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Normal Hematopoietic Progenitor Subsets Have Distinct Reactive Oxygen Species, BCL2 and Cell-Cycle Profiles That Are Decoupled from Maturation in Acute Myeloid Leukemia

View Article: PubMed Central - PubMed

ABSTRACT

In acute myeloid leukemia (AML) quiescence and low oxidative state, linked to BCL2 mitochondrial regulation, endow leukemic stem cells (LSC) with treatment-resistance. LSC in CD34+ and more mature CD34− AML have heterogeneous immunophenotypes overlapping with normal stem/progenitor cells (SPC) but may be differentiated by functional markers. We therefore investigated the oxidative/reactive oxygen species (ROS) profile, its relationship with cell-cycle/BCL2 for normal SPC, and whether altered in AML and myelodysplasia (MDS). In control BM (n = 24), ROS levels were highest in granulocyte-macrophage progenitors (GMP) and CD34− myeloid precursors but megakaryocyte-erythroid progenitors had equivalent levels to CD34+CD38low immature-SPC although they were ki67high. BCL2 upregulation was specific to GMPs. This profile was also observed for CD34+SPC in MDS-without-excess-blasts (MDS-noEB, n = 12). Erythroid CD34− precursors were, however, abnormally ROS-high in MDS-noEB, potentially linking oxidative stress to cell loss. In pre-treatment AML (n = 93) and MDS-with-excess-blasts (MDS-RAEB) (n = 14), immunophenotypic mature-SPC had similar ROS levels to co-existing immature-SPC. However ROS levels varied between AMLs; Flt3ITD+/NPM1wild-type CD34+SPC had higher ROS than NPM1mutated CD34+ or CD34− SPC. An aberrant ki67lowBCL2high immunophenotype was observed in CD34+AML (most prominent in Flt3ITD AMLs) but also in CD34− AMLs and MDS-RAEB, suggesting a shared redox/pro-survival adaptation. Some patients had BCL2 overexpression in CD34+ ROS-high as well as ROS-low fractions which may be indicative of poor early response to standard chemotherapy. Thus normal SPC subsets have distinct ROS, cell-cycle, BCL2 profiles that in AML /MDS-RAEB are decoupled from maturation. The combined profile of these functional properties in AML subpopulations may be relevant to differential treatment resistance.

No MeSH data available.


Influence of ROS levels on lineage fate and viability in normal progenitors.Sorting scheme of normal BM CD34+CD38high cells separated into CD45RA+ and CD45RA−subsets, followed by gating into 4 different populations based on differential DCF staining (A). Sorted cells were seeded onto methylcellulose media supplemented with cytokines. After 2 week culture at 37°C in vitro, colonies were scored using an inverted light microscope and recorded as a percentage of the total colony yield for FACS-sorted control CD34+CD38high CD45RA−(B) and CD34+CD38high CD45RA+ (C) progenitors with different levels of DCF staining. Total colony yield as percentage of input cell number was also determined (D). Colony forming unit assay data is from three independent experiments. Colonies were scored as erythroid (e), macrophage (m), granulocyte (g), granulocyte-macrophage (gm), or granulocyte-erythroid-macrophage mixed (gemm). Viability was determined by Annexin V and 7-AAD staining of normal BM labelled with HSPC-specific mAb. Initial gating was performed on SSClow CD45intCD117+ cells. The pooled viability data from 7 control BM sample are shown (E). The effects of redox modification on DCF staining and cell viability in CD34+CD38high progenitors, is shown from two independent experiments (F). BM cells were treated overnight with the pro-oxidant BSO (100μM) or left untreated (control).
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pone.0163291.g003: Influence of ROS levels on lineage fate and viability in normal progenitors.Sorting scheme of normal BM CD34+CD38high cells separated into CD45RA+ and CD45RA−subsets, followed by gating into 4 different populations based on differential DCF staining (A). Sorted cells were seeded onto methylcellulose media supplemented with cytokines. After 2 week culture at 37°C in vitro, colonies were scored using an inverted light microscope and recorded as a percentage of the total colony yield for FACS-sorted control CD34+CD38high CD45RA−(B) and CD34+CD38high CD45RA+ (C) progenitors with different levels of DCF staining. Total colony yield as percentage of input cell number was also determined (D). Colony forming unit assay data is from three independent experiments. Colonies were scored as erythroid (e), macrophage (m), granulocyte (g), granulocyte-macrophage (gm), or granulocyte-erythroid-macrophage mixed (gemm). Viability was determined by Annexin V and 7-AAD staining of normal BM labelled with HSPC-specific mAb. Initial gating was performed on SSClow CD45intCD117+ cells. The pooled viability data from 7 control BM sample are shown (E). The effects of redox modification on DCF staining and cell viability in CD34+CD38high progenitors, is shown from two independent experiments (F). BM cells were treated overnight with the pro-oxidant BSO (100μM) or left untreated (control).

Mentions: The relationship between ROS levels and immunophenotypic normal progenitors (CD34+CD38high fraction) was further assessed by colony assays using purified progenitor subsets from control BM (n = 3). CD34+CD38highCD45RA+ cells (GMP-enriched) and CD34+CD38highCD45RA−cells (CMP/MEP-enriched), were sorted into four populations denoted DCFlow, DCFint1, DCFint2 and DCFhigh (Fig 3A) and assayed for colony output after 14 days culture in vitro. Results from CD45RA−cells showed that mixed (CFU-GEMM) and erythroid (CFU-E) potential was limited to DCFlow/DCFint1 fractions and was lost in DCFint2/DCFhigh fractions. Only granulocyte/macrophage colonies were generated in DCFint2 and DCFhigh fractions (Fig 3B). CD45RA+ cells exclusively generated granulocyte (CFU-G) and macrophage (CFU-M) or mixed GM colonies (CFU-GM) with a relative reduction in CFU-GM and increase in CFU-M observed with increasing DCF (DCFint2/DCFhigh fraction) (Fig 3C). Thus, higher ROS in the CD45RA−(CMP/MEP-enriched) compartment correlates with reduced multipotency, loss of erythroid potential and commitment towards GMP while higher ROS in the CD45RA+ (GMP-enriched) compartment correlates with loss of mixed GM potential and higher macrophage potential.


Normal Hematopoietic Progenitor Subsets Have Distinct Reactive Oxygen Species, BCL2 and Cell-Cycle Profiles That Are Decoupled from Maturation in Acute Myeloid Leukemia
Influence of ROS levels on lineage fate and viability in normal progenitors.Sorting scheme of normal BM CD34+CD38high cells separated into CD45RA+ and CD45RA−subsets, followed by gating into 4 different populations based on differential DCF staining (A). Sorted cells were seeded onto methylcellulose media supplemented with cytokines. After 2 week culture at 37°C in vitro, colonies were scored using an inverted light microscope and recorded as a percentage of the total colony yield for FACS-sorted control CD34+CD38high CD45RA−(B) and CD34+CD38high CD45RA+ (C) progenitors with different levels of DCF staining. Total colony yield as percentage of input cell number was also determined (D). Colony forming unit assay data is from three independent experiments. Colonies were scored as erythroid (e), macrophage (m), granulocyte (g), granulocyte-macrophage (gm), or granulocyte-erythroid-macrophage mixed (gemm). Viability was determined by Annexin V and 7-AAD staining of normal BM labelled with HSPC-specific mAb. Initial gating was performed on SSClow CD45intCD117+ cells. The pooled viability data from 7 control BM sample are shown (E). The effects of redox modification on DCF staining and cell viability in CD34+CD38high progenitors, is shown from two independent experiments (F). BM cells were treated overnight with the pro-oxidant BSO (100μM) or left untreated (control).
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Related In: Results  -  Collection

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pone.0163291.g003: Influence of ROS levels on lineage fate and viability in normal progenitors.Sorting scheme of normal BM CD34+CD38high cells separated into CD45RA+ and CD45RA−subsets, followed by gating into 4 different populations based on differential DCF staining (A). Sorted cells were seeded onto methylcellulose media supplemented with cytokines. After 2 week culture at 37°C in vitro, colonies were scored using an inverted light microscope and recorded as a percentage of the total colony yield for FACS-sorted control CD34+CD38high CD45RA−(B) and CD34+CD38high CD45RA+ (C) progenitors with different levels of DCF staining. Total colony yield as percentage of input cell number was also determined (D). Colony forming unit assay data is from three independent experiments. Colonies were scored as erythroid (e), macrophage (m), granulocyte (g), granulocyte-macrophage (gm), or granulocyte-erythroid-macrophage mixed (gemm). Viability was determined by Annexin V and 7-AAD staining of normal BM labelled with HSPC-specific mAb. Initial gating was performed on SSClow CD45intCD117+ cells. The pooled viability data from 7 control BM sample are shown (E). The effects of redox modification on DCF staining and cell viability in CD34+CD38high progenitors, is shown from two independent experiments (F). BM cells were treated overnight with the pro-oxidant BSO (100μM) or left untreated (control).
Mentions: The relationship between ROS levels and immunophenotypic normal progenitors (CD34+CD38high fraction) was further assessed by colony assays using purified progenitor subsets from control BM (n = 3). CD34+CD38highCD45RA+ cells (GMP-enriched) and CD34+CD38highCD45RA−cells (CMP/MEP-enriched), were sorted into four populations denoted DCFlow, DCFint1, DCFint2 and DCFhigh (Fig 3A) and assayed for colony output after 14 days culture in vitro. Results from CD45RA−cells showed that mixed (CFU-GEMM) and erythroid (CFU-E) potential was limited to DCFlow/DCFint1 fractions and was lost in DCFint2/DCFhigh fractions. Only granulocyte/macrophage colonies were generated in DCFint2 and DCFhigh fractions (Fig 3B). CD45RA+ cells exclusively generated granulocyte (CFU-G) and macrophage (CFU-M) or mixed GM colonies (CFU-GM) with a relative reduction in CFU-GM and increase in CFU-M observed with increasing DCF (DCFint2/DCFhigh fraction) (Fig 3C). Thus, higher ROS in the CD45RA−(CMP/MEP-enriched) compartment correlates with reduced multipotency, loss of erythroid potential and commitment towards GMP while higher ROS in the CD45RA+ (GMP-enriched) compartment correlates with loss of mixed GM potential and higher macrophage potential.

View Article: PubMed Central - PubMed

ABSTRACT

In acute myeloid leukemia (AML) quiescence and low oxidative state, linked to BCL2 mitochondrial regulation, endow leukemic stem cells (LSC) with treatment-resistance. LSC in CD34+ and more mature CD34− AML have heterogeneous immunophenotypes overlapping with normal stem/progenitor cells (SPC) but may be differentiated by functional markers. We therefore investigated the oxidative/reactive oxygen species (ROS) profile, its relationship with cell-cycle/BCL2 for normal SPC, and whether altered in AML and myelodysplasia (MDS). In control BM (n = 24), ROS levels were highest in granulocyte-macrophage progenitors (GMP) and CD34− myeloid precursors but megakaryocyte-erythroid progenitors had equivalent levels to CD34+CD38low immature-SPC although they were ki67high. BCL2 upregulation was specific to GMPs. This profile was also observed for CD34+SPC in MDS-without-excess-blasts (MDS-noEB, n = 12). Erythroid CD34− precursors were, however, abnormally ROS-high in MDS-noEB, potentially linking oxidative stress to cell loss. In pre-treatment AML (n = 93) and MDS-with-excess-blasts (MDS-RAEB) (n = 14), immunophenotypic mature-SPC had similar ROS levels to co-existing immature-SPC. However ROS levels varied between AMLs; Flt3ITD+/NPM1wild-type CD34+SPC had higher ROS than NPM1mutated CD34+ or CD34− SPC. An aberrant ki67lowBCL2high immunophenotype was observed in CD34+AML (most prominent in Flt3ITD AMLs) but also in CD34− AMLs and MDS-RAEB, suggesting a shared redox/pro-survival adaptation. Some patients had BCL2 overexpression in CD34+ ROS-high as well as ROS-low fractions which may be indicative of poor early response to standard chemotherapy. Thus normal SPC subsets have distinct ROS, cell-cycle, BCL2 profiles that in AML /MDS-RAEB are decoupled from maturation. The combined profile of these functional properties in AML subpopulations may be relevant to differential treatment resistance.

No MeSH data available.