Jean D, Harbison M, McConkey D J, Ronai Z, Bar-Eli M
Department of Cell Biology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
J Biol Chem. 1998 Sep 18;273(38):24884-90. doi: 10.1074/jbc.273.38.24884.
cAMP response element-binding protein (CREB) and activating transcription factor 1 (ATF-1), members of the CREB/ATF family, have been implicated in cAMP- and calcium-induced transcriptional activation. We have previously demonstrated that quenching of CREB-associated proteins in metastatic melanoma cells by a dominant-negative CREB (KCREB) that is mutated within its DNA-binding domain decreased their radiation resistance, and their tumorigenic and metastatic potential in nude mice. As the induction of apoptosis by diverse exogenous signals is dependent on the elevation of intracellular Ca2+, the purpose of this study was to determine the role of CREB and its associated proteins in apoptosis using KCREB. We used thapsigargin (Tg), which inhibits endoplasmic reticulum-dependent Ca2+-ATPase and thereby increases cytosolic Ca2+, to induce apoptosis. MeWo human melanoma cells were transfected with the KCREB expression vector and subsequently analyzed for their susceptibility to Tg-induced apoptosis. Here we demonstrate that expression of KCREB in MeWo cells rendered them susceptible to Tg-induced apoptosis. Tg treatment induced phosphorylation of CREB and possibly ATF-1 transcription factors. Treatment with Tg induced CRE-dependent transcription in parental cells, whereas this activation was reduced in the KCREB-transfected cells. In addition, CAT activity driven by the CRE-dependent promoter was inhibited in parental MeWo cells cotransfected with increasing concentrations of KCREB in a dose-dependent manner. We did not observe any changes in Bcl-2 or Bcl-2-related proteins (Bcl-x, Bax, and Bad) in control or KCREB-transfected cells before or after treatment with Tg. Collectively, these data indicate that CREB and its associated proteins act as survival factors for human melanoma cells, and hence contribute to the acquisition of the malignant phenotype.
环磷酸腺苷反应元件结合蛋白（CREB）和激活转录因子1（ATF-1）属于CREB/ATF家族成员，与环磷酸腺苷（cAMP）和钙诱导的转录激活有关。我们之前已经证明，通过在其DNA结合域内发生突变的显性负性CREB（KCREB）来抑制转移性黑色素瘤细胞中与CREB相关的蛋白，可降低其辐射抗性以及在裸鼠中的致瘤和转移潜能。由于多种外源性信号诱导的细胞凋亡依赖于细胞内钙离子（Ca2+）浓度的升高，本研究旨在利用KCREB确定CREB及其相关蛋白在细胞凋亡中的作用。我们使用毒胡萝卜素（Tg）来诱导细胞凋亡，它可抑制内质网依赖性Ca2+ -ATP酶，从而增加胞质Ca2+浓度。将KCREB表达载体转染入MeWo人黑色素瘤细胞，随后分析其对Tg诱导凋亡的敏感性。在此我们证明，MeWo细胞中KCREB的表达使其对Tg诱导的凋亡敏感。Tg处理诱导了CREB以及可能还有ATF-1转录因子的磷酸化。用Tg处理可诱导亲本细胞中依赖于CRE的转录，而在转染了KCREB的细胞中这种激活作用减弱。此外，在与浓度不断增加的KCREB共转染的亲本MeWo细胞中，由CRE依赖性启动子驱动的氯霉素乙酰转移酶（CAT）活性以剂量依赖性方式受到抑制。在用Tg处理之前或之后，我们在对照细胞或转染了KCREB的细胞中均未观察到Bcl-2或Bcl-2相关蛋白（Bcl-x、Bax和Bad）有任何变化。总体而言，这些数据表明CREB及其相关蛋白作为人黑色素瘤细胞的存活因子，从而有助于获得恶性表型。
