Cell Cycle Check Points

项目2，细胞周期检查点，将重点放在检查点对紫外线诱导的DMAdamage的反应。 由于细胞周期检查点增强了DNA损伤的修复，检查点功能缺陷增加了 紫外线诱导的染色体畸变会产生“突变子”表型。紫外线诱导并激活p53， 诱导G1期停滞或细胞凋亡，它激活ATR诱导S检查点内的反应，从而减缓 复制启动子的起始，并触发p38依赖的G2延迟。量化指标将建立 正常人黑素细胞对论文检查点功能的功能能力以及 黑色素瘤细胞系显示出功能缺陷。目标1中的研究将表达显性-负的P53等位基因 确定是否需要P53信号来阻止生长或诱导生长 对紫外线损伤作出反应的细胞凋亡。AIM 2的研究将确定黑素细胞是否使用 RAD17/ATR/Chk1信号通路抑制紫外线损伤后复制子的启动 敲除由Timless和Timless-2组成的复制叉状保护复合体的表达 相互作用的蛋白使S检查点内对紫外线的反应失活。Aim 3的研究将决定 突变的B-Raf和N-Ras癌基因在正常人黑素细胞中的表达可产生 G2检查点功能。选择性失活p16和ARF的黑素细胞系也将被监测 确定CDKN2A/INK4A基因座的遗传和体细胞突变如何影响检查点反应 造成DNA损伤。微阵列技术将被用来定义基础基因表达的签名， 预测黑色素瘤细胞株的G1和G2检查点功能以及这些信号是否区分黑色素瘤 来自淋巴结和内脏转移的线条。在目标4中建立了G2检查点函数的数学模型 将被开发来测试蛋白质表达水平的变化如何影响反应结果。一个 目标4中将开发紫外线断裂的计算模型，以测试DNA修复和细胞周期 检查点功能协作以防止紫外线诱导的染色体异常。项目2将 计数正常人黑素细胞、黑色素瘤细胞系和黑素细胞的细胞周期检查点功能 通过黑色素瘤基因改变确定检查点功能缺陷是否会产生 染色体突变子表型增强紫外线诱导的恶性进展。

Project 2, Cell Cycle Checkpoints, will focus on checkpoint responses to UV-induced DMAdamage. Because cell cycle checkpoints enhance the repair of DNA damage, defects in checkpoint function enhance UV-induced chromosomal aberrations arid produce a"mutator" phenotype. UV induces and activates p53, which induces G1 arrest or apoptosis, it activates ATR to induce an intra-S checkpoint response to slow the ate of replicon initiation, and it triggers a p38 kinase-dependent G2 delay. Quantitative metrics will establish the functional capacities of normal human melanocytes for thesis checkpoint functions and whether melanoma lines display functional defects. Studies in Aim 1will express a dominant-negative p53 allele in normal human melanocytes to determine whether p53 signaling is required to arrest growth or induce apoptosis in response to UV damage. Studies in Aim 2 will determine whether melanocytes use the Rad17/ATR/Chk1 signaling pathway to inhibit replicon initiation in response to UV damage and whether knockdown of expression of a replication fork-protection complex composed of Timeless and Timeless- interacting protein inactivates the intra-S checkpoint response to UV. Studies in Aim 3 will determine whether expression of mutant B-Raf and N-Ras oncogenes in normal human melanocytes produces an attenuation of G2 checkpoint function. Melanocyte lines with selective inactivation of p16 and ARF will also be monitored to determine how inherited and somatic mutations in the CDKN2A/INK4A locus affect checkpoint responses to DNA damage. Microarray technology will be used to define signatures of basal gene expression that predict G1 and G2 checkpoint functions in melanoma lines and whether the signatures distinguish melanoma lines from lymph node and visceralmetastases. In Aim 4 a mathematical model of G2 checkpoint function will be developed to test how variation in levels of protein expression affect response outcomes. A computational model of UV-clastogenesis will be developed in Aim 4 to test how DNA repair and cell cycle checkpoint functions collaborate to protect against UV-induced chromosomal aberrations. Project 2 will enumerate cell cycle checkpoint function in normal human melanocytes, melanoma lines, and melanocytes with melanomagenic genetic alterations to determine whether defects in checkpoint function produce a chromosomal mutator phenotype to enhance UV-induced malignant progression.