MITOMYCIN SIGNALING TO AND FROM P53 DURING THE CELL CYCLE

细胞周期中丝裂霉素对p53的信号传导在DNA损伤的反应中，p53蛋白被翻译后修饰，促进细胞周期生长停滞（G1/S和G2/M过渡）或细胞凋亡。许多野生型p53靶基因已被确定；其中包括GADD45， p21/Waf1, bax, 14-3-3， mdm2和IGF-BP3。为了使学生和研究界能够更多地了解p53的信号通路，我们将研究以下领域。1)提供为期三个月的研究模块，使学生掌握p53相关细胞周期研究的复杂性。目的2)确定不同化疗药物引起的DNA损伤是否能够诱导p53靶基因的差异激活。我们将研究丝裂霉素C （MC）和两个MC衍生物的作用。MC引起双链断裂，而两种衍生物则不会。我们还将比较喜树碱和Zeocin的作用，它们会导致不同类型的DNA损伤。利用新型荧光探针实时RT-PCR检测多种靶点的基因表达水平。目的3)确定DNA损伤诱导的p53靶基因的差异激活是否受细胞周期调控。我们打算利用实时RT-PCR检测细胞周期中不同时间对喜树碱、Zeocin和丝裂霉素的p53应答基因mRNA表达情况。细胞周期馏分将使用离心洗脱分离。目的4)确定所选靶基因的差异表达是否对应于p53体内和体外DNA结合活性的差异。选择基因的p53结合位点区域的保护将在细胞核中使用体内LM-PCR足迹监测差异基因激活过程。体外p53的DNA结合活性将通过EMSA检测，使用培养细胞的核提取物和DNA损伤剂处理的细胞周期组分。目的5)检查p53的特异性翻译后修饰是否负责p53介导的差异基因激活和DNA结合活性。在特定药物处理的片段中，p53的翻译后修饰显示出mRNA表达和p53 DNA结合活性的明显差异，将通过Western blot分析使用特异性磷酸化和乙酰化形式的p53抗体来检查。经过修饰的乙酰化位点将发生突变，用于诱导型p53系统。

Mitomycin Signaling to and from p53 During the Cell Cycle; In response to DNA damage the p53 protein is post-translationally modified and promotes either cell cycle growth arrest (at both the G1/S and G2/M transitions) or apoptosis. Many wild-type p53 target genes have been identified; a few of these include GADD45, p21/Waf1, bax, 14-3-3, mdm2 and IGF-BP3. To enable students and the research community to understand more about the signaling pathways to and from p53 we will investigate the following areas. Aim 1) Provide a three month research module that enables students to grasp the complexities of p53 related cell cycle research. Aim 2) Determine if DNA damage caused by different chemotherapeutic drugs is able to induce differential activation of p53 target genes. We will examine the effects of mitomycin C (MC) and two MC derivatives. MC causes double strand breaks and the two derivatives do not. We will also compare the effects of camptothecin and Zeocin, which causes different types of DNA damage. Analysis of the gene expression level of multiple targets will be monitored using real-time RT-PCR probed with novel fluorescent probes. Aim 3) Determine if DNA damage induced differential activation of p53 target genes is cell cycle regulated. We intend to use real-time RT-PCR to examine the mRNA expression from p53 responsive genes at different times during the cell cycle in response to camptothecin, Zeocin and the mitomycins. Cell cycle fractions will be isolated using centrifugal elutriation. Aim 4) Determine if differential expression of select target gen corresponds to a difference in the in vivo and in vitro DNA binding activity of p53. Protection of the p53 binding site regions of select genes will be monitored in nuclei during differential gene activation using in vivo LM-PCR footprinting. The in vitro DNA binding activity of p53 will be examined by EMSA using nuclear extract from cultured cells and cell cycle fractions treated with DNA damaging agents. Aim 5) Examine if specific post-translational modifications of p53 are responsible for differential p53 mediated gene activation and DNA binding activity. The post-translational modification of p53 in specific drug treated fractions shown to exhibit overt differences in mRNA expression and p53 DNA binding activity will be examined by Western blot analysis using antibodies to specific phosphorylated and acetylated forms of p53. The acetylated sites that are modified will be mutated for use in the inducible p53 system.