Genomics & Proteomics：揭开癌症遗传学之谜

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Genomics & Proteomics：揭开癌症遗传学之谜 近几十年来，研究人员一直把癌症看作是一种遗传疾病，许多人都把研究重点放在确定突变分子生物学和癌症之间的关系上。自上世纪80年代以来，在关注基于序列的可遗传的变化方面的研究取得了一些进展。同时，表观遗传学领域也日益显露出勃勃生机，该领域关注的是与序列无关的那些遗传变化。南加州大学Keck医学院Norris肿瘤综合中心PeterLaird副教授认为，表观遗传学研究领域是一匹真正的黑马。他说：“许多研究

  近几十年来，研究人员一直把癌症看作是一种遗传疾病，许多人都把研究重点放在确定突变分子生物学和癌症之间的关系上。自上世纪80年代以来，在关注基于序列的可遗传的变化方面的研究取得了一些进展。同时，表观遗传学领域也日益显露出勃勃生机，该领域关注的是与序列无关的那些遗传变化。

  南加州大学Keck医学院Norris肿瘤综合中心PeterLaird副教授认为，表观遗传学研究领域是一匹真正的黑马。他说：“许多研究人员还没有真正的从事表观遗传学的研究。”在基因组中除了DNA和RNA序列以外，还有许多调控基因的信息，它们虽然本身不改变基因的序列，但是可以通过基因修饰，蛋白质与蛋白质、DNA和其它分子的相互作用，而影响和调节遗传的基因的功能和特性，并且通过细胞分裂和增殖周期影响遗传，这些都属于表观遗传学研究的范畴。

  PeterLaird表示，表观遗传学帮我们解释了许多疑问，例如，为何肝细胞的功能与脑细胞的不一样，虽然它们都是由同一个受精卵发育衍生而来的。因此，在控制细胞行为，包括与癌症有关的控制细胞行为方面，表观遗传学扮演了重要的角色。实际上，许多表观遗传学领域早期发表的文章都试图阐述肿瘤中发现的表观遗传变异情况。

  但是直到20世纪90年代，研究人员才开始研究癌症细胞的甲基化变化。令人惊讶的是，人们并没有去研究癌症和这些变化之间的因果关系，是Laird等人首开先河，决定更直接的去研究这种因果关系。例如，在Laird的研究中，他选取了非癌性细胞，包括DNA甲基化作用，然后思考这些变化在癌症中的效应。

  Laird说：“我开始研究这些甲基化作用，分析它们仅仅是副产品，还是真正的原因所在。我在1995年发表的一篇文章中阐述了我的研究结果：抑制小鼠的甲基化作用，就能完全阻止人结肠直肠癌小鼠模型形成肠息肉。”
  聚焦DNA甲基化

  DNA甲基化是表观遗传学的重要组成部分，在维持正常细胞功能、遗传印记、胚胎发育以及人类肿瘤发生中起着重要作用，是目前新的研究热点之一。随着对甲基化研究的不断深入，各种各样甲基化检测方法被开发出来以满足不同类型研究的要求。

  通常来说，DNA甲基化发生在DNA启动子区域的胞嘧啶残基上，尤其是在CpG岛上，绝大多数正常细胞中的基因是不会形成甲基化胞嘧啶核苷酸的，目前认为CpG岛甲基化导致转录抑制是恶性肿瘤发生的重要机制之一，细胞中的甲基转移酶催化了甲基化的形成。

  约翰霍普金斯大学肿瘤学教授StephenBaylin表示，在每一种人类癌症疾病中，都能发现超甲基化基因，包括液体肿瘤和固体肿瘤。然而，研究甲基化和癌症之间的关系，不在于癌症细胞是否有甲基化基因，而是要考虑与癌症有关的基因的数量以及不同的甲基化变异类型。
  因为知道了这些基因的准确位置，就可以采用一种敏感方式――针对肿瘤检测的一种极其有效的生物标记策略――去进行检测。这种策略对风险评估也很有用，因为可以在癌症发育的前期阶段，看到肿瘤上的表观变化。Laird认为，DNA甲基化作为一种早期检测理论，还有许多优点。通过检测体液如血液中的肿瘤DNA，能观察到异常的DNA甲基化模式。但是检测甲基化也并非易事。在上世纪70年代的SouthernBlotting实验中，早期检测方法需要依靠甲基化敏感限制性酶，如HpaII。

  在上世纪80年代期间，研究的主要方法是PCR和克隆，两者都不涉及甲基化内容，因此很快就被弃用了。90年代，Laird开发了两类技术去检测肿瘤细胞中的甲基化模式，MethylLight和COBRA，两者都是以甲基化胞嘧啶的脱氨作用（将胞嘧啶转化为尿嘧啶残基）以及通过分析方法鉴定修改的序列为基础。

  结合重硫酸盐限制性分析法(COBRA法)是由Laird于90年代晚期开发的。这种方法对DNA样品进行重亚硫酸盐处理及PCR扩增，处理后原甲基化的胞嘧啶被保留，而非甲基化的胞嘧啶变为胸腺嘧啶。随后用限制性内切酶对转化后PCR产物切割的特性，识别原标本DNA的甲基化状况。最终对非甲基化敏感的序列进行分析，COBRA方法中使用的限制性酶可以对重硫酸盐处理后诱导的序列变化进行识别。1999年，Laird开发了第二种方法MethylLight，用重亚硫酸盐处理待测DNA片段，然后用特异性实时荧光定量PCR(TaqManPCR)反应进行扩增，识别完全甲基化的序列。Methy—Light是一种高度敏感的分析方法，能够在未甲基化等位基因超出1万倍的情况下检测到甲基化的等位基因；非常精确地检测特定DNA甲基化模式的相对丰度，只需少量的模板DNA。

  Laird表示，需要不断改进相关技术，提高灵敏度，才能更好的对癌症细胞中的甲基化模式进行研究。幸运的是，加州的Illumina公司为此开发了特定的分析方法。Illumina公司在早期的分析方法金门分析法（TheGoldenGateAssay）基础上改良了新的甲基化分析方法。主要差异在于：GoldenGate分析是三探针设计，而甲基化分析是四探针设计。

  Illumina公司的甲基化产品经理VivianZhang说：“就工作流程来说，它们基本上是相同的，只需要对上游的DNA样本进行重亚硫酸盐转变，然后将其加入GoldenGate工作流程平台即可。”


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



The principle of Illumina’s Methylation Assay, the most recent tool for determining the methylation pattern of CpG islands, is illustrated here. See text for detailed explanation. (Source: Illumina Corp.)
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.

Going through changes
DNA methylation occurs on cytosine residues in DNA promoter regions, especially in CpG islands, which, in most genes in most normal cells, are free of methylated cytosine nucleotides. The methylation event is catalyzed by cellular enzymes called DNA methyltransferases, which are commonly found in cells.
“You can find hypermethylated genes in every type of human cancer that I am aware of, including liquid and solid tumors,” says Stephen Baylin, MD, professor of oncology and medicine and associate director of laboratory research at The Johns Hopkins University, Baltimore, Md. However, he says, in terms of the relationship between methylation and cancer, it is not a matter of whether the cancer cells have methylated genes or not, but rather that the number of genes involved and patterns of methylation vary between cancers.
“Because you know exactly where to look in these genes, it makes detecting this in a sensitive way an extremely productive biomarker strategy for tumor detection,” says Baylin. And he goes on to explain that this strategy would also be good for risk assessment because the epigenetic changes seen in tumors can occur early in the pre-stages of cancer development.
“There’s a lot of promise for DNA methylation as an early detection mechanism,” says Laird. “One could look at abnormal DNA methylation patterns by detecting DNA released from tumors in body fluids such as blood.” But detecting a methylation event is not so easy. Earlier detection methods relied on digestion by a methylation-sensitive restriction enzyme such as HpaII, followed by Southern Blotting. That was in the 1970s.
During the 1980s, the methodology shifted to PCR and cloning, both of which erased the methylation events, so these were quickly scrapped. In the 1990s, however, Laird developed two



click to enlarge
The Swi/Snf complex performs its chromatin-remodeling duties, freeing up the DNA for its interaction with activated glucocorticoid receptors (GR). (Source: Gordon Hager, PhD)
technologies for determining methylation patterns in cancer cells, Methyl Light and COBRA, both of which were based on the deamination of methylated cytosines—which converts these cytosines to uracil residues—and identification of the converted sequence by analytical methods.
COBRA—which stands for COmbined Bisulfite Restriction Analysis—was developed by Laird in the late 1990s. The method involves bisulfite treatment of the DNA sample, PCR amplification of the treated DNA, and finally, a restriction analysis that is not methylation-sensitive. The restriction enzyme used in COBRA has a recognition sequence that is affected by the change in sequence induced by bisulfite treatment. In 1999, Laird published a second method, Methyl Light, which involves treating DNA with bisulfite and then amplifying it using specific TaqMan-based PCR reactions to recognize fully methylated versions of the sequence.
Laird says that further advancements based on this technology need to have even greater ability to look at methylation patterns in cancer cells and with greater sensitivity. Luckily, companies like Illumina, San Diego, Calif., have created assays specifically for this purpose. The Methylation Assay Illumina created is an adaptation of one of their earlier assays, The Golden Gate Assay. The main difference: The Golden Gate Assay is a three-probe design, whereas The Methylation Assay is a four-probe design.
“As far as workflow is concerned, they are basically the same…You simply do a bisulfite conversion [of the sample DNA] upfront and then feed that into The Golden Gate workflow platform, which all of our customers are familiar with,” says Vivian Zhang, methylation product manager at Illumina.
Here’s how it works. The bisulfite treatment converts unmethylated cytosine residues to uracil, but leaves methylated cytosines unchanged. Two pairs of probes called allele-specific oligonucleotides (ASO) and locus-specific oligonucleotides (LSO) are designed for each CpG island in the genome under study, giving the assay high specificity when they hybridize to the treated DNA. A major advantage of The Methylation Assay is that it can multiplex up to 1,536 CpG sites in a single run, allowing it to rival the gold standard for methylation profiling, methylation-specific PCR, which can only measure one gene at a time.
Because the ASO and LSO probes hybridize very near each other in the genome, extension occurs followed by ligation, which links the two probes together and makes them targets for PCR amplification. The ligated products are then amplified by PCR using fluorescently-labeled common primers, and the PCR products are hybridized to Illumina’s Bead array technology, which bears the complementary sequence for hybridizing to the PCR product. The fluorescence from each bound product is measured, and the readout can be used to determine whether a CpG site is fully methylated, semi-methylated, or unmethylated. For The Methylation Assay, the readout is called beta, which is calculated from the fluorescent signal ratio of Cy3 (green) to Cy 5 (red), where all green signal means unmethylated and all red, fully methylated.
Epigenetic targets
Although there are methods for accurately detecting a specific methylated gene, this in no way proves causation of cancer. “When you see the methylation in these genes, you have to take some further steps to show that these genes are causative or have a high degree of function,” says Baylin. In other words, researchers have to prove that the disruption of this function, either genetically through mutation, or epigenetically through DNA methylation or chromatin modification, causes the initiation or progression of cancer. And that’s a big challenge in the field of epigenetics.
The next major challenge is learning how to reverse aberrant gene expression in cancer cells. “There is the potential for using some epigenetic therapy strategies to turn those [tumor suppressor] genes on again,” says Baylin. And, he says, major pharma is getting very interested in working with academics to see whether this approach will work as a cancer therapy.
In fact, the US Food and Drug Administration (FDA) has approved anticancer agents that work by inhibiting epigenetic mechanisms. “We are now looking to use knowledge about the relationship between DNA methylation and cancer development to specifically and effectively treat cancer,” says David Bearrs, PhD, vice president and chief scientist at SuperGen, Salt Lake City, Utah. And with the acquisition of MGI Pharma’s decitabine, SuperGen is making this treatment option a reality.
Decitabine targets DNA methyltransferases— the enzymes that catalyze the addition of a methyl group to the C-5 position on cytosine residues. The drug is a cytosine analog that can become incorporated into DNA in replicating cells in place of normal cytosine. Because it is modified, this analog (2-deoxy, 5-azacytosine) cannot be methylated by the DNA methyltransferase as it comes down the DNA strand. “Our goal is to try to understand how we might be able to affect the methyltransferase-maintenance pathway, even the de novo methylation of cytosine residues during cancer, as a treatment for cancer,” says Bearrs.

With drugs like decitabine, he says, there is the possibility of having pleiotropic effects on tumor cells. That is, targeting more than one protein or pathway. In particular, the expression of tumor-suppressor genes—which is turned off through DNA methylation—has been targeted by hypomethylating agents like decitabine. “So if we can turn those genes back on by inhibiting DNA methylation in cancer cells, we think that we could have a positive effect on reversing the tumorigenic phenotypes.”

So far, the FDA has only indicated decitabine for myelodysplastic syndrome—a precursor to myelogenous leukemia. However, Bearrs says the application of decitabine should not be limited to liquid tumors, and that drugs similar to decitabine are already being investigated for solid tumors. He predicts that these investigational hypomethylating agents will likely be used in combination with a class of compounds known as histone deacetylase inhibitors, which block the epigenetic event, histone deacetylation—another regulatory mechanism that can activate the expression of specific genes.