Maxwell S A, Kurzrock R, Parsons S J, Talpaz M, Gallick G E, Kloetzer W S, Arlinghaus R B, Kouttab N M, Keating M J, Gutterman J U
Cancer Res. 1987 Mar 15;47(6):1731-9.
An altered c-abl gene product (P210bcr-abl) possessing associated tyrosine protein kinase activity was recently been reported in several blast chronic myelogenous leukemia (CML) cell lines. We have examined different morphological types of leukocytes directly obtained from patients at the blast crisis stage of CML for expression of P210bcr-abl tyrosine protein kinase activity. Phosphorylation of P210bcr-abl in an immune complex kinase assay using an anti-v-abl peptide serum was observed in blast cells from four Philadelphia chromosome (Ph1)-positive CML patients in blast crisis. P210bcr-abl protein kinase activity was detected regardless of whether the blast cells were of myeloid, lymphoid, or undifferentiated morphology. P210bcr-abl protein kinase activity was not detected in immune complexes either from leukocytes of four Ph1-negative CML patients in blast crisis, of five acute myelogenous leukemia patients, or in the promyelocytic cell line HL-60. Mature myeloid cells are associated with an inhibitory factor for not only P210bcr-abl protein kinase activity, but also protein kinases in general. Therefore, analyses of Ph1-positive benign phase CML myeloid cells, the majority of which are well differentiated, could not be successfully performed. The inhibition of P210bcr-abl protein kinase activity is not a specific property of mature cells from CML patients since granulocytes from a normal volunteer also demonstrated a similar effect. However, extracts of Ph1-positive cultured B-lymphocytes from a patient in benign phase demonstrated active P210bcr-abl protein indicating that the P210bcr-abl protein is expressed in an enzymatically active form in the earlier phases of CML. In addition to the previously reported P210 and P190 abl-related proteins, a novel Mr 53,000 protein was found to undergo phosphorylation at serine and tyrosine in immune complex kinase assays of two blast crisis CML cell lines (K562 and EM2) and in samples from blast crisis patients in which P210bcr-abl was detected. Peptide mapping by the Cleveland technique suggested that Mr 53,000 protein is unrelated to P210bcr-abl. Immune complex kinase assays of K562 cells with an anti-src serum (GD-11) yielded active c-src kinase and a Mr 50,000 phosphorylated protein, both of which were resistant to alkaline hydrolysis. Peptide mapping suggested that Mr 53,000 protein is related to Mr 50,000 protein which is precipitated with P210bcr-abl as an Mr 300,000 protein complex.
最近在几种急变期慢性粒细胞白血病（CML）细胞系中报道了一种具有相关酪氨酸蛋白激酶活性的改变的c-abl基因产物（P210bcr-abl）。我们检测了直接从处于CML急变期的患者获取的不同形态类型的白细胞中P210bcr-abl酪氨酸蛋白激酶活性的表达情况。在使用抗v-abl肽血清进行的免疫复合物激酶测定中，观察到来自4例处于急变期的费城染色体（Ph1）阳性CML患者的原始细胞中P210bcr-abl发生了磷酸化。无论原始细胞是髓系、淋巴系还是未分化形态，均检测到P210bcr-abl蛋白激酶活性。在4例处于急变期的Ph1阴性CML患者的白细胞、5例急性髓性白血病患者的白细胞以及早幼粒细胞系HL-60的免疫复合物中均未检测到P210bcr-abl蛋白激酶活性。成熟髓细胞不仅与P210bcr-abl蛋白激酶活性的抑制因子有关，而且与一般的蛋白激酶抑制因子有关。因此，对Ph1阳性慢性期CML髓细胞（其中大多数已充分分化）的分析未能成功进行。对P210bcr-abl蛋白激酶活性的抑制不是CML患者成熟细胞的特异性特性，因为来自一名正常志愿者的粒细胞也表现出类似的效应。然而，来自一名处于慢性期患者的Ph1阳性培养B淋巴细胞提取物显示出有活性的P210bcr-abl蛋白，这表明P210bcr-abl蛋白在CML的早期阶段以酶活性形式表达。除了先前报道的P210和P190 abl相关蛋白外，在两种急变期CML细胞系（K562和EM2）以及检测到P210bcr-abl的急变期患者样本的免疫复合物激酶测定中，发现一种新的分子量为53,000的蛋白在丝氨酸和酪氨酸处发生磷酸化。用克利夫兰技术进行的肽图谱分析表明，分子量为53,000的蛋白与P210bcr-abl无关。用抗-src血清（GD-11）对K562细胞进行免疫复合物激酶测定产生了活性c-src激酶和一种分子量为50,000的磷酸化蛋白，二者均对碱水解有抗性。肽图谱分析表明，分子量为53,000的蛋白与分子量为50,000的蛋白有关，后者与P210bcr-abl以分子量为300,000的蛋白复合物形式沉淀。
