Polymerase Chain Reaction

PCR is one technique that has revolutionized the applicability of gene-based technologies to clinical medicine. It permits the identification and amplification of DNA sequences from nearly any source, as long as part of the sequence is known. Therefore, any known DNA sequence can be detected in any sample containing genetic material, including tumors.

Initially, two oligonucleotide primers (short pieces of synthetic DNA with sequences designed to stick to regions flanking the DNA sequences to be amplified) are designed and synthesized chemically. DNA containing the target sequence to be amplified is mixed together with oligonucleotide primers (often referred to as oligos), free nucleotides, and heat-stable DNA polymerases. Each cycle of PCR requires heating the mixture to melt double-stranded DNA into two single strands, cooling to anneal or “stick” primers to the template DNA to be amplified, synthesis of a new complementary strand from free nucleotides, and finally, melting and annealing to separate new from old strands of DNA. Each cycle results in doubling of the initial template quantity. After 10 cycles of PCR, the original template DNA is amplified approximately 100 times, and after 20 cycles, approximately 1 million times. Once a PCR product is separated on a gel, DNA fragments of expected size are collected, and the nucleotide sequence is checked to verify amplification of the correct target.

PCR can be used to detect or quantify particular DNA sequences in a sample, for example, to detect the presence of viral sequences or to determine whether a tumor sample contains known sequences or a known genetic alteration. PCR products can also be used to generate purified DNA of known sequences for use in the construction of novel DNA molecules. Perhaps most important, PCR is now used routinely to detect new mutations in amplified or deleted regions of tumor DNA and new mutations in known or putative tumor suppressor genes and oncogenes.

DNA Construction and Gene Expression

Once DNA products are isolated from PCR experiments, their function can be studied. One simple way to study the function of a gene is to insert its encoding DNA into a cell. The transcriptional machinery of the cell can then transcribe the DNA to RNA, which is then translated to protein. If the product is an oncogene, for example, introduction of its DNA into cells may induce unrestrained proliferation of these cells.

Transfection

Polynucleotides may be introduced into cells in a number of ways. The most primitive and least effective way involves simply incubating “naked” DNA (or other types of nucleotides) in cell culture, where they are eventually taken up by cells and transcribed. Other techniques require DNA construction and insertion of the DNA into a cloning vector through the use of restriction enzymes and ligation techniques. Plasmids gain easy access into most types of cells, and the DNA carried within a plasmid can be replicated and transcribed, its RNA translated, and eventually the protein product of the gene expressed within a given cell. The process of incorporating DNA into cells growing in tissue culture is called transfection. By using transfection techniques, the function of a gene can be gleaned by observing the phenotypic changes that occur in a cell after transfection with a certain type of nucleotide construct.

Viral transfection or, more appropriately, viral infection of cells is particularly useful with animal cells and is a very efficient means of delivering genes to the cells in question. Viral vectors contain powerful promoters that drive expression of the genes incorporated into them. The three most commonly used viral vectors are retroviruses, herpesviruses, and adenoviruses. Retroviruses integrate into the host genome and become permanent in all progeny. The other vectors do not.

Gene Expression in Mice

To verify that a gene defect is the cause of a disease, it is necessary to evaluate the in vivo biologic consequences of genetic alterations. This can be accomplished by using transgenic or knockout mice. Such genetic manipulation of disease-associated genes results in genetically engineered models (GEMs) of disease.⁴ Genetic manipulation in mice is now fairly sophisticated, and gene expression can be induced or halted in animals with remarkable temporal and cell-specific specificity.

Transgenic Mice

Transgenic mice are created by introducing a desired DNA sequence, referred to as a transgene, into fertilized mouse eggs, which are subsequently placed in the oviducts of pseudopregnant female mice. Progeny conceived through this process are derived from the altered oocyte and contain the transgene in all cells in their body. Transgenes contain promoter and regulatory sequences that direct gene expression in one or more tissue types. Transgenes may also include inducible promoter regions so that expression of the transgene can be temporally and spatially controlled by administration of an inducing agent. In most cases, the foreign DNA does not disrupt cellular function, and expression of the transgene occurs only in tissues that use the transgene promoter sequence. Progeny are screened by analyzing the mouse tissue for expression of the desired transgene.

With this technology, human genes can be transferred to animals and their ability to cause disease ascertained. By linking the gene of interest with a tissue-specific promoter, expression can be restricted to a particular organ or cell type. Transgenic mice can also be used to measure the activity of signal transduction pathways or other molecular functions of a cell. For example, p53 activity can be measured by introducing the consensus sequence for p53 binding upstream of the firefly luciferase gene, thereby inducing transcription of the luciferase gene upon p53 binding. The luciferase gene converts a substrate called luciferin into light, and the photons emitted from the reaction can be measured directly by a camera. Emission of light from living organisms is known as bioluminescence. In the p53/luciferase scenario, mice that produce luciferase after p53 binding will convert luciferin into a bioluminescence signal, and this signal serves as an indirect measurement of the DNA binding activity of p53.⁵ The potential applications of transgene technology in mice are virtually unlimited, and glioma mouse models are beginning to use transgenic animals to measure the proliferation rates of tumors, measure the status of tumor signal transduction pathways, and study a host of other events that occur during the genesis of glioma.

Knockout Mice

Creation of knockout mice, in which genes are mutated or disrupted, results in a loss of function. The technology is similar to but more elaborate than transgenic protocols because the gene to be ablated, along with its flanking regions, must be characterized. First, a DNA construct is designed that has sequence homology with the flanking regions of one or more exons of the target gene. In addition to the homologous sequences, the new construct usually includes a gene encoding drug resistance or sensitivity. Once a construct is generated, it is transfected into murine embryonic stem cells (derived from mouse embryos) and then selected for expression of the foreign DNA by using the appropriate drug-based selection strategy. Because of the homology between the vector and the gene being targeted, some cells incorporate the foreign DNA construct via homologous recombination, with the complementary sequences either disrupting or completely replacing the target gene.

Transfected embryonic stem cells are propagated in culture, analyzed to verify the presence of the desired DNA construct, and then injected into developing mouse blastocysts. Once implanted into a pseudopregnant female, the blastocysts continue to develop normally and give rise to chimeric progeny in which some cells and tissues are derived from the original blastocyst and others are derived from the genetically altered embryonic stem cells. All cells derived from embryonic stem cells, including some gametes, have the gene of interest disrupted or replaced with a nonfunctional sequence. Matings between these genetically manipulated animals and wild-type mice result in a few offspring heterozygous for the disrupted gene. Subsequent mating of two heterozygotes results in a percentage of progeny homozygous for the mutation and therefore deficient in expression of the targeted gene.

Knockout mice provide a definitive method for establishing in vivo gene function; loss of a function after deletion of a gene is direct evidence that the gene is responsible for that function. Unlike the transgenic situation, in which the DNA construct is present in every cell but expressed in only certain cell types, in a knockout mouse the gene of interest is missing from every cell, but only cells or tissues in which the gene in question would normally be expressed are phenotypically affected.

Conditional Knockouts

Conditional knockout mice are created by introducing agents that selectively remove genetically engineered sequences from mouse chromosomes.⁴ In one such system, known as Cre-lox, the knockout mice are created as described earlier, except that palindromic loxP sites flank the target gene DNA construct. The Cre recombinase protein identifies loxP sites, and the DNA sequence between flanking loxP sites can be excised by the Cre protein to yield a functional knockout. The advantage of this system is that Cre expression can be spatially restricted to certain cell types through the use of tissue-specific promoters. Moreover, Cre constructs may be engineered so that Cre expression is induced only when certain drugs are administered to an animal expressing the Cre transgene. Such temporal and spatial control of knockout genes enables realistic modeling of carcinogenesis because this system can re-create a multistep biologic situation in which specific genes are deleted or overexpressed at specific junctures during tumor progression.

Somatic Cell Gene Transfer

Somatic cell gene transfer involves the transfer of DNA to nongermline cells, often using retroviruses engineered to contain specific genes.⁴ The concept of somatic cell gene transfer dates back to the discovery of Rous sarcoma virus (RSV) by Rous. Injection of RSV into the organs of several different types of animals results in the formation of tumors because of transfer of the v-src oncogene to somatic cells. However, RSV-based somatic cell gene transfer has some serious disadvantages: (1) there is no control over the type of cells infected by the virus and (2) the use of RSV somatic cell gene transfer allows the examination of only one oncogene.

One somatic cell gene transfer system that obviates these problems is the RCAS/tv-a system.⁴ This system combines transgene technology with viral infection to achieve cell-specific, gene-specific somatic cell transfer of a number of different oncogenes. Replication-competent avian leukemia virus (ALV) family splice acceptor (RCAS) vectors can be engineered to contain specific genes. Moreover, the RCAS virus specifically infects cells that express the ALV type a receptor tv-a. Thus, in transgenic animals engineered to express tv-a only in certain cell types, RCAS vectors can be used to deliver oncogenes in a cell type–specific manner.

Fluorescence In Situ Hybridization

Cancer, especially in its most malignant forms, progresses by accumulating multiple errors in the genome of affected organisms. Many of the genetic errors occurring in cancer specifically affect genes that normally function to repair damaged DNA. As a result of errors in DNA damage response mechanisms, the entire genome of an affected organism becomes prone to error, thereby leading to a phenomenon called genomic instability. One consequence of genomic instability is that damaged genomic DNA is prone to single- and double-strand breaks. At a chromosomal level, these breaks can result in very large losses, gains, or translocations of chromosomal regions. These chromosomal areas encode genes or regulatory elements of genes involved in cancer initiation and progression, and identification of these abnormal chromosomal regions is important for a general understanding of cancer biology. As a consequence, cytogenetics, which encompasses the study of chromosomal structure, has become a central part of cancer biology.⁶

Before the advent of molecular cytogenetic techniques, the primary method for studying chromosomal aberrations was direct visualization of chromosomes during the metaphase period of the cell cycle. Chromosome condensation during this time allows the highly compact metaphase chromosomes to be directly visualized with light microscopy. Chromosomal spreads—metaphase or interphase chromosomes placed on glass slides—can distinguish unique properties of individual chromosomes and allows chromosome regions to be identified. Aberrant chromosome structures, such as those found in Down’s syndrome, can therefore be readily identified by looking at chromosome spreads. This technique allowed proper enumeration of diploid human chromosomes and also identification of the chromosome numerical aberrations observed in Kleinfelter’s and Down’s syndromes. However, gross cytogenetics fails to provide the resolution required to identify the small regions of DNA amplification or deletion that are frequently encountered in neoplastic diseases.

Molecular cytogenetics is an amalgamation of molecular biology and the conventional cytogenetic methods described earlier. The prototypical molecular cytogenetic technique, fluorescence in situ hybridization (FISH), is now widely used for the identification of chromosomal aberrations in cancer genomes. FISH techniques can easily identify large chromosomal abnormalities such as rearrangements and deletions; by using modern FISH techniques investigators can occasionally identify single nucleotide abnormalities in chromosomes. FISH requires the use of a probe, a synthetic oligonucleotide sequence that corresponds to a known region of the genome. The probe is then hybridized to the target DNA (usually genomic DNA of the researcher’s interest). During synthesis of the probe, modified nucleotides containing fluorophores or antigenic sites are used to create a modified oligonucleotide. Oligonucleotide probes modified with fluorophores can be visualized directly because of the fluorescence emitted by the probes; alternatively, antigen-modified oligonucleotides can be visualized by immunologic or enzymatic reactions. After synthesis of the probe, both the probe and target DNA are denatured and then mixed to allow binding of the probe and target DNA. Structurally normal target DNA will exhibit a signal at the expected site in the chromosome, whereas rearranged or deleted target DNA will exhibit a signal in an aberrant location or no signal at all, respectively.

Array Platform Technology

Modern molecular genetics is aided by the availability of high-throughput systems that permit concurrent analysis of thousands of genes or gene products. A DNA microarray is one such tool and consists of solid platforms that contain thousands of spots of immobilized complementary DNA (cDNA) or genomic DNA oligonucleotides. The identity of each spot on the array is known and the DNA is labeled with fluorescent tags. In a typical experiment, genomic DNA or cDNA from experimental samples, called “probes,” are labeled with fluorescent tags that differ from the immobilized “target” DNA on the microarray. The sum of the fluorescent signal obtained from hybridizing the probe and target represents the relative abundance of either the probe or target. Because a laser scanner can analyze the fluorescent signals from array experiments, each experiment can be performed in a relatively short time. DNA microarrays can provide enormously useful information about the expression levels of DNA.

Comparative Genomic Hybridization Arrays

Although FISH has significantly advanced our progress in the discovery of abnormal target genes and chromosomes, the technique is imperfect for solid tumors in that it requires preparation of tumor metaphase chromosomal spreads that do not differ markedly from normal tissue. Because advanced solid tumors acquire many chromosomal abnormalities, it is often impossible to interpret tumor chromosomal spreads with accuracy inasmuch as chromosomes in solid tumors are often altered beyond recognition. To combat this problem, a variant of FISH called comparative genomic hybridization (CGH) was invented in the early 1990s.⁶ CGH obviates the need for chromosome spreads by using differentially labeled test and reference genomic DNA oligonucleotide samples, which can then be applied onto genomic DNA microarrays. CGH arrays permit unprecedented ability to evaluate chromosomal abnormalities in cancer. In particular, this technique is especially sensitive for the identification of changes in DNA copy number, such as the increases in gene copy number that occur at the epidermal growth factor receptor (EGFR) locus in a significant number of glioblastomas.

Single Nucleotide Polymorphism Genotyping Arrays

FISH and CGH techniques improve the analysis of gross chromosomal abnormalities (especially changes in copy number) that occur in cancer. However, comprehensive analysis of cancer genetics must address other specific events such as deleterious point mutations and the sequential loss of tumor suppressor alleles (loss of heterozygosity [LOH]) because these events are key contributors to the initiation and progression of cancer. Large-scale identification of point mutations and LOH requires the ability to scan large swaths of tumor genomes with high resolution.

Single nucleotide polymorphism (SNP) analysis is a powerful molecular genetic tool capable of identifying changes in DNA copy number, point mutations, and LOH.⁶ SNPs are nonfunctional point mutations in the genome that occur at a frequency of about 1% in the human genome. They occur at a frequency of one SNP every few hundred base pairs of genomic DNA. Because many SNPs have been sequenced, they can be used to compare the haplotypes of cancer genomes with other nontumor DNA from the same individual. Moreover, because the genomic location of SNPs is known, changes in the SNP genotype of tumor DNA identify genomic regions that can be further analyzed by PCR to detect precise DNA aberrations (point mutations, deletions, insertions, and so on) that underlie the changes in SNP haplotype. Furthermore, because of the availability of microarray platforms, up to 500,000 SNPs can be analyzed in a single experiment, thus allowing excellent genomic resolution.

Gene Expression Arrays

CGH, FISH, and SNP techniques identify changes in genomic DNA. Although these platforms can be used to make inferences about gene expression, they do not provide direct data about the transcription of these genes from DNA to messenger RNA (mRNA). The importance of studying changes in gene expression is aptly represented by the expression pattern of vascular endothelial growth factor (VEGF) during the genesis of glioma. Although the VEGF gene is rarely mutated in glioblastoma multiforme (GBM), VEGF mRNA and protein levels are often markedly elevated, which leads to an increase in tumor angiogenesis and virulence.⁷

Gene expression arrays are now widely available. In practice, gene expression array techniques are identical to the CGH and SNP arrays, except that cDNA (transcribed from mRNA) is most frequently used as a substrate for hybridization. Currently, up to 19,000 unique genes can be analyzed for their expression levels in a single array experiment.

Applying Molecular Genetic Tools to Glioma Analysis

Despite the many technologic advances in the molecular characterization of gliomas, there was, until recently, a dearth of data linking molecular genetic changes to clinical outcomes for gliomas. These shortcomings partially resulted from studies that relied on single molecular genetic techniques and a small number of glioma samples. Recently, however, a number of studies have used a combination of molecular genetic and other techniques to analyze large samples of GBM, and the results from these studies are promising.

Phillips and colleagues recently used both gene expression and CGH profiling of high-grade gliomas to explore genes that correlate with patient survival.⁸ Using gene expression microarrays, they identified three gene clusters that correlate with patient survival. Consistent with these findings, CGH analysis of patients with shorter survival showed frequent loss of the phosphatase and tensin homologue from chromosome 10 (PTEN) tumor suppressor locus and gains at the EGFR and phosphatidylinositol-3′-kinase (PI3K) oncogene loci in comparison to patients with longer survival.

Perhaps the most comprehensive single genetic analysis study of human gliomas to date comes from the efforts of The Cancer Genome Atlas (TCGA), a national initiative whose goal is to identify and catalogue the wide array of genetic aberrations that cause cancer.⁹ TCGA uses a number of genetic tools, including CGH, SNP genotyping, PCR genotyping, gene expression arrays, microRNA profiling, and DNA methylation arrays, to characterize a cancer specimen. Using 206 GBM samples in a pilot study, the group has identified hitherto unknown genetic aberrations that probably contribute to glioma genesis. In particular, they identified novel deletions of NF1 and PARK2 in glioma samples and characterized NF1