MUTATIONS CAUSED BY LINE 1 TRANSPOSONS IN BREAST CANCER

乳腺癌细胞在引起临床疾病之前必须积累一系列的基因改变。导致乳腺癌DNA损伤的机制尚不清楚。LINE-1 （L1Hs）转位是一种诱变过程，尚未被研究作为乳腺癌的原因。人类基因组平均包含10万个L1Hs序列；大多数是无功能的，但30-60可能有转位的能力。活性元件通过一个涉及RNA中间体的过程进行自我扩增，并将一个新的L1Hs插入基因组。当插入到一个敏感的基因位点时，结果是一个新的突变。已经描述了由L1Hs插入引起的疾病的例子。我们假设L1Hs逆转录转位在散发性或遗传性乳腺癌或两者的发展中是一个重要的突变机制。有几条证据支持这一假设。1)很大一部分遗传性乳腺癌是BRCA1或BRCA2基因突变的结果。BRCA1/2蛋白参与DNA双链断裂（DSB）的修复。L1Hs转位涉及在整合位点形成DSB；在DSB修复通路改变的细胞中，转位率可能会增加。2) L1Hs的转运需要L1Hs编码蛋白的转录和翻译。分化的细胞和大多数恶性细胞类型不转录或翻译L1Hs。相比之下，L1Hs第一个开放阅读框编码的蛋白p40存在于大多数乳腺癌和乳腺癌来源的细胞培养系中。这表明在正常细胞中抑制L1Hs转位的控制在乳腺癌中已经失活。3)有报道称，包括1例乳腺癌患者在内的癌症患者存在体细胞L1Hs转位，并在乳腺癌细胞系中观察到逆转录转位。如果L1Hs转位是乳腺癌的重要前奏，那么从患者肿瘤细胞中分离的DNA将具有从同一患者正常细胞中分离的DNA中不存在的L1Hs插入位点。这些位点被称为LINE插入二态性，或lid。我们将使用一种称为L1显示的新技术，在从乳腺癌患者获得的成对血液和肿瘤DNA样本中寻找lid的存在。将散发性和遗传性乳腺癌患者的相对频率与正常人的相对频率进行比较。研究人群中lid频率的显著差异将提示L1Hs转位在乳腺癌发展中的作用。这也将提示在高危患者中抑制L1Hs编码的逆转录酶可能降低其乳腺癌的发病率。

Breast cancer cells must accumulate a series of genetic alterations before causing clinical disease. The mechanisms responsible for causing DNA damage in breast cancer are not known. LINE-1 (L1Hs) transposition is a mutagenic process that has not yet been investigated as a cause of breast cancer. The average human genome contains 100,000 L1Hs sequences; most are nonfunctional but 30-60 are potentially capable of transposition. Active elements amplify themselves by a process that involves an RNA intermediate and inserts a new L1Hs into the genome. When insertion occurs into a sensitive genetic locus the result is a new mutation. Examples of disease caused by L1Hs insertions have been described. We hypothesize that L1Hs retrotransposition is an important mechanism of mutagenesis in the development of either sporadic or hereditary breast cancer, or both. Several lines of evidence support this hypothesis. 1) A large fraction of hereditary breast cancers are the result of mutations in either the BRCA1 or BRCA2 genes. The BRCA1/2 proteins are involved in the repair of DNA double strand breaks (DSB). L1Hs transposition involves the formation of a DSB at the integration site; transposition rates may be enhanced in cells with altered pathways of DSB repair. 2) Trasposition by L1Hs requires the transcription and translation of L1Hs-encoded proteins. Differentiated cells, and most malignant cell types, do not transcribe or translate L1Hs. In contrast, p40, the protein encoded by the L1Hs first open reading frame, is present in most breast cancers and breast cancer-derived cell culture lines. This suggests that the controls that inhibit L1Hs transposition in normal cells have been deactivated in breast cancers. 3) Somatic L1Hs transposition in individuals with cancer, including a patient with breast cancer, have been reported, and retrotransposition has been observed in a breast cancer cell line. If L1Hs transposition is an important prelude to breast cancer, DNA isolated from patients' tumor cells will have L1Hs insertion sites that are not present in DNA isolated from the same patients' normal cells. These sites are called LINE Insertion Dimorphisms, or LIDs. We will use a new technique called L1 display to look for the presence of LIDs in paired blood and tumor DNA samples obtained from breast cancer patients. The relative frequency of LIDs in patients with sporadic and hereditary breast cancers will be compared with the frequency of LIDs in normal individuals. A significant difference in the frequency of LIDs in the study populations will suggest a role for L1Hs transposition in the development of breast cancer. It will also suggest that inhibiting the L1Hs encoded reverse transcriptase in high-risk patients may lower their incidence of breast cancer.