Identification of Philadelphia-negative granulocyte-macrophage colony-forming units generated by stroma-adherent cells from chronic myelogenous leukemia patients.
Chronic myelogenous leukemia (CML) is a clonal disorder of the hematopoietic stem cell characterized by the coexistence of Philadelphia-negative (Ph-) with Ph+ progenitors. CML progenitor cells have been shown to be defective in adherence to marrow stroma. The present study investigated at the cytogenetic level marrow-derived CML clonogenic cells generated from the stroma-adherent cell fraction. On direct cytogenetic analysis, the overall mean (+/- SEM) percentage of Ph- metaphases was 3% +/- 1%. Mononuclear marrow cells from CML patients (n = 18) were incubated with mafosfamide (100 micrograms/mL) or control medium, seeded onto marrow stromal layers and allowed to adhere (2 hours, 37 degrees C). After a short-term (3-day) liquid culture, the cells were harvested, incorporated in methyl-cellulose, and individual colonies were analyzed by single colony karyotyping. The mean (+/- SEM) percentage of Ph- colonies generated from the stroma-adherent fraction was 35% +/- 6%. As compared with marrow colony-forming unit granulocyte-macrophage plated before any manipulation, the mean (+/- SEM) percentage of Ph- clones was significantly increased by stroma adherence (35% +/- 6% v 15% +/- 4%, P < or = .005) and mafosfamide (100 micrograms/mL) incubation of marrow cells before stroma adherence (58% +/- 9% v 35% +/- 6%, P < or = .005). An additive effect was observed by combining mafosfamide treatment and stroma adherence. Single-colony transfer experiments showed that 50% +/- 4% stroma-adherent and 70% +/- 4% stroma-adherent mafosfamide-treated progenitors gave rise to secondary colonies. To further characterize the stroma-adherent fraction, experiments were performed in which CD34+ marrow cells were used. The mean (+/- SEM) output of progenitors generated by 10,000 CD34+, stroma-adherent cells was 888 +/- 188 and 570 +/- 258 for untreated and mafosfamide-treated cells, respectively. Individual colonies were analyzed by single-colony karyotyping and fluorescent in situ hybridization using a biotinylated cosmid DNA probe that hybridize to abl oncogene. The CD34+, stroma-adherent fraction contained 38% +/- 14% (untreated) and 56% +/- 18% (mafosfamide-treated) (P < or = .025) Ph- progenitors. In conclusion, the present data show the possibility to select Ph- clones that (1) have a maintained capability of stroma adherence, (2) are mafosfamide resistant, (3) are derived from the CD34+ fraction, and (4) have high-replating potential.
Carlo-Stella
C
C
Department of Hematology, University of Parma, Italy.
Mangoni
L
L
Piovani
G
G
Garau
D
D
Almici
C
C
Rizzoli
V
V
D000328:Adult; D000368:Aged; D015703:Antigens, CD / Q000032:analysis; D018952:Antigens, CD34; D001853:Bone Marrow / Q000473:pathology*; D002448:Cell Adhesion; D002469:Cell Separation; D005260:Female; D016044:Fusion Proteins, bcr-abl / Q000235:genetics; D006098:Granulocytes / Q000166:cytology; D006412:Hematopoietic Stem Cells / Q000473:pathology*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics / Q000473:pathology*; D008264:Macrophages / Q000166:cytology; D008297:Male; D008875:Middle Aged
D015703:Antigens, CD; D018952:Antigens, CD34; D016044:Fusion Proteins, bcr-abl