Understanding ALT activation in C. elegans and human cells

描述(申请人提供)：端粒保护人类染色体免受降解、融合和其他酶的攻击。在复制周期中，端粒变短，因此随着细胞分裂而受到侵蚀。这种机制限制了细胞分裂的次数，代表了一条强大的肿瘤抑制途径。癌细胞为了获得永生，必须绕过复制相关的端粒缩短，并通过激活两种可能的端粒维持机制中的任何一种来做到这一点。大多数癌细胞启动端粒酶途径，依赖于端粒酶催化亚单位的上调，端粒酶是一种逆转录酶，专门延长染色体末端的TTAGGG重复序列。因此，端粒酶活性是大多数癌细胞的标志，端粒酶是密集研究和靶向方法的主题。一类癌细胞亚群，通常来自间充质细胞，在端粒酶活性缺失的情况下维持其端粒。这些细胞通过激活另一种端粒延长途径(ALT)来避免端粒缩短。ALT是端粒之间重组的结果，导致DNA合成，从而导致长度增加，从而避免极端短的端粒。虽然已经知道ALT需要重组，但ALT是如何激活的，ALT是如何维持的，以及为什么一些肿瘤类型更喜欢激活ALT而不是端粒酶，目前还完全不清楚。此外，有证据表明ALT可以被激活作为抵抗端粒酶抑制的机制，指出在靶向端粒长度机制成为一种有效和广泛应用的癌症治疗之前，必须了解并抑制端粒酶和ALT。在此之前，没有实验模型可以在受控环境中诱发ALT。上一次赠款期间的进展为线虫和哺乳动物细胞提供了这样的模型。在这次更新应用的三个具体目标中，有人建议利用这些模型来加深我们对ALT激活和调节的理解。首先，将破译线虫ALT激活的分子步骤，并研究ALT诱导后端粒复合体的变化。其次，重点将放在RTEL1上，这是一种ALT的调节因子，在过去的赠款期间的研究中出现，作为ALT维持的核心因素。第三，将对复制分叉停滞导致有利于ALT激活的染色质环境的假设进行调查。综上所述，这项提议旨在利用哺乳动物和线虫中ALT激活的模型来理解这一重要途径。

DESCRIPTION (provided by applicant): Telomeres protect human chromosomes from degradation, fusion, and other enzymatic attack. During the replicative cycle telomeres shorten and therefore erode as a function of cell division. This mechanism limits the number of times cells can divide, representing a powerful tumor-suppressive pathway. Cancer cells, in order to acquire immortality, have to circumvent replication-associated telomere shortening and do so by activating either one of two possible telomere maintenance mechanisms. Most cancer cells switch on the telomerase pathway, dependent on the upregulation of the catalytic subunit of telomerase, a reverse transcriptase that specifically elongates the TTAGGG repeats at chromosome ends. Consequently, telomerase activity is a hallmark of most cancer cells and telomerase is the subject of intense research and targeting approaches. A subgroup of cancer cells, frequently of mesenchymal origin, maintains their telomeres in the absence of telomerase activity. These cells manage to avoid telomere shortening by activating Alternative Lengthening of Telomeres pathways (ALT). ALT is an outcome of recombination between telomeres, leading to DNA synthesis and consequently to length gains, avoiding critically short telomeres. While it is known that recombination is required for ALT, it is entirely unclear how ALT is activated, how ALT is maintained and why some tumor types prefer to activate ALT instead of telomerase. Furthermore, evidence is emerging that ALT can be activated as a resistance mechanism to telomerase inhibition, pointing out that both telomerase and ALT have to be understood and inhibited, before the targeting of telomere length mechanisms can become an effective and widely used cancer therapy. Previously no experimental models existed where ALT can be induced in a controlled environment. Progress during the last grant period has provided such models in nematodes as well as in mammalian cells. In the three specific aims of this renewal application, it is proposed to take advantage of these models to further our understanding of ALT activation and regulation. First, the molecular steps of ALT activation in C. elegans will be deciphered and changes in the telomeric complex upon ALT induction will be investigated. Second, the focus will be on RTEL1, a regulator of ALT that emerged from research during the past grant period, as a central factor in ALT maintenance. Third, the hypothesis will be investigated that replication fork stalling leads to a chromatin environment that favors ALT activation. In summary, this proposal is designed to take advantage of models for ALT activation in mammals and nematodes to understand this essential pathway.