Spangler R, Sytkowski A J
Laboratory for Cell and Molecular Biology, New England Deaconess Hospital, Boston, MA 02215.
Blood. 1992 Jan 1;79(1):52-7.
The proto-oncogene c-myc has been identified as an early response gene for erythropoietin (Epo) in transformed murine erythroleukemia cells. Epo activation of c-myc in these cells requires protein kinase C. We now show the fidelity of this signaling pathway in normal erythroid cells isolated from the spleens of phenylhydrazine-treated mice. Mouse spleen cells rich in erythroid progenitors were washed free of endogenous Epo and then incubated in the absence of Epo. Subsequent addition of Epo for 1 hour led to a dramatic elevation of c-myc transcript. Addition of the protein synthesis inhibitor cycloheximide did not prevent the c-myc response, thus identifying c-myc as an Epo early response gene in normal cells. We used this c-myc response as a reporter for signals initiated by the Epo receptor. Using a series of inhibitors with known specificities and established rank-orders of potency for different kinases, we determined that the c-myc response to Epo was blocked with the following rank order: staurosporine much greater than H7 greater than sangivamycin greater than H8. This sequence is identical to that obtained using transformed cells and is diagnostic of a protein kinase C-dependent signal. Because direct activation of protein kinase by phorbol esters does not induce terminal differentiation of normal cells, the pathway to c-myc established by these studies must represent one part of a signal transduction mechanism.
原癌基因c-myc已被鉴定为转化的小鼠红白血病细胞中促红细胞生成素（Epo）的早期反应基因。在这些细胞中，Epo对c-myc的激活需要蛋白激酶C。我们现在证明了从苯肼处理的小鼠脾脏中分离出的正常红系细胞中该信号通路的保真度。富含红系祖细胞的小鼠脾脏细胞被洗涤以去除内源性Epo，然后在无Epo的情况下孵育。随后添加Epo 1小时导致c-myc转录本显著升高。添加蛋白质合成抑制剂环己酰亚胺并不能阻止c-myc的反应，因此确定c-myc为正常细胞中的Epo早期反应基因。我们将这种c-myc反应用作Epo受体引发信号的报告指标。使用一系列具有已知特异性和针对不同激酶已确定的效力等级顺序的抑制剂，我们确定c-myc对Epo的反应被以下效力等级顺序阻断：星形孢菌素远大于H7大于放线菌素大于H8。该序列与使用转化细胞获得的序列相同，并且是蛋白激酶C依赖性信号的诊断指标。由于佛波酯对蛋白激酶的直接激活不会诱导正常细胞的终末分化，这些研究建立的通向c-myc的途径必定代表信号转导机制的一部分。
