Understanding ALT activation in C. elegans and human cells

描述（由申请人提供）：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，端粒缩短，因此作为细胞分裂的功能而腐蚀。这种机制限制了细胞分裂的次数，代表了一种强大的肿瘤抑制途径。癌细胞为了获得永生，必须通过激活两种可能的端粒维持机制中的一种来绕过与复制相关的端粒缩短。大多数癌细胞开启端粒酶途径，依赖于端粒酶催化亚基的上调，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，端粒酶是激烈的研究和靶向方法的主题。癌细胞的一个亚群，通常起源于间充质细胞，在端粒酶活性缺失的情况下维持它们的端粒。这些细胞通过激活端粒选择性延长途径（ALT）来避免端粒缩短。ALT是端粒之间重组的结果，导致DNA合成，从而增加长度，避免端粒极度短。虽然已知ALT需要重组，但ALT是如何被激活的，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，这些都是完全不清楚的。此外，越来越多的证据表明，ALT可以被激活作为端粒酶抑制的一种抵抗机制，指出在端粒长度靶向机制成为有效和广泛应用的癌症治疗之前，端粒酶和ALT都必须被理解和抑制。以前没有实验模型可以在受控环境中诱导ALT。在上次拨款期间取得的进展已经在线虫和哺乳动物细胞中提供了这种模型。在这个更新应用的三个具体目的中，我们提出利用这些模型来进一步了解ALT的激活和调控。首先，我们将破译秀丽隐杆线虫中ALT激活的分子步骤，并研究ALT诱导后端粒复合物的变化。其次，重点将放在RTEL1上，RTEL1是ALT的调节因子，在过去的资助期研究中出现，是ALT维持的核心因素。第三，我们将研究复制叉停滞导致有利于ALT激活的染色质环境的假设。总之，本提案旨在利用哺乳动物和线虫的ALT激活模型来了解这一重要途径。

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.