Selection of myeloid progenitors lacking BCR/ABL mRNA in chronic myelogenous leukemia patients after in vitro treatment with the tyrosine kinase inhibitor genistein.
Chronic myelogenous leukemia (CML) is a clonal disorder of the hematopoietic stem cell characterized by a chimeric BCR/ABL gene giving rise to a 210-kD fusion protein with dysregulated tyrosine kinase activity. We investigated the effect of genistein, a protein tyrosine kinase inhibitor, on the in vitro growth of CML and normal marrow-derived multi-potent (colony-forming unit-mix [CFU-Mix]), erythroid (burst-forming unit-erythroid [BFU-E]), and granulocyte-macrophage (colony-forming unit-granulocyte-macrophage [CFU-GM]) hematopoietic progenitors. Continuous exposure of CML and normal marrow to genistein induced a statistically significant and dose-dependent suppression of colony formation. Genistein doses causing 50% inhibition of CML and normal progenitors were not significantly different for CFU-Mix (27 mumol/L v 23 mumol/L), BFU-E (31 mumol/L v 29 mumol/L), and CFU-GM (40 mumol/L v 32 mumol/L v 32 mumol/L). Preincubation of CML and normal marrow with genistein (200 mumol/ L for 1 to 18 hours) induced a time-dependent suppression of progenitor cell growth, while sparing a substantial proportion of long-term culture-initiating cells (LTC-IC) from CML (range, 91% +/- 9% to 32% +/- 3%) and normal marrow (range, 85% +/- 8% to 38% +/- 9%). Analysis of individual CML colonies for the presence of the hybrid BCR/ABL mRNA by reverse transcription-polymerase chain reaction (RT-PCR) showed that genistein treatment significantly reduced the mean +/- SD percentage of marrow BCR/ABL+ progenitors both by continuous exposure (76% +/- 18% v 24% +/- 12%, P < or = .004) or preincubation (75% +/- 16% v 21% +/- 10%, P < or = .002) experiments. Preincubation with genistein reduced the percentage of leukemic LTC-IC from 87% +/- 12% to 37% +/- 12% (P < or = .003). Analysis of individual colonies by cytogenetics and RT-PCR confirmed that genistein-induced increase in the percentage of nonleukemic progenitors was not due to suppression of BCR/ABL transcription. Analysis of nuclear DNA fragmentation by DNA gel electrophoresis and terminal deoxynucleotidyl transferase assay showed that preincubation of CML mononuclear and CD34+ cells with genistein induced significant evidence of apoptosis. These observations show that genistein is capable of (1) exerting a strong antiproliferative effect on CFU-Mix, BFU-E, and CFU-GM while sparing the more primitive LTC-IC and (2) selecting benign hematopoietic progenitors from CML marrow, probably through an apoptotic mechanism.
Carlo-Stella
C
C
Department of Hematology, University of Parma, Italy.
Dotti
G
G
Mangoni
L
L
Regazzi
E
E
Garau
D
D
Bonati
A
A
Almici
C
C
Sammarelli
G
G
Savoldo
B
B
Rizzo
M T
MT
Rizzoli
V
V
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D017209:Apoptosis / Q000187:drug effects*; D016465:Bone Marrow Purging / Q000379:methods*; D002455:Cell Division / Q000187:drug effects; D053938:DNA Fragmentation; D004273:DNA, Neoplasm / Q000032:analysis; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D016044:Fusion Proteins, bcr-abl / Q000037:antagonists & inhibitors / Q000235:genetics*; D019833:Genistein; D006412:Hematopoietic Stem Cells / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D007529:Isoflavones / Q000494:pharmacology*; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000201:enzymology / Q000235:genetics / Q000473:pathology*; D008297:Male; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000032:analysis / Q000235:genetics*; D014411:Neoplastic Stem Cells / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D010766:Phosphorylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D012333:RNA, Messenger / Q000032:analysis*; D012334:RNA, Neoplasm / Q000032:analysis*; D014407:Tumor Cells, Cultured / Q000187:drug effects; D014410:Tumor Stem Cell Assay
D004273:DNA, Neoplasm; D004791:Enzyme Inhibitors; D007529:Isoflavones; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D019833:Genistein; D016044:Fusion Proteins, bcr-abl