Mutagenesis as a novel target for cancer prevention

描述（由申请人提供）：本项目的总体目标是应用诱变基本机制的新知识直接测试癌症的体细胞突变假设，并将此知识转化为临床有用的方案，以减少日光相关皮肤癌的发病率。DNA突变通常被认为在癌症的发展中具有病因学作用，但直到最近才能直接检查致癌物暴露诱导的细胞生理学中诱变和表观遗传改变的相对贡献。我们假设，在活跃分裂的皮肤细胞的DNA中由紫外线（UV）诱导的突变与皮肤癌的发生有因果关系，降低这种突变的频率将降低由UV诱导的皮肤癌的风险。现在有大量的证据表明，几乎所有的突变诱导的紫外线在真核细胞中依赖于基因产物编码的REV1和REV3基因。我们进一步表明，针对这些基因（REV1）的基因特异性核酶的mRNA几乎废除紫外线诱变。为了验证我们的假设，我们建议将新开发的REV3缺失转基因小鼠暴露于UV。我们预计，与对照动物相比，这些小鼠患皮肤癌的风险将大大降低。此外，我们建议将我们的数据转化为预防紫外线诱导的皮肤癌的实用方法。我们将开发和优化在培养细胞中表达的基因特异性核酶，使用几种不同的系统将它们靶向细胞质或细胞核。然后，我们建议将这样的核酶表达载体送入小鼠皮肤细胞。这些核酶将特异性地靶向REV 1 mRNA，该蛋白质可以协调UV损伤DNA的诱变复制所需的几种蛋白质的活性。小鼠和适当的对照将暴露于已知会诱发皮肤癌的UV方案。我们的假设预测，降低这种蛋白质的水平将减少皮肤细胞对紫外线的诱变反应，并将导致皮肤癌的发病率大大降低。如果是这样的话，这些数据将为开发类似的策略以降低人类皮肤癌的发病率提供实验依据。最重要的是，这种策略作为紫外线照射后和疾病症状出现前的预防措施应该是成功的。

DESCRIPTION (provided by applicant): The overall aim of this project is to apply new knowledge of fundamental mechanisms of mutagenesis to directly test the somatic mutation hypothesis of cancer, and to translate this knowledge into a clinically useful regimen to reduce the incidence of sunlight-associated skin cancer. Mutations in DNA are generally considered to have an etiologic role in the development of cancer, but until recently it has not been possible to directly examine the relative contribution of mutagenesis and epigenetic alterations in cellular physiology induced by carcinogen exposure. We hypothesize that mutations induced by ultraviolet light (UV) in the DNA of actively dividing skin cells are causally involved in skin carcinogenesis, and reducing the frequency of such mutations will reduce the risk of skin cancer induced by UV. There is now abundant evidence that virtually all mutations induced by UV in eukaryotic cells are dependent on the gene products encoded by the REV1 and REV3 genes. We showed further that targeting the mRNA of one of these genes (REV1) with gene-specific ribozymes virtually abolishes UV mutagenesis. To test our hypothesis, we propose to expose newly-developed REV3-depleted transgenic mice to UV. We anticipate that these mice will have a greatly reduced risk of developing skin cancer compared with control animals. Further, we propose to translate our data into a practical method to prevent UV-induced skin cancer. We will develop and optimize gene-specific ribozymes expressed in cultured cells using several different systems to target them to the cytoplasm or to the nucleus. Then, we propose to deliver such ribozyme expression vectors into mouse skin cells. These ribozymes will specifically target REV1 mRNA, a protein that may coordinate the activity of several proteins that are required for the mutagenic replication of UVdamaged DNA. The mice and the appropriate controls will be exposed to UV protocols that are known to induce skin cancer. Our hypothesis predicts that reducing the level of this protein will reduce the mutagenic response of skin cells to UV, and will result in a greatly reduced incidence of skin cancer. If so, these data will provide the experimental rationale to develop similar strategies to reduce the incidence of skin cancer in humans. Most importantly, such a strategy should be successful as a preventive measure after UV exposure and prior to appearance of disease symptoms.