p38 MAP kinase in early thymocyte development

描述(申请人提供)：p38 MAP激酶(MAPK)通路在诱导DNA双链断裂(DSB)的应激刺激(例如电离辐射)下被激活，并在诱导细胞周期检查点(主要是G2/M)以促进DNA修复中发挥作用。当T细胞在胸腺中进行V(D)J重组时，也会在发育过程中产生DSB。V(D)J介导的双链断裂(编码和信号末端)触发DNA损伤反应和DNA修复，与电离辐射产生的双链断裂相似。虽然编码末端的连接是形成功能性TCR的关键，但修复切除的DNA片段两侧的信号末端也是重要的，以防止DNA片段随机整合到基因组中和基因组的不稳定性。我们先前已经证明，在TCR2的V(D)J重组过程中，p38MAPK在双阴性(DN)3胸腺细胞中被激活，并有助于诱导G2/M细胞周期检查点。此外，p38MAPK还可提高DN3胸腺细胞的存活率。我们最近的研究表明，p38MAPK在一个未被鉴定的残基(Ser389)上使GSK32磷酸化，这种磷酸化使GSK32失活。GSK32的失活与存活率的增加有关。我们认为，p38MAPK是通过V(D)J介导的DSB引发的DNA损伤反应(ATM激活)在DN3胸腺细胞中激活的，不仅通过诱导细胞周期检查点来促进信号末端的修复和防止信号末端整合，而且还通过灭活GSK32来促进生存。我们将研究是否：1)在DN3胸腺细胞中p38MAPK的激活是由ATM介导的V(D)J介导的DSB(目标1)所触发的DNA损伤反应的结果；2)p38MAPK通过磷酸化和失活GSK32向DN3胸腺细胞提供生存信号(目标2)；3)激活p38MAPK促进信号末端的修复并减少信号末端片段在基因组中的整合(目标3)。公共卫生相关性：DNA损伤和基因组不稳定显然与恶性肿瘤的发展有关。为了防止基因组不稳定，细胞通常会经历细胞周期检查点(G1/S和G2/M检查点)，这些检查点会推迟正常的周期，以便有时间进行DNA修复。尽管紫外线和电离辐射以及化疗药物是DNA损伤的主要诱因，但发育中的T和B细胞在经历相应的T细胞受体(TCR)或BCR基因重组的同时，仍存在基因组不稳定性。在这个过程中，V(D)J重组，即带有非保护性双链断裂(信号端)的DNA片段从基因组中被切除。这些信号末端DNA片段对T和B细胞可能是一种危险，因为它们可以攻击其他双链DNA，并可以随机整合到基因组中，导致潜在的恶性肿瘤。T细胞和B细胞白血病的发生率相对较高，通常与染色体易位、缺失和插入有关，这些易位、缺失和插入会影响特定原癌基因的表达或活性。因此，在T和B细胞受体的产生过程中建立检查点对于维持基因组的稳定性是很重要的。我们建议研究p38 MAPK通路在阻止TCRb V(D)J重组导致的未成熟胸腺细胞(DN3胸腺细胞)中信号末端片段整合到基因组中的作用，通过促进细胞周期检查点的诱导(主要是G2/M)并提供生存。P38MAPK通路的药物抑制剂目前正在进行临床试验。如果我们的假设是正确的，这些抑制剂应该主要在T细胞发育高度活跃的婴儿中避免使用。此外，可能影响这一途径的基因突变可能决定了发生T细胞恶性肿瘤的相对较高的易感性。

DESCRIPTION (provided by applicant): The p38 MAP kinase (MAPK) pathway is activated in response to stress stimuli (e.g. ionizing irradiation) that induce DNA double stranded breaks (DSBs) and plays a role in the induction of cell cycle checkpoints (primarily G2/M) to facilitate DNA repair. DSBs are also generated during the development of T cells while undergoing V(D)J recombination in the thymus. V(D)J-mediated DSBs (coding and signal ends) trigger a DNA damage response and DNA repair similar to those induced by DSBs generated by ionizing irradiation. Although joining of coding ends is critical for the formation of functional TCR, repairing of signal ends flanking the excised DNA fragment is also important to prevent integration of the DNA fragments randomly in the genome and genomic instability. We have previously shown that p38 MAPK is activated in double negative (DN)3 thymocytes undergoing V(D)J recombination of the TCR2 and contributes to the induction of a G2/M cell cycle checkpoint. In addition, p38 MAPK also provides survival to DN3 thymocytes. Our recent studies show that p38 MAPK phosphorylates GSK32 at a non-previously characterized residue (Ser389) and this phosphorylation inactivates GSK32. Inactivation of GSK32 is associated with increased survival. We propose that p38 MAPK is activated in DN3 thymocytes by the DNA damage response (Atm activation) triggered by V(D)J-mediated DSBs and contributes to repairing of signal ends and prevention of signal end integration not only by inducing a cell cycle checkpoint, but also by promoting survival through inactivation of GSK32. We will investigate whether: 1) activation of p38 MAPK in DN3 thymocytes is mediated by ATM as a result of the DNA damage response triggered by V(D)J-mediated DSBs (Aim 1); 2) p38 MAPK provides survival signals to DN3 thymocytes by phosphorylating and inactivating GSK32 (Aim 2); 3) activation of p38 MAPK promotes the repair of signal ends and diminishes integration of signal end fragments in the genome (Aim 3). Public Health Relevance: DNA damage and genomic instability are clearly associated with the development of malignancies. To prevent genomic instability, cells normally undergo cell cycle checkpoints (G1/S and G2/M checkpoints) that delay the normal cycle to allow time for DNA repair to occur. Although UV and ionizing irradiation as well as chemotherapeutic drugs are the major inducers of DNA damage, genomic instability is continuously present in developing T and B cells while undergoing recombination of their corresponding T cell receptor (TCR) or BCR genes. During this process, denominated V(D)J recombination, fragments of DNA with non-protected double stranded breaks (signal ends) are excised from the genome. These signal end DNA fragments can be a hazard for T and B cells since they can attack other duplex DNA and can randomly integrate into the genome, causing potential malignancies. The frequency of T and B cell leukemias is relatively high and often these malignancies have been associated with chromosomal translocations, deletions and insertions that affect the expression or activity of specific proto-oncogenes. The establishment of checkpoints during the generation of T and B cell receptors is therefore important to maintain genomic stability. We propose to investigate the role that the p38 MAP kinase pathway has in preventing integration in the genome of signal ended fragments resulting from TCRb V(D)J recombination in immature thymocytes (DN3 thymocytes), by promoting the induction of a cell cycle checkpoint (primarily G2/M) and also providing survival. Pharmaceutical inhibitors of the p38 MAPK pathway are currently in clinical trials. If our hypothesis is correct, these inhibitors should be avoided primarily in infants where T cell development is highly active. In addition, genetic mutations that may affect this pathway could determine a relatively higher susceptibility to develop T cell malignancies.