Mechanisms of UV-induced DNA damage responses and carcinogenesis in skin

 描述（由申请人提供）：太阳紫外线引起的皮肤癌的数量大于所有其他类型癌症的总和。需要更好地了解潜在的生物学，以开发新的方法来解决这个问题。 紫外线照射瞬间产生两种结构不同的DNA损伤：环丁烷嘧啶二聚体（CPD）和6-4光产物（6-4PP）。复制细胞通过激活ATR激酶及其下游靶点Chk 1对这些有害损伤迅速作出反应。然而，目前尚不清楚这些病变是否对DNA复制和/或ATR-Chk 1激活有不同的影响。为了解决这些问题，在目标1中，我们将通过将病变特异性光解酶与多参数流式细胞术相结合来产生具有一种、两种或两种DNA病变类型都没有的细胞。拟议的研究将测试的假设，支持的初步数据，有显着的差异，这两种损伤类型的DNA复制和UV-DNA损伤反应的机制和影响。 人类流行病学研究和小鼠体内数据表明，摄入咖啡因（一种非选择性ATR抑制剂）可预防紫外线相关的皮肤癌。事实上，我们估计在美国每年有260，000例皮肤癌是通过摄入含咖啡因的饮料来预防的。咖啡因的这种癌症预防作用可以通过对其机制的更好理解来进一步优化，包括ATR是否应该在UV后立即抑制或在UV后很长时间内抑制。在目标2中，我们将确定ATR抑制抑制UV致癌作用的机制。我们将测试的假设，即立即（而不是延迟）ATR抑制通过阻断易错病变旁路减少皮肤中的突变负担。 ATR途径的激活需要将10种以上的蛋白质募集到DNA损伤位点。理论上，靶向这些蛋白质中的任何一种来抑制ATR途径是可能的，其对DNA损伤敏感的能力可能存在差异。然而，目前阻断该途径的方法仅限于靶向ATR激酶活性的ATP竞争性抑制剂 或Chk 1。为了确定该途径中的其他药物机制，我们进行了基于细胞的高通量筛选，并确定了几种通过ATP竞争以外的机制抑制ATR途径的小分子。一种化合物（“ARPIN”）显示出一种任何其他抑制剂都不具有的重要机制：阻断Chk 1在损伤后从染色质中释放。在目标3中，我们将使用ARPIN的这种独特的生物学效应作为工具来剖析Chk 1通常从染色质释放以执行其效应子功能的基本机制。 这三个提出的目标将显著推进我们对复制检查点功能的基本理解，表征该途径的机制性新抑制剂，并提供可以直接翻译的见解。 以进一步扩大已经广泛的公共卫生影响。这些研究将与五位科学家高度合作，他们的专业知识将促进拟议目标的关键创新方面。

 DESCRIPTION (provided by applicant): The number of solar UV-induced skin cancers is greater than all other types of cancers combined. A better understanding of the underlying biology is needed to develop novel approaches to address this problem. UV irradiation instantaneously generates two structurally distinct types of DNA lesion: cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PP). Replicating cells rapidly respond to these deleterious lesions by activating ATR kinase and its downstream target Chk1. However, it is not clear whether these lesions have different effects on DNA replication and/or ATR-Chk1 activation. To address these questions, in Aim 1, we will generate cells that have one, both, or neither type of DNA lesion by combining lesion-specific photolyases with multi-parameter flow cytometry. The proposed studies will test the hypothesis, supported by preliminary data, that there are striking differences in the mechanisms and impact of these two lesion types on DNA replication and UV-DNA damage responses. Human epidemiological studies and mouse in vivo data demonstrate that intake of caffeine (a non- selective ATR inhibitor) prevents UV-associated skin cancers. Indeed, we estimate that 260,000 skin cancers are prevented annually in the U.S. by caffeinated beverage intake. This cancer-preventive effect of caffeine could be further optimized by an improved understanding of its mechanism including whether ATR should be inhibited immediately after UV or long after UV. In Aim 2, we will determine the mechanism by which ATR inhibition suppresses UV carcinogenesis. We will test the hypothesis that immediate (not delayed) ATR inhibition reduces the mutation burden in skin by blocking error-prone lesion bypass. ATR pathway activation requires recruiting more than 10 proteins to a site of DNA damage. Theoretically, it is possible to target any of these proteins to inhibit the ATR pathway, with likely differences in the ability to sensitize to DNA damage. However, current approaches to block this pathway are limited to ATP-competitive inhibitors that target the kinase activity of ATR or Chk1. To identify other druggable mechanisms in this pathway, we performed a cell-based high-throughput screen and identified several small molecules that inhibit the ATR pathway through mechanisms other than ATP competition. One compound ("ARPIN") displayed an important mechanism not shared by any other inhibitor: blockage of Chk1 release from chromatin following damage. In Aim 3, we will use this unique biological effect of ARPIN as a tool to dissect the fundamental mechanism by which Chk1 is normally released from chromatin in order to carry out its effector functions. The three proposed Aims will markedly advance our fundamental understanding of how the replication checkpoint functions, characterize a mechanistically novel inhibitor of the pathway, and provide insight that can be directly translated to further augment an already extensive public health impact. The studies will be highly collaborative with five scientists whose expertise will facilitate critical innovative aspects of te proposed Aims.