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Isolation of a highly quiescent subpopulation of primitive leukemic cells in chronic myeloid leukemia.
Blood 1999; 94(6):2056-64Blood

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+Show Affiliations

Terry Fox Laboratory, BC Cancer Agency, Vancouver, British Columbia, Canada.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

10477735

Citation

Holyoake, T, et al. "Isolation of a Highly Quiescent Subpopulation of Primitive Leukemic Cells in Chronic Myeloid Leukemia." Blood, vol. 94, no. 6, 1999, pp. 2056-64.
Holyoake T, Jiang X, Eaves C, et al. Isolation of a highly quiescent subpopulation of primitive leukemic cells in chronic myeloid leukemia. Blood. 1999;94(6):2056-64.
Holyoake, T., Jiang, X., Eaves, C., & Eaves, A. (1999). Isolation of a highly quiescent subpopulation of primitive leukemic cells in chronic myeloid leukemia. Blood, 94(6), pp. 2056-64.
Holyoake T, et al. Isolation of a Highly Quiescent Subpopulation of Primitive Leukemic Cells in Chronic Myeloid Leukemia. Blood. 1999 Sep 15;94(6):2056-64. PubMed PMID: 10477735.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Isolation of a highly quiescent subpopulation of primitive leukemic cells in chronic myeloid leukemia. AU - Holyoake,T, AU - Jiang,X, AU - Eaves,C, AU - Eaves,A, PY - 1999/9/9/pubmed PY - 1999/9/9/medline PY - 1999/9/9/entrez SP - 2056 EP - 64 JF - Blood JO - Blood VL - 94 IS - 6 N2 - 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. SN - 0006-4971 UR - https://www.unboundmedicine.com/medline/citation/10477735/Isolation_of_a_highly_quiescent_subpopulation_of_primitive_leukemic_cells_in_chronic_myeloid_leukemia_ L2 - http://www.bloodjournal.org/cgi/pmidlookup?view=long&pmid=10477735 DB - PRIME DP - Unbound Medicine ER -