title: Isolation of a highly quiescent subpopulation of primitive leukemic cells in chronic myeloid leukemia.
abstract: Chronic myeloid leukemia (CML) is characterized by an increased proliferative activity of the leukemic progenitors that produce an elevated number of mature granulocytes. Nevertheless, cell cycle-active agents, even in very high doses, are alone unable to eradicate the leukemic clone, suggesting the presence of a rare subset of quiescent leukemic stem cells. To isolate such cells, we first used Hoechst 33342 and Pyronin Y staining to obtain viable G(0) and G(1)/S/G(2)/M fractions of CD34(+) cells by fluorescence-activated cell sorting (FACS) from 6 chronic-phase CML patients' samples and confirmed the quiescent and cycling status of the 2 fractions by demonstration of expected patterns of Ki-67 and D cyclin expression. Leukemic (Ph(+)/BCR-ABL(+)) cells with in vitro progenitor activity and capable of engrafting immunodeficient mice were identified in the directly isolated G(0) cells. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that many leukemic CD34(+) G(0) cells also expressed BCR-ABL mRNA. CD34(+) from 8 CML patients were also labeled with carboxyfluorescein diacetate succinimidyl diester (CFSE) before being cultured (with and without added growth factors) to allow viable cells that had remained quiescent (ie, CFSE(+)) after 4 days to be retrieved by FACS. Leukemic progenitors were again detected in all quiescent populations isolated by this second strategy, including those exposed to a combination of flt3-ligand, Steel factor, interleukin-3, interleukin-6, and granulocyte colony-stimulating factor. These findings provide the first direct and definitive evidence of a deeply but reversibly quiescent subpopulation of leukemic cells in patients with CML with both in vitro and in vivo stem cell properties.
authors.affiliation: Terry Fox Laboratory, BC Cancer Agency, Vancouver, British Columbia, Canada.
authors.forename: T
authors.initials: T
authors.lastname: Holyoake
authors.forename: X
authors.initials: X
authors.lastname: Jiang
authors.forename: C
authors.initials: C
authors.lastname: Eaves
authors.forename: A
authors.initials: A
authors.lastname: Eaves
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000032:analysis; D001854:Bone Marrow Cells / Q000473:pathology*; D002453:Cell Cycle; D002469:Cell Separation; D002478:Cells, Cultured; D056741:Cyclin D; D016213:Cyclins / Q000032:analysis; D005434:Flow Cytometry; D016044:Fusion Proteins, bcr-abl / Q000032:analysis / Q000235:genetics; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000473:pathology; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000097:blood* / Q000235:genetics / Q000473:pathology*; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured; D014916:Whole-Body Irradiation
chemical_list: D018952:Antigens, CD34; D056741:Cyclin D; D016213:Cyclins; D016044:Fusion Proteins, bcr-abl