by Jeffrey M. Perkel
The classical view of cancer was of a progressive series of mutations conferring growth advantages, such that the cell is no longer constrained by the biochemical limitations of other, normal cells. No growth factor, no problem.
Today, that perspective has changed. For one thing, the cancer stem cell hypothesis suggests that not every tumor progenitor is equally capable of creating new tumors, an idea with dramatic implications for anti-cancer therapy. In addition, researchers have come to appreciate the critical role of epigenetics—non-sequence-based, but no-less-heritable factors—in development in general, and cancer in particular.
Epigenetic changes include chemical modifications of both DNA and histones (the protein scaffolds about which nuclear DNA winds), as well as the expression of non-coding RNAs. DNA methylation, for instance, is typically associated with decreased gene expression, and cancerous cells often alter DNA methylation patterns to up- or downregulate genes, such as the tumor-suppressors APC and p16, which might otherwise inhibit cancer progression.1
Naturally, drug developers and diagnostics firms have taken note. Drugs like decitabine (Dacogen), an analog of the DNA nucleotide cytosine, specifically target the DNA methyltransferases that induce these epigenetic changes. Recently, Germany biotech firm Epigenomics won European approval to market its Epi proColon diagnostic for colon cancer screening. The test uses PCR to detect methylated septin-9 gene sequences in human plasma.
For basic researchers looking to identify the next biomarker or inhibitor, tools are key. Fortunately, there exists a wide array of products2 to help.
As an application of DNA methylation assays, "I think oncology is fairly highly rated," says ActiveMotif Product Manager Kyle Hondorp. "Right now, people are trying to look at differences, to take samples from different specimens and compare and contrast and start to build profiles, and so the application of these tools and techniques to do that is really valuable."
DNA methylation refers to the chemical coupling of a methyl group to the cytosine residue in CpG dinucleotides by DNA methyltransferases, creating 5-methylcytosine (5-meC). Often, these dinucleotides cluster to form CpG islands, which frequently serve regulatory roles and may be several hundreds to thousands of nucleotides in length.
The problem is, DNA methylation is more or less invisible to most molecular biology methods (other than restriction endonuclease digestion). For instance, says Sallie Cassel, marketing director for antibodies and immunoassay kits at Millipore, "when you [PCR] amplify DNA that has been methylated, you lose the methylation mark." How then can you detect it?
The answer, in most cases, is bisulfite conversion. Assays based on bisulfite conversion, Cassel says, represent "the lion's share of the market."
In a bisulfite conversion, methylated cytosine residues remain unchanged, whereas unmethylated cytosine is chemically altered to uracil. Thus, the sequence A(meC)GTTCC would, following bisulfite treatment, become ACGTTUU, a difference easily distinguishable by sequencing, PCR, and melting point analyses.
Commercial bisulfite conversion kits include the MethylDetector™ Bisulfite Modification Kit from ActiveMotif, the DNA Methylation Detection Kit from BioChain, Life Technologies' MethylCode Bisulfite Conversion Kit, Millipore's CpGenome Fast DNA Modification Kit, Qiagen's EpiTect Bisulfite Kits, and Zymo Research's EZ DNA Methylation™ kits.
According to BioChain Chief Technical Officer Dongyuan Xia, one popular approach to testing the DNA following conversion is PCR. "When the DNA is methylated, it is not converted; if it is not methylated, the sequence changes. So if you design a primer that targets the unconverted sequence, [amplification] means the sequence was methylated."
This type of PCR is called methylation-specific PCR (MSP), and both Millipore and Roche support it.
Millipore's two-dozen or so CpG WIZ amplification kits employ separate PCR primer pairs to amplify the modified and unmodified forms of specific genes, such as the tumor suppressors p16 and VHL. Because bisulfite conversion modifies the sequence of unmethylated regions, these primers can distinguish the different forms via their ability to hybridize to the sequence, the results of which can be visualized on a DNA gel.
Users can also use methylation-specific high-resolution melting (MS-HRM) analyses to assess methylation content of bisulfite-converted DNA. Like MSP, MS-HRM is a PCR-based assay, but its primers are designed to amplify both methylated and unmethylated sequences equally, in contrast to MSP, says Alex Pierson, a technical services consultant for LightCycler® systems at Roche Applied Science. Sequence differences between the two amplified forms produce distinct melting profiles, which can be detected and quantified in the Roche LightCycler 480 via the release of intercalated fluorescent dyes during amplification and comparison to standard curves.
According to Pierson, MSP can typically detect methylation differences of as little as 1% of a sample (that is, one methylated copy in 100 unmethylated forms). "For MS-HRM, people can get down to 0.1%," he says.
Other bisulfite-based methods provide nucleotide-level resolution. These include Illumina's Infinium and GoldenGate methylation assays and next-generation DNA sequencing (a method called BS-Seq). Qiagen's PyroMark systems use Pyrosequencing (the same fundamental chemistry Roche uses in its 454 next-generation sequencers) to read bisulfite converted DNA.
Pyrosequencing couples the release of pyrophosphate during DNA synthesis to a luciferase reaction. As nucleotides are sequentially added to the reaction, the resulting light emission is charted, producing a Pyrogram from which the starting sequence may be read.
According to Peter Urbitsch, director of global business applications at Qiagen, PyroMark "tells you precisely the percentage of methylation" at each site in a sample. "This is especially important," he adds, "because [methylation] is not a yes or no answer; the level of methylation can change during the course of disease."
But Robert Brazas, global product manager for arrays and reagents at Roche NimbleGen, says that in many cases, nucleotide-level methylation detection is unnecessary, because methylation occurs over large regions, and any given CpG may or may not be methylated in any given sample. "If you are looking at a particular nucleotide, you may detect it being methylated in one sample, but not in all of them," he says.
Other methylation analyses avoid the use of bisulfite conversion. The Methyl-Profiler™ kits from SABiosciences (a Qiagen company), for instance, use restriction enzymes and real-time PCR to distinguish methylated from unmethylated regions of unconverted DNA in each of 24 or 96 genes in pathway or disease-focused gene panels. Thirteen panels currently exist, including panels for tumor suppressor genes in general, or for genes implicated in breast, gastric, liver, lung, prostate, and colon cancers in particular.
The Methyl-Profiler process involves four restriction enzyme digestions: one cuts only in the presence of methylation, one cuts only in the absence of methylation, one reaction contains both enzymes, and one contains none. These four reactions are then loaded into a 96- or 384-well PCR plate, each well of which contains one of either 24 or 96 gene-specific primer pairs. The resulting amplification patterns indicate the extent of methylation across each region, says Jeffrey Hung, senior marketing director for SABiosciences.
"The primers are targeting specific CpG island regions," Hung explains; the analysis "will tell you whether or not a region is unmethylated, hypermethylated, or partially methylated for each gene in the panel."
Another bisulfite-free option uses enrichment to purify methylated DNA prior to analysis using either antibodies (a process called methyl-DNA immunoprecipitation, or MeDIP) or methyl-CpG-binding domain (MBD)-containing proteins. ActiveMotif's MethylCollector Ultra kit uses two MBDs to capture fragmented, methylated DNA from its unmethylated counterparts, a process called MIRA (Methylated CpG Island Recovery Assay). A companion assay, the UnMethylCollector kit, selects for unmethylated DNA.
These enriched DNAs can then be analyzed, either target-by-target (using PCR) or globally via next-gen sequencing or microarrays. Sequencing, says Hondorp, is growing as a downstream detection application. But microarrays also remain popular, and Agilent Technologies, Illumina, Affymetrix, and Roche NimbleGen all offer off-the-shelf products for methylation analysis.
NimbleGen's 3x720K CpG Island Plus RefSeq Promoter Arrays (available for human, mouse, and rat research), for instance, contain three identical, 720,000-element microarrays per slide, each of which tiles each gene promoter and CpG island at 100-base resolution. "It's a great balance between genome content while at the same time increasing sample throughput three-fold and decreasing the cost per sample," says Brazas,.
Of course, not all DNA methylation work focuses on the DNA itself. Methylation is an enzyme-catalyzed process, and some researchers are interested in identifying specific inhibitors.
ActiveMotif's DNMT Activity/Inhibition Assay, released in January, is a plate-based, colorimetric assay of DNA-methyltransferase (DNMT) activity in cell extracts. According to Hondorp, the assay quantifies an extract's ability to methylate a universal substrate based on detection of the resulting methylated DNA, first by polyhistidine-tagged MBDs, and then by enzyme-linked anti-His-tag antibodies.
"If somebody is trying to do DNA methylation studies and looking for inhibitors," says Hondorp, "that is one way to have an end-result."
References
1 R. Humeniuk et al., "Molecular targets for epigenetic therapy of cancer," Curr Pharma Biotechnol, 10:161-5, 2009.
2J.M. Perkel, "DNA methylation analysis," Biocompare.com, 5 October 2009.