Weinstein-Oppenheimer C, Steelman L S, Algate P A, Blalock W L, Burrows C, Hoyle P E, Lee J T, Moye P W, Shelton J G, Franklin R, McCubrey J A
Department of Microbiology and Immunology, Brody School of Medicine at East Carolina University, Greenville, NC 27858, USA.
Leukemia. 2000 Nov;14(11):1921-38. doi: 10.1038/sj.leu.2401926.
The effects of deregulated Raf activation on the growth and differentiation of hematopoietic cells were investigated. The cytokine-dependent murine myeloid FDC-P1 and human erythroleukemic TF-1 cell lines were transformed to grow in response to deregulated Raf expression in the absence of exogenous cytokines. The conditionally active Raf proteins were regulated by beta-estradiol as cDNAs containing the Raf catalytic, but lacking negative-regulatory domains, were ligated to the hormone binding domain of the estrogen receptor (deltaRaf:ER). Continuous deltaRaf expression prevented apoptosis in the absence of exogenous cytokines and altered the morphology of the FD/deltaRaf:ER cells as they grew in large aggregated masses (>100 cells) whereas the parental cytokine-dependent FDC-P1 cells grew in smaller grape-like clusters (< 10 cells). FD/deltaRaf-1:ER cells growing in response to Raf activation displayed decreased levels of the Mac-2 and Mac-3 molecules on their cell surface. In contrast, when these cells were cultured in IL-3, higher levels of these adhesion molecules were detected. Expression of activated Raf oncoproteins also abrogated cytokine dependency and prevented apoptosis of TF-1 cells. Moreover, the differentiation status of these Raf-responsive cells was more immature upon Raf activation as culture with the differentiation-inducing agent phorbol 12 myristate 13-acetate (PMA) and beta-estradiol resulted in decreased levels of the CD11b and CD18 integrin molecules on the cell surface. In contrast when the Raf-responsive cells were induced to differentiate with PMA and GM-CSF, in the absence of deltaRaf:ER activation, increased levels of the CD11b and CD18 molecules were detected. Retinoic acid (RA) inhibited 3H-thymidine incorporation in response to GM-CSF. Interestingly, Raf activation counterbalanced the inhibition of DNA synthesis caused by RA but not PMA. Thus deregulated Raf expression can alter cytokine dependency, integrin expression and the stage of differentiation. These Raf-responsive cell lines will be useful in elucidating the roles of the MAP kinase cascade on hematopoietic cell differentiation and malignant transformation.
研究了Raf激活失调对造血细胞生长和分化的影响。细胞因子依赖性小鼠髓系FDC-P1和人红白血病TF-1细胞系在无外源性细胞因子的情况下，因Raf表达失调而转化为可生长状态。条件性激活的Raf蛋白受β-雌二醇调控，因为含有Raf催化结构域但缺乏负调控结构域的cDNA与雌激素受体的激素结合结构域相连（δRaf:ER）。持续的δRaf表达在无外源性细胞因子时可防止细胞凋亡，并改变FD/δRaf:ER细胞的形态，它们会聚集成大的团块生长（>100个细胞），而亲本细胞因子依赖性FDC-P1细胞则以较小的葡萄状簇生长（<10个细胞）。因Raf激活而生长的FD/δRaf-1:ER细胞在其细胞表面显示Mac-2和Mac-3分子水平降低。相反，当这些细胞在IL-3中培养时，可检测到这些黏附分子的水平更高。激活的Raf癌蛋白的表达也消除了细胞因子依赖性，并防止了TF-1细胞的凋亡。此外，这些对Raf有反应的细胞在Raf激活后分化状态更不成熟，因为用分化诱导剂佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）和β-雌二醇培养会导致细胞表面CD11b和CD18整合素分子水平降低。相反，当在无δRaf:ER激活的情况下用PMA和GM-CSF诱导对Raf有反应的细胞分化时，可检测到CD11b和CD18分子水平升高。视黄酸（RA）抑制GM-CSF诱导的3H-胸腺嘧啶掺入。有趣的是，Raf激活可抵消RA而非PMA对DNA合成的抑制作用。因此，Raf表达失调可改变细胞因子依赖性、整合素表达和分化阶段。这些对Raf有反应的细胞系将有助于阐明MAP激酶级联在造血细胞分化和恶性转化中的作用。
