Mohammad R M, Maki A, Pettit G R, al-Katib A M
Division of Hematology and Oncology, Karmanos Cancer Institute, Wayne State University, Detroit, Mich, USA.
Enzyme Protein. 1996;49(5-6):262-72. doi: 10.1159/000468636.
It has been previously reported that Bryostatin 1 (Bryo1) induces differentiation of the human acute lymphoblastic leukemia (ALL) cell line, Reh, to a monocytoid B-cell stage. In this study we demonstrate that a novel protein, ubiquitin COOH-terminal hydrolase (UCH-L1), is associated with this differentiation. Reh cells were treated with 200 nmol/l of Bryo1 for 72 h and analyzed for changes in morphology, surface immunophenotype, acid phosphatase and terminal deoxynucleotidyl transferase. Protein patterns of the parent and differentiated cells, by two-dimensional polyacrylamide gel electrophoresis (2D PAGE), were studied. Bryo1-treated cells expressed morphologic, phenotypic and enzymatic features of the monocytoid B-cell stage. The UCH-L1 enzyme (MW-pl 34-5.3) was detected by 2 D PAGE in the differentiated, but not in parent cells. The presence of UCH-L1 in the Bryo1-treated cells was further confirmed by immunoblotting of 2 D PAGE using UCH-L1 polyclonal antibody. Ubiquitin expression was studied in parent and Bryo1-treated cells and was compared with 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated cells. Both agents, TPA and Bryo1, increased the level of ubiquitin expression as detected by flow cytometry. Sodium borohydride, an inhibitor of UCH-L1, inhibited the Bryo1-induced differentiating effect on Reh cells. To date, the mechanism by which Bryo1, exerts its B-cell differentiating effect is not fully understood. This study shows that UCH-L1 expression may play a major role in Bryo1-induced differentiation in pre-B-ALL.
先前有报道称，苔藓抑素1（Bryo1）可诱导人急性淋巴细胞白血病（ALL）细胞系Reh分化为单核样B细胞阶段。在本研究中，我们证明一种新型蛋白质，泛素羧基末端水解酶（UCH-L1）与这种分化相关。用200 nmol/l的Bryo1处理Reh细胞72小时，并分析其形态、表面免疫表型、酸性磷酸酶和末端脱氧核苷酸转移酶的变化。通过二维聚丙烯酰胺凝胶电泳（2D PAGE）研究亲本细胞和分化细胞的蛋白质图谱。经Bryo1处理的细胞表现出单核样B细胞阶段的形态、表型和酶学特征。通过2D PAGE在分化细胞中检测到UCH-L1酶（分子量-等电点为34-5.3），而在亲本细胞中未检测到。使用UCH-L1多克隆抗体对2D PAGE进行免疫印迹进一步证实了经Bryo1处理的细胞中存在UCH-L1。研究了亲本细胞和经Bryo1处理的细胞中的泛素表达，并与经12-O-十四烷酰佛波醇-13-乙酸酯（TPA）处理的细胞进行了比较。通过流式细胞术检测，TPA和Bryo1这两种试剂均增加了泛素表达水平。硼氢化钠，一种UCH-L1抑制剂，抑制了Bryo1对Reh细胞的诱导分化作用。迄今为止，Bryo1发挥其B细胞分化作用的机制尚未完全明确。本研究表明，UCH-L1表达可能在Bryo1诱导的前B-ALL分化中起主要作用。
