Carlo-Stella C, Dotti G, Mangoni L, Regazzi E, Garau D, Bonati A, Almici C, Sammarelli G, Savoldo B, Rizzo M T, Rizzoli V
Department of Hematology, University of Parma, Italy.
Blood. 1996 Oct 15;88(8):3091-100.
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.
慢性粒细胞白血病（CML）是一种造血干细胞的克隆性疾病，其特征是存在一种嵌合性BCR/ABL基因，该基因可产生具有失调酪氨酸激酶活性的210-kD融合蛋白。我们研究了染料木黄酮（一种蛋白酪氨酸激酶抑制剂）对CML以及正常骨髓来源的多能（集落形成单位混合[CFU-Mix]）、红系（爆式红系集落形成单位[BFU-E]）和粒-巨噬系（粒-巨噬细胞集落形成单位[CFU-GM]）造血祖细胞体外生长的影响。将CML和正常骨髓持续暴露于染料木黄酮可诱导集落形成受到具有统计学意义的剂量依赖性抑制。对于CFU-Mix（27 μmol/L对23 μmol/L）、BFU-E（31 μmol/L对29 μmol/L）和CFU-GM（40 μmol/L对32 μmol/L），导致CML和正常祖细胞50%抑制的染料木黄酮剂量无显著差异。用染料木黄酮（200 μmol/L，处理1至18小时）对CML和正常骨髓进行预孵育可诱导祖细胞生长的时间依赖性抑制，同时使CML（范围为91%±9%至32%±3%）和正常骨髓（范围为85%±8%至38%±9%）中的大部分长期培养起始细胞（LTC-IC）得以保留。通过逆转录-聚合酶链反应（RT-PCR）分析单个CML集落中杂交BCR/ABL mRNA的存在情况，结果显示，无论是通过持续暴露（76%±18%对24%±12%，P≤0.004）还是预孵育（75%±16%对21%±10%，P≤0.002）实验，染料木黄酮处理均显著降低了骨髓中BCR/ABL+祖细胞的平均±标准差百分比。用染料木黄酮预孵育可使白血病LTC-IC的百分比从87%±12%降至37%±12%（P≤0.003）。通过细胞遗传学和RT-PCR对单个集落进行分析证实，染料木黄酮诱导的非白血病祖细胞百分比增加并非由于BCR/ABL转录受到抑制。通过DNA凝胶电泳和末端脱氧核苷酸转移酶测定分析核DNA片段化情况，结果显示用染料木黄酮对CML单核细胞和CD34+细胞进行预孵育可诱导明显的凋亡证据。这些观察结果表明，染料木黄酮能够（1）对CFU-Mix、BFU-E和CFU-GM发挥强大的抗增殖作用，同时保留更原始的LTC-IC；（2）可能通过凋亡机制从CML骨髓中选择良性造血祖细胞。
