Svingen P A, Tefferi A, Kottke T J, Kaur G, Narayanan V L, Sausville E A, Kaufmann S H
Department of Oncology, Mayo Medical School, Rochester, Minnesota 55905, USA.
Clin Cancer Res. 2000 Jan;6(1):237-49.
The tyrphostin AG957 (NSC 654705) inhibits p210bcr/abl, the transforming kinase responsible for most cases of chronic myelogenous leukemia (CML). The present studies were performed to determine the fate of AG957-treated cells and assess the selectivity of AG957 for CML myeloid progenitors. When K562 cells (derived from a patient with blast crisis CML) were treated with AG957, dose- and time-dependent p210bc/abl down-regulation was followed by mitochondrial release of cytochrome c, activation of caspase-9 and caspase-3, and apoptotic morphological changes. These apoptotic changes were inhibited by transfection with cDNA encoding dominant negative caspase-9 but not dominant-negative FADD or blocking anti-Fas antibodies. In additional experiments, a 24-h AG957 exposure caused dose-dependent inhibition of K562 colony formation in soft agar. To extend these studies to clinical samples of CML, peripheral blood mononuclear cells from 10 chronic phase CML patients and normal controls were assayed for the growth of hematopoietic colonies in vitro in the presence of increasing concentrations of AG957. These assays demonstrated selectivity of AG957 for CML progenitors, with median IC50s (CML versus normal) of 7.3 versus >20 microM AG957 in granulocyte colony-forming cells (P < 0.001), 5.3 versus >20 microM in granulocyte/macrophage colony-forming cells (P < 0.05), and 15.5 versus > 20 microM in erythroid colony-forming cells (P > 0.05). The adamantyl ester of AG957 (NSC 680410) down-regulated p210bcr/abl in K562 cells and inhibited granulocyte colony formation in CML specimens at lower concentrations without enhanced toxicity in normal progenitors. These observations not only demonstrate that AG957-induced p210bcr/abl down-regulation is followed by activation of the cytochrome c/Apaf-1/caspase-9 pathway but also indicate that this class of kinase inhibitor exhibits selectivity worthy of further evaluation.
酪氨酸磷酸化抑制剂AG957（NSC 654705）可抑制p210bcr/abl，这是一种导致大多数慢性粒细胞白血病（CML）病例的转化激酶。进行本研究以确定经AG957处理的细胞的命运，并评估AG957对CML髓系祖细胞的选择性。当用AG957处理K562细胞（源自一名处于急变期CML的患者）时，p210bc/abl呈剂量和时间依赖性下调，随后细胞色素c从线粒体释放、caspase-9和caspase-3激活以及出现凋亡形态学改变。这些凋亡改变可通过转染编码显性负性caspase-9的cDNA来抑制，但不能通过转染显性负性FADD或阻断抗Fas抗体来抑制。在另外的实验中，24小时的AG957暴露导致软琼脂中K562集落形成呈剂量依赖性抑制。为了将这些研究扩展至CML的临床样本，对10例慢性期CML患者和正常对照的外周血单个核细胞在存在浓度递增的AG957的情况下进行体外造血集落生长检测。这些检测证明了AG957对CML祖细胞的选择性，在粒细胞集落形成细胞中，AG957的中位IC50（CML对正常）为7.3 μM对>20 μM（P<0.001），在粒细胞/巨噬细胞集落形成细胞中为5.3 μM对>20 μM（P<0.05），在红系集落形成细胞中为15.5 μM对>20 μM（P>0.05）。AG957的金刚烷基酯（NSC 680410）可下调K562细胞中的p210bcr/abl，并在较低浓度下抑制CML标本中的粒细胞集落形成，而对正常祖细胞无毒性增强作用。这些观察结果不仅证明AG957诱导的p210bcr/abl下调之后会激活细胞色素c/Apaf-1/caspase-9途径，还表明这类激酶抑制剂具有值得进一步评估的选择性。
