 0 Ca2+ bathing medium. Ca2+ was then added to the extracellular solution eliciting a rise in [Ca2+]i due to Ca2+ influx through open store-operated Ca2+ channels. The amplitude of the peak Ca2+ response to CPA, was measured as the difference between the ratio at the peak and the mean ratio of 1 min baseline before the peak. SOCE amplitude was measured as the difference between the peak and the mean ratio of 1 min baseline recorded before the readdition of Ca2+. Extension of sER was assessed by electron microscopy (EM) followed by analysis by ImageJ software. Data are expressed as mean +/- SD.
Results
Resting levels of Ca2+ of CALR-mutated CD34+ cells were not statistically different from those of JAK2-mutated cells, but higher compared to those of HS. CALR-mutated CD34+ cells released more Ca2+ from ER stores compared to JAK2-mutated cells (60.6 nM ± 30.9 vs 26.7 nM ± 24.9, respectively, P=0.038) and compared to HS (27.6 ± 19.8 nM, P=0.041 compared to CALR-mutated cells). SOCE of CALR-mutated CD34+ cells (318.5 nM ± 206.1) was slightly increased compared to JAK2-mutated patients (242.8 ± 146.8) and lower than SOCE of CD34+ cells of HS (104.3 nM ± 66.5), without reaching a statistical significance. At EM evaluation, the extension of vescicles of the sER was higher in CALR-mutated CD34+ cells compared to JAK2-mutated cells (0.82 vs 0.24 mcm, respectively, P=0.0001) and to HS-cells (0.101 mcm, P=0.0007).
Summary
Our results show that CALR mutation in CD34+ cells of patients with PMF is associated with a rearrangement of intracellular Ca++ balance, with a significant difference in Ca++ release compared to that observed both in JAK2-mutated- and HS-CD34+ cells. In keeping with the higher Ca2+ release, we observed an increased extension of s-ER. Considering the pivotal role of Ca2+ in regulating many cell functions, including proliferation and differentiation, these results seem to suggest that CALR mutation could be implicated in the myeloproliferative phenotype that characterizes PMF patients carrying CALR mutations by interfering with the signaling role of Ca2+.
Keyword(s): Calcium, CD34, Mutation, Myelofibrosis
Session topic: Novel insights into the mechanisms involved in MPNs
Type: Oral Presentation
Presentation during EHA20: From 14.06.2015 08:45 to 14.06.2015 09:00
Location: Room Strauss 2
Background
Mutations of the JAK2, CALR, and MPL are detectable in the hematopoietic cells of about 90% of patients with myelofibrosis. Whereas JAK2 and MPL mutations are represented by point mutations resulting in a gain of function of the gene, CALR mutations are mainly represented by either a 52-bp deletion or a 5 bp insertion, resulting in a novel C-terminal peptide sequence. This domain regulates the Ca2+-binding activity of the protein and the two mutations are predicted to modify the Ca2+ storage capacity in the endoplasmic reticulum (ER). The ER-Ca2+ content of a cell depends on the balance between the so-called Store Operated Calcium Entry (SOCE) and the ER-dependent Ca2+ release and, in turn, it regulates many important cell functions, such as proliferation, apoptosis and gene expression. In physiologic conditions, most of the ER-bound Ca2+ is stored in the smooth-ER (s-ER). Little is known about the effects of the mutations on intracellular Ca2+ homeostasis in CD34+ cells of patients with myelofibrosis.
Aims
We have investigated the intracellular Ca2+ release and SOCE in circulating CD34+ cells of patients with MF with either the V617FJAK2 mutation (n=7) or a mutation of the CALR gene (n=7), and of healthy subjects (HS, n=4). We have also studied the ultrastructural extension of the s-ER of CD34+ cells of the same subjects.
Methods
After loading CD34+ cells with 4 mcM fura-2 acetoxymethyl ester, depletion of intracellular Ca2+ stores was induced by adding 10 mcM cyclopiazonic acid (CPA) to a 0 Ca2+ bathing medium. Ca2+ was then added to the extracellular solution eliciting a rise in [Ca2+]i due to Ca2+ influx through open store-operated Ca2+ channels. The amplitude of the peak Ca2+ response to CPA, was measured as the difference between the ratio at the peak and the mean ratio of 1 min baseline before the peak. SOCE amplitude was measured as the difference between the peak and the mean ratio of 1 min baseline recorded before the readdition of Ca2+. Extension of sER was assessed by electron microscopy (EM) followed by analysis by ImageJ software. Data are expressed as mean +/- SD.
Results
Resting levels of Ca2+ of CALR-mutated CD34+ cells were not statistically different from those of JAK2-mutated cells, but higher compared to those of HS. CALR-mutated CD34+ cells released more Ca2+ from ER stores compared to JAK2-mutated cells (60.6 nM ± 30.9 vs 26.7 nM ± 24.9, respectively, P=0.038) and compared to HS (27.6 ± 19.8 nM, P=0.041 compared to CALR-mutated cells). SOCE of CALR-mutated CD34+ cells (318.5 nM ± 206.1) was slightly increased compared to JAK2-mutated patients (242.8 ± 146.8) and lower than SOCE of CD34+ cells of HS (104.3 nM ± 66.5), without reaching a statistical significance. At EM evaluation, the extension of vescicles of the sER was higher in CALR-mutated CD34+ cells compared to JAK2-mutated cells (0.82 vs 0.24 mcm, respectively, P=0.0001) and to HS-cells (0.101 mcm, P=0.0007).
Summary
Our results show that CALR mutation in CD34+ cells of patients with PMF is associated with a rearrangement of intracellular Ca++ balance, with a significant difference in Ca++ release compared to that observed both in JAK2-mutated- and HS-CD34+ cells. In keeping with the higher Ca2+ release, we observed an increased extension of s-ER. Considering the pivotal role of Ca2+ in regulating many cell functions, including proliferation and differentiation, these results seem to suggest that CALR mutation could be implicated in the myeloproliferative phenotype that characterizes PMF patients carrying CALR mutations by interfering with the signaling role of Ca2+.
Keyword(s): Calcium, CD34, Mutation, Myelofibrosis
Session topic: Novel insights into the mechanisms involved in MPNs
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