Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation

Abstract Leukemia initiating cells (LICs) in acute lymphoblastic leukemia (ALL) have been considered to be organized in a similar hierarchical fashion as in hematopoietic differentiation and acute myeloid leukemia (AML). Following this hierarchical concept, only few immature leukemia cells at the apex of a differentiation tree possess self renewal and differentiation potential and are capable of propagating leukemia upon transplantation onto immunodeficient mice. This view however has been challenged by recent findings showing that LICs in ALL are not restricted to few phenotypically defined cellular subsets but that both immature and mature B-cell precursor (BCP)-ALL cells are equally able to reconstitute leukemia in vivo, thus indicating a stochastic model for LICs in ALL. However, identification and characterization of a distinct leukemia initiating cell population in ALL remains still elusive although of key clinical importance since these cells giving rise to the disease need to be eradicated to cure the patient. To further characterize LICs in BCP-ALL, we assessed leukemia initiating activities of cellular ALL subpopulations corresponding to distinct cell cycle phases using our NOD/SCID/huALL xenotransplantation model. Patient derived xenograft B-cell precursor ALL samples were stained, sorted according to cell cycle phases (i.e. G0/G1 and G2/M) and transplanted onto recipient animals. Interestingly, transplantation of all four sorted cellular leukemia sub-fractions led to engraftment and development of leukemia showing leukemia initiating capacity irrespective of cell cycle stage. However, cells isolated from early phases of the cell cycle (G0/G1) led to earlier leukemia engraftment in the recipients in contrast to cells from later cell cycle phases with cellular G2/M sub-fractions constantly showing the longest time from transplantation until leukemia engraftment. In order to define additional biological features characteristic for leukemia initiating cells in ALL, we investigated the metabolic activity of cellular sub-fractions which had been functionally characterized with respect to their NOD/SCID mouse repopulating activities. For this purpose, levels of reactive oxygen species (ROS) were analyzed by detection of oxidation-specific fluorescence of chloromethyl-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) in patient derived xenograft ALL samples. Most interestingly, low ROS levels indicating inferior metabolic activity were identified in leukemia cell fractions of early G0/G1 cell cycle phases, which also showed increased NOD/SCID repopulating activity in functional assays. In contrast, leukemic cell populations of late G2/M cell cycle phases characterized by prolonged engraftment and lower leukemia initiating capacity, displayed increased metabolic activity as reflected by high ROS levels. Vice versa, analysis of cell cycle distribution with respect to metabolic activity revealed that leukemia cells characterized by low ROS levels (10% lowest) are allocated to early cell cycle whereas the majority of cells with high levels of ROS (10% highest) are shifted towards later cell cycle phases, denoting that the ALL cell’s oxidative state is indicative for its leukemia initiating activity. Taken together, in ALL cells of all cell cycle phases possess the ability to engraft and reconstitute leukemia upon transplantation into recipient mice. Thus, all ALL cells harbor leukemia initiating capacities irrespective of cell cycle phases, a finding which is in line with recent data showing that LICs in ALL are frequently and ubiquitously found. Most importantly, despite LIC activity in all cellular sub-fractions of the cell cycle, a higher leukemia repopulating activity was identified in ALL cells which display low ROS levels and originate from G0/G1 subpopulations indicating that leukemia initiating cells in ALL are enriched in early cell cycle phases and characterized by low metabolic activity. In conclusion, our data indicate that leukemia initiating potential in BCP-ALL is not restricted to distinct cellular subpopulations but that all cells show LIC activity with cells in early cell cycle and low metabolic activity representing the driving leukemia initiating cell compartment. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Physcion blocks cell cycle and induces apoptosis in human B cell precursor acute lymphoblastic leukemia cells by downregulating HOXA5

Biomedicine & Pharmacotherapy ◽

10.1016/j.biopha.2017.07.149 ◽

2017 ◽

Vol 94 ◽

pp. 850-857 ◽

Cited By ~ 13

Author(s):

Fei Gao ◽

Wenjun Liu ◽

Qulian Guo ◽

Yongqi Bai ◽

Hong Yang ◽

...

Keyword(s):

Cell Cycle ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Lymphoblastic Leukemia ◽

Leukemia Cells ◽

Cell Precursor ◽

Human B Cell

Download Full-text

Bafilomycin A1 Targets Both Autophagy and Apoptosis Pathways in Pediatric t(1;19) Pre-B Acute Lymphoblastic Leukemia Cells

Blood ◽

10.1182/blood.v124.21.3699.3699 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3699-3699

Author(s):

Na Yuan ◽

Lin Song ◽

Suping Zhang ◽

Weiwei Lin ◽

Yan Cao ◽

...

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

Western Blotting ◽

Bone Marrow Cells ◽

Lymphoblastic Leukemia ◽

Leukemia Cells ◽

Primary Cells ◽

Bafilomycin A1 ◽

Low Concentration

Abstract The t (1; 19) subtype leukemia accounts for a quarter of pre-B acute lymphoblastic leukemia (ALL) and up to 5% of all ALL patients. Despite plausible remission rate, current treatment regimen on the pediatric pre-B ALL is associated with side effects and CNS relapse, which poses the need for more effective and safer drugs. Bafilomycin A1 (Baf-A1) is known as an inhibitor of late phase of autophagy by inhibiting fusion between autophagosomes and lysosomes as well as by inhibiting lysosomal degradation. Here we show that Baf-A1 of low concentration (1 nM) effectively and specifically inhibits and kills the pre-B ALL cells. E2A/Pbx1 fusion gene positivepre-B ALL 697 cells were used for In vitro experiments. The results of flow cytometry analysis and western blotting showed that Baf-A1 induced cell cycle arrest and proliferation inhibition of ALL cells by upregualting cell cycle negative regulators and downregulating cell cycle positive regulators. In contrast, AML and CML cell lines were insensitive to Baf-A1 treatment. Western blotting and confocal observation on protein LC3 also showed that Baf-A1 at 1 nM blocked basal autophagic flux. Baf-A1 treatment activated mTOR signaling and induced the formation of Becn1–Bcl-2 complex to inhibit the induction of autophagy. Furthermore, apoptosis was induced in ALL cells treated with Baf-A1 for 72 h. However, procaspase-3 and poly-(ADP-ribose) polymerase (PARP) were not cleaved in these cells. We observed that AIF relocalized to the nucleus after 72h Baf-A1 treatment by confocal and immunoblotting. Knockdown of AIF significantly attenuated apoptosis induced by Baf-A1. These data suggest that Baf-A1 targets mitochondria membrane to trigger apoptosis via AIF pathway. In the in vivo experiment, Baf-A1 treatment extended survival and improved pathology of 697 xenograft mice, and significantly reduced the E2A/PBX1 positive leukemia cells in the bone marrow of mice. In vivomouse toxicity assay confirms Baf-A1 as a safe compound. The bone marrow cells of pre-B ALL leukemia patients were sorted against CD133+CD19+ markers, and treatment with Baf-A1 induced a clear inhibition on the CD133+CD19+ primary cells with a significant increased cell death in the sorted B-ALL patient samples. Conversely, Baf-A1 had no inhibitory effect on the bone marrow cells isolated from acute myeloid leukemia patients and healthy people. In summary, Baf-A1 treatment at low concentration effectively and specifically inhibited autophagy by activating mTOR and inducing beclin1-Bcl-2 interaction and induced AIF-dependent apoptosis in t (1; 19) pre-B ALL 697 cells. In the pre-B ALL xenograft mouse model, Baf-A1 specifically targets the leukemia cells while sparing normal cells. More importantly, Baf-A1 potently inhibits and kills the primary cells from pediatric pre-B ALL patients both at initial diagnosis and relapse without compromising normal human hematopoietic cells, all proposing Baf-A1 as a promising drug candidate for this pre-B ALL. Disclosures No relevant conflicts of interest to declare.

Download Full-text