Human Molecular Genetics, 2001, Vol. 10, No. 7 669-675
© 2001 Oxford University Press
Unraveling human cancer in the mouse: recent refinements to modeling and analysis
Departments of Pediatrics and Medicine, UCSD Cancer Center, UCSD School of Medicine, 9500 Gilman Drive, Mail Code 0627, La Jolla, CA 92093, USA
Received 17 January 2001; Revised and Accepted 29 January 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMODELING CANCER IN MICEANALYZING MOUSE MODELS OF...CONCLUSIONREFERENCES |
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The ability to manipulate the mouse genome has made the mouse the primary mammalian genetic model organism. It has been possible to model human cancer in the mouse by overexpressing oncogenes or inactivating tumor suppressor genes, and these experiments have provided much of our in vivo understanding of cancer. However, these transgenic approaches do not always completely and accurately model human carcinogenesis. Recent developments in transgenic and knockout approaches have improved the accuracy of modeling somatic cancer in the mouse and analyzing the genomic instability that occurs in murine tumors. It is possible to use retroviral gene delivery, chromosome engineering and inducible transgenes to selectively manipulate the genome in a more precise spatial and temporal pattern. In addition, the development of powerful cytogenetic tools such as spectral karyotyping, fluorescence in situ hybridization and comparative genome hybridization have improved our ability to detect chromosomal rearrangements. Finally, global patterns of gene expression can be determined by microarray analysis to decipher complex gene patterns which occur in cancers. Several of these advances in mouse modeling of human cancer are discussed in this review.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMODELING CANCER IN MICEANALYZING MOUSE MODELS OF...CONCLUSIONREFERENCES |
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The mouse has been one of the most informative experimental systems for dissecting genetic factors important for mammalian cancer, the consequence of transgenic and knockout techniques that have been developed primarily for the mouse. Using transgenic technology, it is possible to overexpress virtually any gene in a tissue-specific fashion. Applying the powerful technology of gene targeting in embryonic stem (ES) cells, it is also possible to inactivate any gene, or to make more subtle genetic modifications to a gene. With respect to cancer, transgenic mice have been used to overexpress putative oncogenes in a variety of tissues for nearly 20 years and, for the past 10 years, gene-targeting methods have been used to inactive tumor suppressor loci. These studies have resulted in most of our current understanding of cancer in vivo.
Although these techniques have provided great insight, they do not fully model the vast majority of cases of cancer. In general, cancer arises because individual somatic cells sustain a series of sequential mutations that provide a growth or survival advantage, escape from senescence and the onset of genomic instability, thus perpetuating genetic changes that ultimately result in tumor formation. Standard transgenic and knockout techniques do not faithfully model such changes. Transgenic techniques generally result in a somewhat uniform overexpression of a given oncogene in a particular tissue. Germline knockouts in the heterozygous state lead to some rare forms of familial cancers. However, progression to homozygosity often occurs too slowly in mice to result in tumor formation. In many cases, homozygous loss of such tumor suppressors results in embryonic lethality, and the role in cancer cannot be evaluated.
Several recent advances in the modeling of cancer in mice have resulted from the use of novel transgenic and targeting techniques to provide a more faithful representation of the sequential changes which occur in somatic cells during the genesis of human cancer. In addition, new techniques have been developed to investigate mechanisms of genomic instability in the mouse. Several of these new approaches are briefly highlighted in this review (Fig. 1).
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MODELING CANCER IN MICE |
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TOPABSTRACTINTRODUCTIONMODELING CANCER IN MICEANALYZING MOUSE MODELS OF...CONCLUSIONREFERENCES |
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It is now possible to make genetic modifications in the mouse that result in tighter control of gene expression in somatic cells. A number of novel approaches involving bacterial recombinases and recombination recognition sites have been developed to inactivate genes or whole chromosomal regions that harbor tumor suppressor loci in somatic cells of mice. Inducible transgenes and viral expression systems have recently been employed to overexpress oncogenes in the mouse.
Conditional gene inactivation with Cre recombinase
The bacteriophage P1 Cre/loxP recombinase system is currently the most widely used method to conditionally disrupt gene expression. Cre recognizes and catalyzes recombination between 34 bp loxP sites. When the mouse genome is engineered to possess such loxP sites, recombination can be controlled in a precise spatial and temporal pattern by using well-defined promoters to drive Cre after pro-nuclear injection using standard transgenic approaches. These loxP sites can flank an exon, exons or the entire gene and is termed a floxed allele. Cre can be expressed in mice to convert a functional allele to a null allele after recombination occurs. Thus, by controlling the expression of Cre it is possible to control when the knockout is produced. Now that a large number of characterized promoters are available, it is possible to express Cre in a variety of tissues and cells in the mouse, making modeling of cancer simple and efficient (1). It is also possible to use viral systems or inducible systems to deliver Cre to specific regions of a tissue or to more finely control Cre expression (see below).
Cre mediated excision of a number of genes has been used to avoid the embryonic lethal phenotype of null alleles of some tumor suppressor genes. For example, homozygous knockouts of Nf2, Apc, Brca1 and Rb display such lethal phenotypes. When these genes were somatically inactivated in specific targeted cell types and tissues, cancer phenotypes produced in the mouse were similar to those seen in humans. Nf2 is the mouse homolog of the human gene mutated in neurofibromatosis type II. A Schwann cell specific knockout of Nf2 was produced by mating mice with a floxed allele of Nf2 with mice expressing Cre under the control of the P0 promoter (2). These mice developed neurofibromas similar to the human disorder. Apc is the mouse homolog of a human tumor suppressor gene that is inactivated in familial adenomatous polyopsis (FAP). The conditional inactivation of a floxed allele of Apc in the colon of mice by adenoviral transduction leads to the development of adenomas similar to those seen in FAP (3). Brca1 is the mouse homolog of a familial gene important for human breast and ovarian cancer. A breast cancer model was produced by conditionally knocking out Brca1 in the mammary epithelia in the adult using mouse mammary tumor virus-long terminal repeat Cre or whey acidic protein Cre transgenic mice. Brca1 inactivation in the mammary epithelium resulted in breast cancer after a long latency (4). Loss of p53 in these mice resulted in accelerated mammary tumor development and these tumors displayed genetic instability. Rb is the mouse homolog of a human tumor suppressor gene mutated in familial retinoblastoma. A conditional double knockout of p53 and Rb was produced specifically in glial cells (5). These mice developed highly aggressive medulloblastoma that can occur in children with Rb mutations. These studies have shown the value of using Cre mediated excision of tumor suppressors to model human tumorigenesis in the mouse. Such mouse models can now be used to understand cancer development and to screen anti-cancer treatments.
Further refinements to the Cre loxP system will be needed to reduce the leaky and varied expression of Cre, as well as the toxicity observed in some cases after elevated expression of Cre. Some of these problems can be overcome with the use of bacterial artificial chromosomes (BACs) to insulate the transgene from integration effects, or by knocking in Cre to endogenous loci. Such modifications will result in more tightly controlled gene inactivation that will more faithfully model human cancer.
Chromosome engineering
Germline or somatic mutations in tumor suppressor genes of the mouse can be used to model cancer when the tumor suppressor gene is known. However, recurrent chromosomal aberrations or deletions are often seen in many cancers, and these arise at the somatic level. For example, a mutation often occurs somatically in one allele of a tumor suppressor locus, followed by loss of heterozygosity (LOH) or mutation of the other allele. It is often difficult to identify the causative gene in such cases, and it has been difficult to recapitulate such events in a model system.
If a hemizygous condition can be created for an unknown tumor suppressor, the probability of generating the critical somatic mutation event on the other allele is greatly increased. Recently, Allan Bradley and colleagues (6) have used loxP/Cre recombinase technology for the generation of very large deletions (up to 4 Mb) and inversions (24 cM) in chromosomes. One loxP insert is tagged with the 5' half of a hypoxanthine phosphoribosyl transferase (hprt) cDNA cassette and another loxP insert is fused to the 3' portion of this hprt cassette. These complimenting loxP sites are then placed at either end of a region of DNA to be deleted in hprt-deficient ES cells such as ABL2.1. Cre-mediated recombination between these loxP sites yields a deletion in which a fully functional hprt allele is reconstituted during recombination, leading to a hypoxanthine amniopterin thymidine (HAT)-resistant ES cell. This strategy provides a means of positively selecting for cells with precise deletions (7).
The generation of these large deletions generates regional haploidy. This can be exploited when used with a wide-range mutagenesis strategy (8). N-ethyl-N-nitrosourea (ENU) is a potent germline mutagen that induces intragenic mutations in spermatogonia with a frequency of 1.5–6 x 10–3, the equivalent of obtaining a mutation in a specific gene in one gamete out of 175–655 screened (9). ENU mutagenized males can be mated with wild-type females to generate males with scattered point mutations. Such males can be mated to females carrying these large genomic deletions. Offspring can then be analyzed for phenotypic expression of cancer (8).
Another strategy devised by the Bradley group is the generation of variable length deletions of the same region of a chromosome (10). A retroviral vector is used to randomly insert the downstream loxP/hprt cassette after the upstream loxP/hprt cassette is inserted in a defined position via homologous recombination. Using this strategy, it is possible to generate variable nested deletions, an approach that could aid in defining more accurately a region in which a tumor suppressor gene lies. This strategy takes advantage of the precision of Cre/loxP recombination and the random tagging of several deletion endpoints with a single retroviral-tagging event (10).
The ability to generate deletions in large regions of mouse chromosomes is a powerful means to uncover recessive tumor suppressors. It is necessary to use ES cells that are deficient for hprt, such as the ABL2.1 cells. These cell lines, loxP insertion constructs and in many cases screened ES lines are available to the community (http://www.imgen.bcm.tmc.edu/molgen/labs/bradley/cell.htm). Therefore, it will be possible for any investigator to use such a strategy to create defined or random deletions. Alternative strategies for generating deletions in ES cells have been developed in the laboratories of John Schimenti (http://lena.jax.org/
jcs/) and Terry Magnuson (11,12).
TV-A/RCAS retroviral gene delivery system
As noted previously, it is possible to use viruses to deliver genes to specific tissues. A novel method for cell- and tissue-specific gene delivery has been developed in the laboratories of Stephen Hughes and Harold Varmus based on avian virus mediated gene delivery to cells and tissues expressing the avian retroviral receptor, tv-a (13,14). Mice do not express the tv-a receptor and are resistant to avian retroviral infection. However, transgenic mice engineered to express tv-a under the control of cell- and tissue-specific promoters render those tissues susceptible to avian retroviral infection. An avian pseudotyped virus that recognizes the tv-a receptor can then be used to deliver genes of interest to these tv-a-expressing cells. In practice the gene of interest is cloned into a Rous sarcoma- based avian proviral vector (RCAS). The maximum size insert that can be used is 2.5 kb, limiting the application to small cDNAs. The proviral vector is transfected into avian viral producer cells in vitro to produce a pseudotyped virus that contains the avian leukosis virus (ALV) coat protein, which recognizes the tv-a receptor. The replication competent virus will produce high-titer virus from these cells and they can be maintained in culture as they infect and reinfect the cells in culture. The virus can then be delivered into the mice by injecting the virus producing cells or by injecting the virus itself (15).
The feasibility of using this system for gene delivery and the study of tumor progression was demonstrated in a study designed to determine the cells that give rise to glial tumors such as astocytomas and oligodendrogliomas (16). The glial fibrillary protein (GFAP) promoter was used to express tv-a in astrocytes of transgenic mice. These mice were infected with virus containing polyoma virus middle T antigen. These mice developed oligodendrogliomas, astrocytomas and mixed gliomas, suggesting that these tumors can form from astrocytes that express GFAP.
An additional advantage of this system is that cells can be infected with multiple genes simultaneously since tv-a receptors are not blocked by viral infection. Such infections with multiple viruses will lead to the production of a heterogenous population of cells expressing different levels and combinations of the genes of interest. This models the in vivo situation seen in heterogeneous tumors, where a number of different genetic lesions can occur within the same tumor mass. The introduction of these genes somatically to a small group of cells at the location of infection also models the situation seen in tumors where a single cell can give rise to a tumor locally. The tv-a retroviral gene delivery system may provide the flexibility needed to model more accurately mammalian cancer progression and tumorigenesis in somatic cells.
Some limitations to this system should be noted. (i) An immune response can occur at the site of virus delivery. (ii) The size of the gene insert is limited to 2.5 kb. (iii) The virus can only infect replicating cells. Thus, somatic introduction of genes into nerve cells and other non-dividing cells is not possible at this time. In the future this restriction may be addressed by pseudotyping the ALV virus with the gag and pol genes from lentiviruses, which are capable of infecting non-replicating cells (17).
The tv-a based retroviral gene delivery system will continue to be a valuable tool in producing murine models of cancer. New target genes can be produced readily and injected anywhere into transgenic mice expressing tv-a. A number of vectors and tv-a expressing transgenic mice that have already been engineered are listed at an updated website (http://rex.nci.nih.gov/RESEARCH/basic/varmus/tva-web/tva2.html).
Inducible transgenes
Standard transgenic approaches have resulted in the global, high-level overexpression of oncogenes, a situation rarely seen in spontaneous tumorigenesis in humans. Recently, inducible systems that achieve tighter spatial and temporal control of gene expression of transgenes have been developed.
The most widely used conditional expression systems employ tetracycline (tet) inducible promoters that were developed in the laboratory of Hermann Bujard (18,19). A tissue-specific promoter is used to express a fusion of the Escherichia coli Tn10Tc protein and HSV VP16 transactivation domain, known simply as ‘tTA’. This protein then binds the Tet operator (TetO) sequence and suppresses expression of the associated transgene in the presence of doxycycline. Suppression is released when doxycycline is cleared from the system. A reverse tTA (rtTA) has also been constructed in which doxycycline treatment activates gene expression. This avoids the dependence for activation on drug clearance kinetics as seen in the tTA system (20). Other systems of conditional expression with different ligand-induced transcriptional regulators have also been developed (21).
These inducible systems have been applied to the study of tumorigenesis. The induction of transgenes can be controlled by the administration of a drug in the animal’s diet. In addition, it is possible to cycle gene expression between on and off states. The SV40 T antigen (T-Ag) was the first oncogene to be analyzed using inducible expression in the mammary gland of mice. T-Ag was induced, and then suppressed in the breast at various times in the adult female. Early in induction, the breast displayed reversible hyperplasia. However, after prolonged induction, T-Ag expression was no longer necessary for maintenance of epithelial hyperplasia and eventual tumor formation (22). Subsequently, several groups used the Tet system to activate oncogenes in specific tissues. For example, c-Myc induction in the epidermis was used to induce papillomas (21), Ha-Ras and Ink4a/p19Arf induction was used to induce melanomas (23) and controlled expression of E2F-1 induced apoptosis in p53 null tumors and mouse embryo fibroblasts (MEFs) (24). These studies suggest that prolonged expression of certain oncogenes results in genetic changes that eventually become independent of the expression of the oncogene (25).
One of the greatest challenges in producing transgenic mice with reproducible inducible gene expression is that cis-acting regulatory elements in the vicinity of insertions influence gene expression. Regions of heterochromatin can also hinder the precision of the system, affecting the ‘leakiness’ of such aberrant expression (18,26). This is an important consideration for the tight control of gene expression. In addition, switching of a gene from a state of expression to dormancy is not always rapid. For example, full induction can take up to a week in the tTA system due to the kinetics of clearance of doxcycline (26). There are also indications that the repeated switching between a state of expression and repression can create leaks in the system and it will become unresponsive to further stimulation (26). Undoubtedly, this system will continue to be refined and exploited in the near future to improve our understanding of oncogenesis.
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ANALYZING MOUSE MODELS OF CANCER |
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TOPABSTRACTINTRODUCTIONMODELING CANCER IN MICEANALYZING MOUSE MODELS OF...CONCLUSIONREFERENCES |
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Once these models of cancer are produced in the mouse, it is necessary to understand the mechanisms of genomic instability that resulted in tumorigenesis. Several methods to measure and document genomic instability have recently been developed and refined in the mouse. Oligonucleotide and cDNA microarrays have been developed to measure global patterns of gene expression. Multicolor fluorescent in situ hybridization (FISH) technologies such as spectral karyotyping (SKY) and standard FISH have been used to examine chromosomal translocations. Finally, comparative genomic hybridization (CGH) and array CGH techniques have been used to determine areas of genomic duplication and deletion in tumors.
Oligonucleotide and cDNA microarrays
It has become possible to catalog the expression differences responsible for tumorigenesis by analyzing global changes at the level of gene expression with microarray technology. Such arrays can monitor the changes in expression of thousands of genes simultaneously. Microarrays are constructed by attaching large numbers of unique oligonucleotides or cDNA clones to substrates such as glass slides. Several thousand of these oligonucleotides or cDNAs that represent a significant fraction of all genes can be ‘spotted’ on a single slide, further simplifying the analysis of complex gene interactions.
RNA probes from tumor and wild-type controls can be labeled with different fluorescent dyes such as Cy3 and Cy5 (visible as green and red, respectively). These differently labeled probes are then simultaneously hybridized to the constructed array(s), and compete for hybridization to the same spots. After stringent washing, the slides are read by a confocal reader and the hybridization signal on each spot is measured. When the differently labeled probes bind equally they appear as a yellow spot, a pseudocolor mixture of the red and green fluors generated by comparative analysis software (Fig. 2, top left panel). The probes that are more highly represented in one RNA sample will bind more effectively than the other and the spot will be represented by one color more than the other. Due to the ability to analyze subtle differences in binding, it is possible to determine both single gene and complex genetic differences in expression between normal and tumor cells (27–29).
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There are several recently published examples of the application of microarray technology in the analysis of gene expression changes that occur during development in the mouse. Much work is also being done in the area of modeling cancer in the mouse. One recent example is the identification of a fusion between PAX3 and FKHR that has transforming properties. This was discovered using cDNA microarrays by comparing global gene expression between cells transfected with either the fusion PAX3/FKHR or PAX3 alone (30). It is hoped that expression analysis of various tumors will provide molecular fingerprints to diagnose cancer and detect similarities and differences in the origin of specific tumors.
SKY and FISH
SKY was developed in the laboratory of Thomas Ried, and has proven to be an invaluable tool in assessing genomic instability by virtue of its ability to detect chromosomal translocations (31,32). SKY is based on labeling flow sorted chromosomes with a chromosome-specific probe using specific combinations of fluorophores (i.e. Cy3, Cy5, Cy5.5, Texas red and Spectrum green) either singly or in combination. These probes are then mixed together and hybridized to metaphase chromosomes. After excitation a unique and specific wavelength is emitted for each chromosome. The spectral emission of a metaphase spread is determined pixel-by-pixel using a spectrophotometer attached to a CCD camera, and this spectral pattern is then translated into chromosome-specific pseudocolors by Fourier transformation. The pictorial output of this analysis represents each chromosome as a different color. Translocation events can be easily identified as a chromosome that consists of more than one color (Fig. 2, top right panel), greatly simplifying the identification of translocations and common breakpoints.
Although it has been used predominantly in human tumors, SKY has proven to be especially useful in mice, since it allows the identification of mouse chromosomes based on color. Telocentric mouse chromosomes are inherently difficult to identify by conventional banding methods. While SKY is very useful it does have limited sensitivity for small rearrangements. SKY cannot visualize translocations that are <1500 kb. A recent comprehensive review by Knutsen and Ried (33) documents the first 300 cases of SKY in identifying new translocations and common breakpoints in a number of tumors published to date.
Conventional FISH can be used to pinpoint the site of a translocation breakpoint after SKY narrows the search to a particular chromosomal band (Fig. 2, bottom right panel). For example, it has been possible to identify common breakpoints in lymphomas of Atm knockout mice (32,34). In these lymphomas, recurrent aberrations were found on chromosome 14, near the site of the T cell receptor 
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locus, suggesting that translocations occurred at the point of V(D)J recombination. This was further supported by FISH using individual probes to the V and C regions (34).
CGH and array CGH
CGH has been used to determine regions of chromosomal amplification and loss. CGH compares DNA copy number variation across a genome. Tumor and non-tumor control genomic DNA are labeled with different fluors and hybridized to the same normal metaphase chromosomes (Fig. 2, bottom left panel). The fluorescent ratio across the chromosomes represents the DNA copy number variation in the tumor relative to normal somatic cells (35). This method has provided important insights into regions of chromosome amplification and loss in a variety of tumors.
However, the resolution of CGH is low since metaphase chromosomes are used. To increase resolution of detection of copy number changes in CGH, cDNA microarrays have recently been used for CGH experiments (array CGH). With array CGH, it is possible to pinpoint areas of copy number variation based on the known chromosomal location of a cDNA, and it also directly identifies candidate genes that may be important for tumor development. This method has been tested by two groups using different techniques: one used varying amounts of 
DNA (36) and the other used tumor cells with known amplifications and deletions and compared them to wild-type cells (37). Both found that array CGH accurately represented DNA copy number.
The use of array CGH has shown great potential in the analysis of complex gene expression patterns in cancerous cells. This method should prove valuable in the analysis of many of the mouse models of cancer that are currently being studied and will give greater insight into the similarities and differences in gene amplification and deletion among the many models that are being studied.
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMODELING CANCER IN MICEANALYZING MOUSE MODELS OF...CONCLUSIONREFERENCES |
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With the imminent completion of the human genome sequence (followed by that of the mouse), it will be possible to identify genes potentially involved in tumorigenesis. The more difficult task will be to prove that such genes are in fact important for human cancer, and mouse models will be one very important tool that will be brought to bear on this question. We have attempted to highlight recent methodologies that have resulted in the improvement of genetic manipulation of the mouse with respect to tumorigenesis, as well as tools developed to demonstrate and analyze genomic instability in these tumors. These powerful tools will advance our ability to use the mouse to model human cancer more precisely. This will further our understanding of the events that lead to the initiation and progression of this devastating group of diseases, as well as provide valid models to test potential treatments.
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ACKNOWLEDGEMENTS |
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The authors wish to thank Ralf Schubert and Jianbo Wang for their critical comments on the manuscript and to apologize for any work that was not cited due to space constraints. We would like to especially thank Thomas Ried for providing the SKY, CGH and FISH images and Brenda Olsen for the microarray image in Figure 2.
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FOOTNOTES |
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+ These authors contributed equally to this work
§ To whom correspondence should be addressed. Tel: +1 858 822 3400; Fax: +1 858 822 3409; Email: awynshawboris@ucsd.edu
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