Cell Cycle Check Points

项目2，细胞周期检查点，将集中在检查点的紫外线诱导的DMadamage的反应。 由于细胞周期检查点增强了DNA损伤的修复，检查点功能的缺陷增强了DNA损伤的修复。 紫外线诱导的染色体畸变产生一种“突变体”表型。UV诱导并激活p53， 诱导G1期阻滞或细胞凋亡，它激活ATR诱导S内检查点反应，以减缓细胞凋亡。 复制子起始的时间，并触发p38激酶依赖的G2延迟。量化指标将建立 正常人黑素细胞的检查点功能的功能能力， 黑色素瘤细胞系显示功能缺陷。目的1中的研究将在 正常人黑素细胞，以确定是否需要p53信号传导来阻止生长或诱导 细胞凋亡对紫外线损伤的反应。目标2中的研究将确定黑素细胞是否使用 Rad 17/ATR/Chk 1信号通路抑制复制子起始对紫外线损伤的反应以及是否 敲低由Timeless和Timeless- 相互作用蛋白使S内检查点对UV的反应失活。目标3中的研究将确定 正常人黑素细胞中突变的B-Raf和N-Ras癌基因的表达产生了一种衰减， G2检查点功能。还将监测具有p16和ARF选择性失活的黑素细胞系 确定CDKN 2A/INK 4A基因座中的遗传和体细胞突变如何影响检查点反应 DNA损伤。微阵列技术将用于确定基础基因表达的特征， 预测黑色素瘤细胞系中的G1和G2检查点功能，以及这些特征是否能区分黑色素瘤 淋巴结和内脏转移的线条。目的4给出了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 在目标4中，将开发一个紫外线断裂的计算模型，以测试DNA修复和细胞周期是如何 检查点功能协作以防止UV诱导的染色体畸变。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中的细胞周期检查点功能 与黑色素基因改变，以确定是否在检查点功能的缺陷产生一个 染色体突变表型，以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.