Genomics & Proteomics：揭开癌症遗传学之谜
新的甲基化分析方法工作原理如下：重亚硫酸盐处理可以将未甲基化的胞嘧啶残基转换为尿嘧啶，而对已经甲基化的胞嘧啶则不起作用。在研究过程中，研究人员特意为基因组中的每一个CpG岛设计了等位基因特异性寡核苷酸(ASO)和基因座特定寡核苷酸(LSO)两对探针，当探针与处理过的DNA杂交时，就会得出很高的特异性分析结果。甲基化分析最大的优势在于，它能在一个反应中与多达1,536个CpG岛反应，实现对甲基化特异性PCR方法的竞争，后者曾被看作甲基化模式的金标准，但它一次只能测量一个基因。
因为ASO和LSO探针在基因组中的杂交非常接近，连接作用之后就会发生扩散，连接作用将两个探针连接在一起，使得它们进行PCR的定向扩增。之后，PCR利用荧光标记的通用引物对连接产物进行扩增，并且将PCR产物与Illumina公司的珠阵列技术杂交。对来自每一个结合产物的荧光进行测量，结果可以用于确定一个CpG位置是完全甲基化，半甲基化，还是未甲基化。甲基化分析的结果称作β，是以荧光信号Cy3（绿色）于Cy5（红色）之比计算得出的结果，如果全部都是绿色信号，表示未甲基化，而全为红色则表示完全甲基化。
表观遗传学的目标
虽然有许多方法可以对一种特异性的甲基化基因进行精确检测，但还不能证明它就是引起癌症的原因。Baylin说道：“当你看到这些基因中的甲基化现象，你必须进一步去证明这些基因就是引起癌症的原因，或者证明它们在这一过程中起了重要作用。”换句话说，研究人员必须证明这些功能的中断，或者是通过遗传上的突变，或者是通过表观上的DNA甲基化或染色质修饰，导致癌症的发生以及进一步恶化。对表观遗传学来说，这是一个巨大的挑战。
另一个主要的挑战是了解癌症细胞中异常基因表达的逆转过程。Baylin认为，采用一些表观治疗策略，使那些肿瘤抑制基因再次出现逆转是可能实现的。目前，一些大型制药公司与学院研究人员一起合作，以确定这种方法是否可以作为癌症治疗的方法。
实际上，美国FDA已经批准了以抑制表观机制为原理的抗癌试剂。SuperGen公司首席科学家DavidBearrs称：“根据DNA甲基化和癌症发育之间的相互关系，我们正在开发特异性的，有效的癌症治疗方法。”SuperGen公司购买了MGI制药公司的骨髓增生异常综合征治疗药物地西他滨（decitabine，Dacogen）的全球独家许可权，期望开发出新的治疗方法。
地西他滨属于DNA甲基转移酶抑制剂，针对的是DNA甲基化酶，该酶催化甲基团使其添加到胞嘧啶残基的C-5位置。地西他滨药物是一种胞嘧啶类似物，在复制细胞中掺入到DNA链中，替代正常的胞嘧啶。经过修饰后，DNA甲基转移酶不能使这种类似物(2-脱氧，5-氮杂胞嘧啶)甲基化。Bearrs称：“我们的目的是去了解，作为一种癌症治疗方法，我们如何影响甲基转移酶持续途径，即使是癌症期间胞嘧啶残基的新生甲基化过程。”
采用地西他滨的类似药物，对肿瘤细胞可能产生多效性。也就是不止针对一种蛋白质或者途径。尤其，采用低甲基化试剂如地西台宾，通过关闭DNA甲基化作用，已经可以定靶于肿瘤抑制基因的表达。Bearrs说：“因此，如果我们能通过抑制DNA甲基化，将癌症细胞中的那些基因逆转过来，我们就能在逆转致癌表型的实验中获得阳性结果。”
因此，FDA只允许地西他滨用于骨髓增生异常综合症的治疗，该疾病是髓性白血病的前体。然而，Bearrs认为，地西他滨不仅仅只限于液态肿瘤的使用，与地西他滨类似的药物已经用于固态肿瘤的研究。他预测，这些研究中的低甲基化试剂很可能与其他的复合物结合使用，如组蛋白脱乙酰基酶抑制剂可以阻断表观事件，而另一种调节机制――组蛋白脱乙酰作用能活化特殊基因的表达。 (生物技术世界)
英文原文：
Genomics & Proteomics
Untangling Cancer Genetics
It’s been a long road, but the role of epigenetics in cancer research is more important than ever.
James Netterwald, PhD, MT (ASCP)
Senior Editor
For the past few decades, researchers have viewed cancer as a genetic disease, with many focused on determining the relationship between the molecular biology of
mutations and cancer. This research, which is focused on sequence-based heritable changes, has only been ongoing since the 1980s. At the same time, the field of epigenetics, which is focused on non-sequence-based heritable changes, was brewing.
“The field of epigenetics was really a dark horse,” says Peter Laird, PhD, associate professor of biochemistry and molecular biology at the University of Southern California, Keck School of Medicine Norris Comprehensive Cancer Center, Los Angeles. “[Epigenetics] was not really actively pursued by many investigators.”
According to Laird, epigenetics helps explain why, for example, our liver cells are not the same, functionally, as our brain cells, despite the fact that both cell types are derived from a single fertilized egg. So, epigenetics plays a role in controlling cell behavior, including that involved in cancer. In fact, many of the early papers in epigenetics tried to describe some of the epigenetic changes found in tumors.
But up until the mid-1990s—when Laird entered the field—investigators were only looking for the presence of methylation changes in cancer cells. Surprisingly, they had not attempted to look for a causal relationship between cancer and these changes. The relationship did not exist until Laird and others decided to look more directly for the cause and effect. For example, in Laird’s studies, he took noncancer cells, induced DNA methylation and then asked: what is the effect of these changes on cancer?
“So I was flipping things around to help me to see if these methylation changes were just mere byproducts or really causal contributors. I published a paper in 1995 showing that when you inhibit the ability of mice to lay down methylation changes, you can almost completely block intestinal polyps from forming in a mouse model for a human colorectal cancer,” says Laird.
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