Blalock W L, Pearce M, Steelman L S, Franklin R A, McCarthy S A, Cherwinski H, McMahon M, McCubrey J A
Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, North Carolina 27858, USA.
Oncogene. 2000 Jan 27;19(4):526-36. doi: 10.1038/sj.onc.1203337.
The Raf/MEK/MAP kinase cascade plays a critical role in transducing growth signals from activated cell surface receptors. Using deltaMEK1:ER, a conditionally-active form of MEK1, we demonstrate the ability of this dual specificity protein kinase to abrogate the cytokine-dependency of the human and murine hematopoietic cells lines TF-1, FDC-P1 and FL5.12. Cytokine-independent cells were obtained from TF-1, FDC-P1 and FL5.12 cells at frequencies of 2.5 x 10(-3), 5 x 10(-5) and 10(-7) respectively, indicating that not all cells expressing deltaMEK1:ER were factor-independent. In general, cells that were converted to a cytokine-independent phenotype displayed a higher level of MAP kinase activity in response to deltaMEK1:ER activation than those that remained cytokine-dependent. deltaME-K1:ER-responsive cells could be maintained long-term in the presence of beta-estradiol as well as the estrogen-receptor antagonist 4-Hydroxy-Tamoxifen and the anti-estrogen ICI 164383. Removal of hormone led to the rapid cessation of cell growth in a manner similar to that observed when cytokine is withdrawn from the parental cells. Treatment of deltaMEKI:ER-responsive cells with a specific and selective inhibitor, PD98059, prevented growth in response to beta-estradiol. GM-CSF mRNA transcripts were detected in the MEK1-responsive cells indicating that the activated deltaMEK1:ER may induce a pathway leading to autocrine proliferation. Treatment of MEK1-responsive cells with an anti-GM-CSF antibody, but not a control antibody, suppressed cell growth. The cell lines described here will be useful for elaborating the ability of the MAP kinase pathway to regulate cell proliferation in hematopoietic cells.
Raf/MEK/MAP激酶级联反应在转导来自活化细胞表面受体的生长信号中起关键作用。使用deltaMEK1:ER（一种MEK1的条件活性形式），我们证明了这种双特异性蛋白激酶能够消除人源和鼠源造血细胞系TF-1、FDC-P1和FL5.12对细胞因子的依赖性。分别以2.5×10⁻³、5×10⁻⁵和10⁻⁷的频率从TF-1、FDC-P1和FL5.12细胞中获得了不依赖细胞因子的细胞，这表明并非所有表达deltaMEK1:ER的细胞都不依赖因子。一般来说，转化为不依赖细胞因子表型的细胞在响应deltaMEK1:ER激活时，其MAP激酶活性水平高于那些仍然依赖细胞因子的细胞。deltaME-K1:ER反应性细胞可以在β-雌二醇以及雌激素受体拮抗剂4-羟基他莫昔芬和抗雌激素ICI 164383存在的情况下长期维持。去除激素导致细胞生长迅速停止，其方式类似于从亲代细胞中撤除细胞因子时观察到的情况。用特异性和选择性抑制剂PD98059处理deltaMEKI:ER反应性细胞，可阻止其对β-雌二醇的反应性生长。在MEK1反应性细胞中检测到GM-CSF mRNA转录本，表明活化的deltaMEK1:ER可能诱导一条导致自分泌增殖的途径。用抗GM-CSF抗体而非对照抗体处理MEK1反应性细胞，可抑制细胞生长。本文所述的细胞系将有助于阐明MAP激酶途径调节造血细胞增殖的能力。
