- Email: [email protected]
Down syndrome mouse models are looking up
Update
TRENDS in Molecular Medicine
such as spinal-cord trauma, Parkinson’s disease and cardiac-muscle atrophy. Any strategy for expansion of stem-cell numbers ex vivo prior to transplantation might have increased risk of generating chromosomal aberrations through defective decatenation checkpoint function. If such aberrations block differentiation along the expected lineage and promote clonal expansion, neoplastic disease might develop as a result of stem-cell therapy. Cancer cell lines in culture often display severe genetic instability and, in two instances, this has been associated with defects in decatenation G2 checkpoint function [20,21]. If the findings by Damelin et al. [5] are confirmed, somatic stem cells might be at increased risk of chromosomal damage during cell division in vivo. Conditions that enhance stem-cell division might increase the yield of daughter cells with oncogenic chromosomal aberrations and the risk of development of cancer. Ex vivo proliferation of stem cells might be associated with attenuation of an important tumor suppressive checkpoint that guards against malignantly entangled chromosomes. Acknowledgements The work performed was supported in part by NIH grants (CA81343 and ES10126). The author thanks a reviewer for providing the reference for uniparental disomy in ES cells.
References 1 Downes, C.S. et al. (1994) A topoisomerase II-dependent G2 cycle checkpoint in mammalian cells. Nature 372, 467–470 2 Deming, P.B. et al. (2001) The human decatenation checkpoint. Proc. Natl. Acad. Sci. U. S. A. 98, 12044–12049 3 Gimenez-Abian, J.F. et al. (2000) Premitotic chromosome individualization in mammalian cells depends on topoisomerase II activity. Chromosoma 109, 235–244 4 Deming, P.B. et al. (2002) ATR enforces the topoisomerase IIdependent G2 checkpoint through inhibition of Plk1 kinase. J. Biol. Chem. 277, 36832–36838 5 Damelin, M. et al. (2005) Decatenation checkpoint deficiency in stem and progenitor cells. Cancer Cell 8, 479–484
Vol.12 No.6 June 2006
237
6 Paulovich, A.G. et al. (1997) When checkpoints fail. Cell 88, 315–321 7 Holm, C. (1994) Coming undone: how to untangle a chromosome. Cell 77, 955–957 8 Haering, C.H. and Nasmyth, K. (2003) Building and breaking bridges between sister chromatids. Bioessays 25, 1178–1191 9 Losada, A. and Hirano, T. (2001) Intermolecular DNA interactions stimulated by the cohesin complex in vitro: implications for sister chromatid cohesion. Curr. Biol. 11, 268–272 10 Holm, C. et al. (1989) DNA topoisomerase II must act at mitosis to prevent nondisjunction and chromosome breakage. Mol. Cell. Biol. 9, 159–168 11 Wang, J.C. (1996) DNA topoisomerases. Annu. Rev. Biochem. 65, 635–692 12 Roca, J. et al. (1994) Antitumor bisdioxopiperazines inhibit yeast DNA topoisomerase II by trapping the enzyme in the form of a closed protein clamp. Proc. Natl. Acad. Sci. U. S. A. 91, 1781–1785 13 Kaufmann, W.K. and Kies, P.E. (1998) DNA signals for G2 checkpoint response in diploid human fibroblasts. Mutat. Res. 400, 153–167 14 Clarke, D.J. et al. (1993) Topoisomerase II inhibition prevents anaphase chromatid segregation in mammalian cells independently of the generation of DNA strand breaks. J. Cell Sci. 105, 563–569 15 Franchitto, A. et al. (2003) The G2-phase decatenation checkpoint is defective in Werner syndrome cells. Cancer Res. 63, 3289–3295 16 Lou, Z. et al. (2005) BRCA1 participates in DNA decatenation. Nat. Struct. Mol. Biol. 12, 589–593 17 Jackman, M. et al. (2003) Active cyclin B1–Cdk1 first appears on centrosomes in prophase. Nat. Cell Biol. 5, 143–148 18 Kaufmann, W.K. et al. (2002) Degradation of ATM-independent decatenation checkpoint function in human cells is secondary to inactivation of p53 and correlated with chromosomal destabilization. Cell Cycle 1, 210–219 19 Cervantes, R.B. et al. (2002) Embryonic stem cells and somatic cells differ in mutation frequency and type. Proc. Natl. Acad. Sci. U. S. A. 99, 3586–3590 20 Doherty, S.C. et al. (2003) Cell cycle checkpoint function in bladder cancer. J. Natl. Cancer Inst. 95, 1859–1868 21 Nakagawa, T. et al. (2004) Identification of decatenation G2 checkpoint impairment independently of DNA damage G2 checkpoint in human lung cancer cell lines. Cancer Res. 64, 4826–4832
1471-4914/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.molmed.2006.04.001
Down syndrome mouse models are looking up Roger H. Reeves Johns Hopkins University School of Medicine, Department of Physiology and McKusick-Nathans Institute for Genetic Medicine, Biophysics 201, 725 North Wolfe Street, Baltimore, MD 21025, USA
A new mouse model of Down syndrome (DS) carries a copy of human chromosome 21 (Hsa21), in addition to a full complement of mouse chromosomes. In terms of the number of trisomic genes represented, this model, known as ‘Tc1’, is closer to the genetic background of DS than any previous model. The Tc1 model not only recapitulates several of the DS features present in other Corresponding author: Reeves, R.H. ([email protected]). Available online 4 May 2006 www.sciencedirect.com
mouse models but also exhibits heart defects that are similar to those that make trisomy 21 the leading cause of congenital heart disease in humans. Many cells in adult Tc1 mice show mosaicism – that is, the Hsa21 is lost from some cells during development – increasing the complexity of analyses using this model. Tc1 mice provide a powerful tool for investigation of the pathogenesis of trisomy 21, and a platform for analysis of similarities and differences in the evolution of gene regulation.
238
Update
TRENDS in Molecular Medicine
Glossary Euploid: a normal or ‘true’ (eu) chromosome number. Microarray: an ordered set of oligonucleotide probes that represent sequences within RNA molecules. It is used to survey a population of RNA to determine what transcripts are represented and at what level they are expressed. Orthologous: a state of homology that represents exact counterparts across species. An orthologous chromosomal segment is a region of, for example, mouse chromosome that is derived from the same ancestral chromosome as the corresponding segment of Hsa21. Roberstonian translocation: the end-to-end fusion of two acrocentric chromosomes to form a new, metacentric chromosome with most of the genetic information from both. Segmental trisomy: a condition in which a contiguous segment of a chromosome is present in three copies. Trisomy: occurrence of a third copy of a complete chromosome.
Down syndrome and trisomic mouse models Trisomy 21 occurs in 1/750 live births, resulting in different phenotypes that are collectively known as Down syndrome (DS). Trisomy (see Glossary) leads to elevated expression of most genes on chromosome 21 (Hsa21) in tissues throughout development, maturation and ageing. Subsets of O80 clinical features occur frequently in individuals with DS [1]. It seems that many aspects of the DS phenotype result from additive effects of multiple-dosage-sensitive genes [2]. The great phenotypic variability among individuals with trisomy 21 suggests that their genetic background (modifier genes) contributes to phenotypic outcomes in DS. Trisomy 21 is among the most-complex genetic insults that are compatible with substantial survival beyond term. Efforts to understand the complex mechanisms by which trisomy affects development and function rely on genetic mouse models. Comparative mapping has identified three highly conserved regions on mouse chromosome (Mmu) 16, Mmu17 and Mmu10 that are orthologous to Hsa21 (Figure 1). Widely used mouse models have segmental trisomy 16 and, consequently, carry a third copy of the orthologs of up to 50% of the genes on Hsa21. Several structural and functional similarities exist between these models and DS, indicating that trisomy for the homologous genes disrupts the same evolutionarily conserved developmental genetic pathways with comparable outcomes. Conserved phenotypes include: (i) short stature and hypoplasia of the craniofacial skeleton and mandible; (ii) multiple brain abnormalities such as small cerebellum, hypocellular dentate gyrus and abnormal
Vol.12 No.6 June 2006
synapse morphology; (iii) pathological outcomes, including age-related degeneration of basal-forebrain cholinergic neurons; and (iv) functional similarities in cognitive skills involving the hippocampus that are correlated, in mice, with deficits in long-term potentiation (LTP) and longterm depression (LTD). Limitations of mouse models Although the demonstration of conserved phenotypes validates the use of mouse models for the study of DS pathogenesis, the models are not perfect. First, the mostrepresentative genetic model of DS, Ts65Dn, contains orthologs of only 50% of Hsa21 genes (Figure 1). Second, Hsa21 might contain regulatory signals in genomic DNA that differ from those in mice, or that are misread when Hsa21 is present in a mouse genetic background. This might lead to different consequences when Hsa21 is overexpressed in mouse as compared with humans [3]. In many cases, these signals might not be recognizable from direct sequence analysis. Third, some patterns of gene expression are likely to vary between mice and humans. Even specific functions of orthologous genes can diverge: the antiviral, interferonresponsive human myxovirus (influenza virus) resistance genes MX1 and MX2 differ in specificity from the corresponding mouse Mx1 and Mx2 genes [4]. These caveats must be considered when evaluating any animal model expressing a gene from another species. In addition, Hsa21 in the Tc1 mouse contains two small gaps so that w8% of known genes on Hsa21 are not present [5]. Nonetheless, the production of a genetic mouse model that is trisomic for 92% of the genes on Hsa21 is clearly a great advance as indicated by the occurrence of DS phenotypes in Tc1 mice that had not been observed in earlier models (see later). Trisomic-gene expression Recent studies have demonstrated that expression of trisomic genes is increased on average w50% in ten tissues in Ts65Dn mice [6,7], confirming a long-held but largely untested assumption regarding the effects of trisomy on transcript levels. Initial analysis of Hsa21 gene expression in the Tc1 mouse was performed by using microarray chips that are specific for human transcripts with RNA from embryonic day (E)14.5 mice. Microarrays provide a broad but insensitive approach for the
Hsa21 (247) Mmu16 (165)
Mmu10 (57) Mmu17 (24)
N.r. (29) Ts65Dn (136) Ts1Cje (83) Ts1Rhr (41)
TRENDS in Molecular Medicine
Figure 1. Gene content of human chromosome 21 (Hsa21) and orthologous regions of mouse chromosome (Mmu) 16, Mmu17 and Mmu10, and widely used mouse models. Hsa21 contains 247 genes. Ts65Dn mice are trisomic for a region of Hsa21 that contains 136 genes. Ts1Cje mice contain a segment with 83 genes and Ts1Rhr mice have three copies of 41 genes from a region of Hsa21 previously known as the ‘critical region’ [2]. Mouse models with segmental trisomy for Hsa21-orthologous regions of Mmu17 and Mmu10 are under development in several laboratories. Abbreviation: n.r., not represented. Gene numbers were adapted from Ref. [16]. www.sciencedirect.com
Update
TRENDS in Molecular Medicine
identification of small changes in gene expression levels that are expected in trisomy. In Tc1 mice, approximately one third (39 in 131) of the Hsa21 genes on the chip gave a significantly elevated signal compared with euploid control RNA. It will be important to analyze gene expression levels with more sensitive methods to determine which human genes, if any, do not respond properly to their human regulatory signals in the developing mouse. Quantitative analysis of protein expression and activity remains to be completed.
DS phenotypes are recapitulated in Tc1 mice It will require much work to determine whether Tc1 mice display the many phenotypes that have been characterized in other mouse models of DS and, thus, assess the applicability of the Tc1 model to DS research, but initial observations are encouraging. Hippocampal function is especially affected in DS, and in Ts65Dn and Ts1Cje mouse models [8–10]. Similar to these mouse models, Tc1 mice display abnormal LTP. Tc1 mice also show decreased performance in a novel hippocampal-based object-recognition test, although no effects have been observed in several other tests related to hippocampal function. Decreased density of cerebellar granule cells occurs in Ts65Dn mice and in the brains of individuals with DS [11], and this has been also observed in Tc1 mice. Similar to Ts65Dn mice, Tc1 mice fail to recapitulate the histopathology of Alzheimer’s disease, which is present in all individuals with DS by the fourth decade. This might indicate that the biochemistry of neuritic plaque and tangle formation requires an absolute passage of time, not just ageing within the lifespan of the mouse. Tc1 mice have small mandibles, which is characteristic of individuals with DS and is observed in other mouse models [12]. An integrated morphometric assessment of the entire skull is still missing for Tc1 mice, but it is reasonable to assume that additional skeletal effects that are analogous to those in individuals with DS and other mouse models occur in these mice. For all phenotypes, individual variability might contribute to different outcomes in Tc1 mice. Further direct comparisons between Tc1 mice and other DS mouse models will be required in additional paradigms. However, these initial results indicate several parallels between important features of DS, which are recapitulated in other DS mouse models, and those observed in the Tc1 model. An important advantage of Tc1 mice over other DS mouse models is the occurrence of heart-septation defects. Trisomy 21 is the leading cause of congenital heart disease [13]. Although rare in Ts65Dn and other mouse models, heart defects occur in most Tc1 mice (a surprising 20% of control mice also showed ventricular septal defects). Two important conclusions from this observation are that: (i) trisomic mice display heart defects similar to those seen in DS; and (ii) at least one dosage-sensitive gene that affects heart septation resides in regions of the genome that are not triplicated in Ts65Dn mice. New mouse models with segmental trisomy in the regions of Mmu17 and Mmu10 that are orthologous to Hsa21 will enable further www.sciencedirect.com
Vol.12 No.6 June 2006
239
understanding of the genetic contributions that produce this effect in Tc1 mice. Limitations of the Tc1 model The Tc1 model is not a perfect model for DS (Box 1). In addition to the caveats mentioned, an important aim of DS research is to develop models that are easily propagated, to encourage more researchers to apply methods from their own fields to DS research. Like Ts65Dn mice, male Tc1 mice are usually sterile. Viable Tc1 mice are propagated at sub-Mendelian frequencies through trisomic females, which provide a trisomic intra-uterine environment and frequently show poor nurturing skills towards newborn pups. Tc1 mice display a high degree of mosaicism – that is, the human chromosome is lost from a subset of cells so that up to 50% of the cells in adult tissues of Tc1 mice no longer contain Hsa21. Previous experiments with chimeric mice that were generated using euploid embryos and mouse embryonic stem cells that contain Hsa21 demonstrated that trisomic cells were present in different Box 1. Outstanding questions † When and where does mosaicism occur during development? Is there a single event with subsequent selection of euploid versus trisomic cells, or is the human chromosome lost from multiple cell lineages at different times in development? † What additional phenotype assessments of Tc1 mice will be needed to compare them with phenotypes that have already been measured in other mouse models? Some assessments include: (i) Morris water maze, which is used to test hippocampal function. Ts65Dn mice show a robust deficit in this test; other models have milder effects that are correlated with the amount of genetic information from Hsa21 (or its mouse homologs) that is present. (ii) Cerebellar structure and function. Cerebellum is small and depauperate of neurons in individuals with DS and mouse models [11], including Tc1 mice. Sensitive tests are needed to assess outcomes of interventions to ameliorate this structural deficit [17]. (iii) Integrated morphometric assessment of the skull [12], which demonstrates direct similarities between DS and several other mouse models [2] should be undertaken in Tc1 mice. The prediction is that Tc1 mice should be more affected than Ts65Dn mice, with the caveat that mosaicism is predicted a priori to attenuate the effect of trisomy. If the frequency of mosaicism can be correlated with the degree of dysmorphology, this assessment might provide important insights into the pathogenesis of this phenotype. (iv) Assessment of LTP and LTD with the same methods that were used for other models [9]. The use of different paradigms for the same measurements in multiple electrophysiological (and behavioral) studies of trisomic mouse models has sometimes led to conflicting results that might reflect differences in methodology as much as or more than real differences in the models. † Which additional DS phenotypes can be studied in Tc1 mice and other models? Reduced tumor frequency in DS has been shown in several epidemiological studies [18]. Along with observations that suggest resistance to atherosclerosis in DS, these should be assessed in Tc1 and other DS models to identify contributing genes and mechanisms. † How are DS phenotypes caused (i.e. why does a slight elevation in the level of a normal gene or genes disrupt development)? Which genes contribute to which phenotypes? How can the effects of trisomy be ameliorated?
Update
240
TRENDS in Molecular Medicine
proportions in different tissues of the same mouse [14]. This was presumed to result from selection against trisomic cells, which differed in different tissues, as proposed earlier for chimeric mice in which one donor embryo contained a complete extra copy of Mmu16 due to a Robertsonian translocation [15]. However, mosaicism in Tc1 mice can only result from loss of Hsa21 that has been inherited from the mother, which was initially present in all cells. This is probably a mechanism by which ‘mosaic DS’ occurs in humans. The timing of Hsa21 loss in Tc1 mice, and whether it occurs once or in multiple cell lineages through development, is unknown. The impact in a given individual is not identical to that in any other individual, even if they have the same percentage of trisomic cells. This variability represents a substantial source of phenotypic variation. There is reasonable inferential evidence that this kind of mosaicism does not normally occur in mouse models and in humans with trisomy 21, but this finding in Tc1 mice suggests that the possibility of mosaicism should be examined more thoroughly in trisomic humans and mice. Concluding remarks The derivation of Tc1 mice has been an epic undertaking spanning O12 years. The effort has led to a great advance in DS research because this compelling model expands the range of phenotypes recapitulated between species with the analogous aneuploid gene set. The Tc1 model also provides an excellent system for the assessment of comparative gene-regulation effects in development. Acknowledgements We thank R. Roper, T. Sussan and C. Epstein for thoughtful suggestions about the manuscript and figures.
References 1 Epstein, C.J. (2001) Down syndrome (trisomy 21). In The Metabolic and Molecular Bases of Inherited Disease (Vol. 1) (Scriver, C.R. et al., eds), pp. 1223–1256, McGraw-Hill 2 Olson, L.E. et al. (2004) A chromosome 21 critical region does not cause specific down syndrome phenotypes. Science 306, 687–690
Vol.12 No.6 June 2006
3 Deutsch, S. et al. (2005) Gene expression variation and expression quantitative trait mapping of human chromosome 21 genes. Hum. Mol. Genet. 14, 3741–3749 4 Goodbourn, S. et al. (2000) Interferons: cell signalling, immune modulation, antiviral response and virus countermeasures. J. Gen. Virol. 81, 2341–2364 5 O’Doherty, A. et al. (2005) An aneuploid mouse strain carrying human chromosome 21 with down syndrome phenotypes. Science 309, 2033–2037 6 Kahlem, P. et al. (2004) Transcript level alterations reflect gene dosage effects across multiple tissues in a mouse model of down syndrome. Genome Res. 14, 1258–1267 7 Lyle, R. et al. (2004) Gene expression from the aneuploid chromosome in a trisomy mouse model of down syndrome. Genome Res. 14, 1268–1274 8 Pennington, B.F. et al. (2003) The neuropsychology of Down syndrome: evidence for hippocampal dysfunction. Child Dev. 74, 75–93 9 Kleschevnikov, A.M. et al. (2004) Hippocampal long-term potentiation suppressed by increased inhibition in the Ts65Dn mouse, a genetic model of Down syndrome. J. Neurosci. 24, 8153–8160 10 Siarey, R.J. et al. (1999) Increased synaptic depression in the Ts65Dn mouse, a model for mental retardation in Down syndrome. Neuropharmacology 38, 1917–1920 11 Baxter, L.L. et al. (2000) Discovery and genetic localization of Down syndrome cerebellar phenotypes using the Ts65Dn mouse. Hum. Mol. Genet. 9, 195–202 12 Richtsmeier, J. et al. (2000) Parallels of craniofacial maldevelopment in Down syndrome and Ts65Dn mice. Dev. Dyn. 217, 137–145 13 Ferencz, C. et al. (1989) Congenital cardiovascular malformations associated with chromosome abnormalities: an epidemiologic study. J. Pediatr. 114, 79–86 14 Shinohara, T. et al. (2001) Mice containing a human chromosome 21 model behavioral impairment and cardiac anomalies of Down’s syndrome. Hum. Mol. Genet. 10, 1163–1175 15 Gearhart, J. et al. (1986) Mouse chimeras composed of trisomy 16 and normal (2N) cells: preliminary studies. Brain Res. Bull. 16, 815–824 16 Antonarakis, S.E. et al. (2004) Chromosome 21 and down syndrome: from genomics to pathophysiology. Nat. Rev. Genet. 5, 725–738 17 Roper, R.J. et al. (2006) Defective cerebellar response to mitogenic Hedgehog signaling in Down syndrome mice. Proc. Natl. Acad. Sci. U. S. A. 103, 1452–1456 18 Yang, Q. et al. (2002) Mortality associated with Down’s syndrome in the USA from 1983 to 1997: a population-based study. Lancet 359, 1019–1025
1471-4914/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.molmed.2006.04.005
Prostaglandins and the colon cancer connection Timothy A. Chan Department of Radiation Oncology and Molecular Radiation Sciences, Sidney Kimmel Cancer Center, Johns Hopkins Hospital, 401 N. Broadway Street, Suite 1440, Baltimore, MD 21231, USA
Colorectal cancer is a leading cause of cancer-related deaths throughout the world. Non-steroidal anti-inflammatory drugs (NSAIDs) are among the few agents that are known to inhibit colorectal tumorigenesis. The mechanisms that underlie this effect are poorly Corresponding author: Chan, T.A. ([email protected]). Available online 2 May 2006 www.sciencedirect.com
understood. Two recent studies have provided some significant insight. Castellone and colleagues showed that prostaglandin E2 modulates the b-catenin signaling axis, a key pathway for colorectal tumorigenesis. Holla and colleagues showed that prostaglandin E2 might act via a nuclear receptor. These findings shed light on the mechanisms that underlie prostaglandin action, and
TRENDS in Molecular Medicine
such as spinal-cord trauma, Parkinson’s disease and cardiac-muscle atrophy. Any strategy for expansion of stem-cell numbers ex vivo prior to transplantation might have increased risk of generating chromosomal aberrations through defective decatenation checkpoint function. If such aberrations block differentiation along the expected lineage and promote clonal expansion, neoplastic disease might develop as a result of stem-cell therapy. Cancer cell lines in culture often display severe genetic instability and, in two instances, this has been associated with defects in decatenation G2 checkpoint function [20,21]. If the findings by Damelin et al. [5] are confirmed, somatic stem cells might be at increased risk of chromosomal damage during cell division in vivo. Conditions that enhance stem-cell division might increase the yield of daughter cells with oncogenic chromosomal aberrations and the risk of development of cancer. Ex vivo proliferation of stem cells might be associated with attenuation of an important tumor suppressive checkpoint that guards against malignantly entangled chromosomes. Acknowledgements The work performed was supported in part by NIH grants (CA81343 and ES10126). The author thanks a reviewer for providing the reference for uniparental disomy in ES cells.
References 1 Downes, C.S. et al. (1994) A topoisomerase II-dependent G2 cycle checkpoint in mammalian cells. Nature 372, 467–470 2 Deming, P.B. et al. (2001) The human decatenation checkpoint. Proc. Natl. Acad. Sci. U. S. A. 98, 12044–12049 3 Gimenez-Abian, J.F. et al. (2000) Premitotic chromosome individualization in mammalian cells depends on topoisomerase II activity. Chromosoma 109, 235–244 4 Deming, P.B. et al. (2002) ATR enforces the topoisomerase IIdependent G2 checkpoint through inhibition of Plk1 kinase. J. Biol. Chem. 277, 36832–36838 5 Damelin, M. et al. (2005) Decatenation checkpoint deficiency in stem and progenitor cells. Cancer Cell 8, 479–484
Vol.12 No.6 June 2006
237
6 Paulovich, A.G. et al. (1997) When checkpoints fail. Cell 88, 315–321 7 Holm, C. (1994) Coming undone: how to untangle a chromosome. Cell 77, 955–957 8 Haering, C.H. and Nasmyth, K. (2003) Building and breaking bridges between sister chromatids. Bioessays 25, 1178–1191 9 Losada, A. and Hirano, T. (2001) Intermolecular DNA interactions stimulated by the cohesin complex in vitro: implications for sister chromatid cohesion. Curr. Biol. 11, 268–272 10 Holm, C. et al. (1989) DNA topoisomerase II must act at mitosis to prevent nondisjunction and chromosome breakage. Mol. Cell. Biol. 9, 159–168 11 Wang, J.C. (1996) DNA topoisomerases. Annu. Rev. Biochem. 65, 635–692 12 Roca, J. et al. (1994) Antitumor bisdioxopiperazines inhibit yeast DNA topoisomerase II by trapping the enzyme in the form of a closed protein clamp. Proc. Natl. Acad. Sci. U. S. A. 91, 1781–1785 13 Kaufmann, W.K. and Kies, P.E. (1998) DNA signals for G2 checkpoint response in diploid human fibroblasts. Mutat. Res. 400, 153–167 14 Clarke, D.J. et al. (1993) Topoisomerase II inhibition prevents anaphase chromatid segregation in mammalian cells independently of the generation of DNA strand breaks. J. Cell Sci. 105, 563–569 15 Franchitto, A. et al. (2003) The G2-phase decatenation checkpoint is defective in Werner syndrome cells. Cancer Res. 63, 3289–3295 16 Lou, Z. et al. (2005) BRCA1 participates in DNA decatenation. Nat. Struct. Mol. Biol. 12, 589–593 17 Jackman, M. et al. (2003) Active cyclin B1–Cdk1 first appears on centrosomes in prophase. Nat. Cell Biol. 5, 143–148 18 Kaufmann, W.K. et al. (2002) Degradation of ATM-independent decatenation checkpoint function in human cells is secondary to inactivation of p53 and correlated with chromosomal destabilization. Cell Cycle 1, 210–219 19 Cervantes, R.B. et al. (2002) Embryonic stem cells and somatic cells differ in mutation frequency and type. Proc. Natl. Acad. Sci. U. S. A. 99, 3586–3590 20 Doherty, S.C. et al. (2003) Cell cycle checkpoint function in bladder cancer. J. Natl. Cancer Inst. 95, 1859–1868 21 Nakagawa, T. et al. (2004) Identification of decatenation G2 checkpoint impairment independently of DNA damage G2 checkpoint in human lung cancer cell lines. Cancer Res. 64, 4826–4832
1471-4914/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.molmed.2006.04.001
Down syndrome mouse models are looking up Roger H. Reeves Johns Hopkins University School of Medicine, Department of Physiology and McKusick-Nathans Institute for Genetic Medicine, Biophysics 201, 725 North Wolfe Street, Baltimore, MD 21025, USA
A new mouse model of Down syndrome (DS) carries a copy of human chromosome 21 (Hsa21), in addition to a full complement of mouse chromosomes. In terms of the number of trisomic genes represented, this model, known as ‘Tc1’, is closer to the genetic background of DS than any previous model. The Tc1 model not only recapitulates several of the DS features present in other Corresponding author: Reeves, R.H. ([email protected]). Available online 4 May 2006 www.sciencedirect.com
mouse models but also exhibits heart defects that are similar to those that make trisomy 21 the leading cause of congenital heart disease in humans. Many cells in adult Tc1 mice show mosaicism – that is, the Hsa21 is lost from some cells during development – increasing the complexity of analyses using this model. Tc1 mice provide a powerful tool for investigation of the pathogenesis of trisomy 21, and a platform for analysis of similarities and differences in the evolution of gene regulation.
238
Update
TRENDS in Molecular Medicine
Glossary Euploid: a normal or ‘true’ (eu) chromosome number. Microarray: an ordered set of oligonucleotide probes that represent sequences within RNA molecules. It is used to survey a population of RNA to determine what transcripts are represented and at what level they are expressed. Orthologous: a state of homology that represents exact counterparts across species. An orthologous chromosomal segment is a region of, for example, mouse chromosome that is derived from the same ancestral chromosome as the corresponding segment of Hsa21. Roberstonian translocation: the end-to-end fusion of two acrocentric chromosomes to form a new, metacentric chromosome with most of the genetic information from both. Segmental trisomy: a condition in which a contiguous segment of a chromosome is present in three copies. Trisomy: occurrence of a third copy of a complete chromosome.
Down syndrome and trisomic mouse models Trisomy 21 occurs in 1/750 live births, resulting in different phenotypes that are collectively known as Down syndrome (DS). Trisomy (see Glossary) leads to elevated expression of most genes on chromosome 21 (Hsa21) in tissues throughout development, maturation and ageing. Subsets of O80 clinical features occur frequently in individuals with DS [1]. It seems that many aspects of the DS phenotype result from additive effects of multiple-dosage-sensitive genes [2]. The great phenotypic variability among individuals with trisomy 21 suggests that their genetic background (modifier genes) contributes to phenotypic outcomes in DS. Trisomy 21 is among the most-complex genetic insults that are compatible with substantial survival beyond term. Efforts to understand the complex mechanisms by which trisomy affects development and function rely on genetic mouse models. Comparative mapping has identified three highly conserved regions on mouse chromosome (Mmu) 16, Mmu17 and Mmu10 that are orthologous to Hsa21 (Figure 1). Widely used mouse models have segmental trisomy 16 and, consequently, carry a third copy of the orthologs of up to 50% of the genes on Hsa21. Several structural and functional similarities exist between these models and DS, indicating that trisomy for the homologous genes disrupts the same evolutionarily conserved developmental genetic pathways with comparable outcomes. Conserved phenotypes include: (i) short stature and hypoplasia of the craniofacial skeleton and mandible; (ii) multiple brain abnormalities such as small cerebellum, hypocellular dentate gyrus and abnormal
Vol.12 No.6 June 2006
synapse morphology; (iii) pathological outcomes, including age-related degeneration of basal-forebrain cholinergic neurons; and (iv) functional similarities in cognitive skills involving the hippocampus that are correlated, in mice, with deficits in long-term potentiation (LTP) and longterm depression (LTD). Limitations of mouse models Although the demonstration of conserved phenotypes validates the use of mouse models for the study of DS pathogenesis, the models are not perfect. First, the mostrepresentative genetic model of DS, Ts65Dn, contains orthologs of only 50% of Hsa21 genes (Figure 1). Second, Hsa21 might contain regulatory signals in genomic DNA that differ from those in mice, or that are misread when Hsa21 is present in a mouse genetic background. This might lead to different consequences when Hsa21 is overexpressed in mouse as compared with humans [3]. In many cases, these signals might not be recognizable from direct sequence analysis. Third, some patterns of gene expression are likely to vary between mice and humans. Even specific functions of orthologous genes can diverge: the antiviral, interferonresponsive human myxovirus (influenza virus) resistance genes MX1 and MX2 differ in specificity from the corresponding mouse Mx1 and Mx2 genes [4]. These caveats must be considered when evaluating any animal model expressing a gene from another species. In addition, Hsa21 in the Tc1 mouse contains two small gaps so that w8% of known genes on Hsa21 are not present [5]. Nonetheless, the production of a genetic mouse model that is trisomic for 92% of the genes on Hsa21 is clearly a great advance as indicated by the occurrence of DS phenotypes in Tc1 mice that had not been observed in earlier models (see later). Trisomic-gene expression Recent studies have demonstrated that expression of trisomic genes is increased on average w50% in ten tissues in Ts65Dn mice [6,7], confirming a long-held but largely untested assumption regarding the effects of trisomy on transcript levels. Initial analysis of Hsa21 gene expression in the Tc1 mouse was performed by using microarray chips that are specific for human transcripts with RNA from embryonic day (E)14.5 mice. Microarrays provide a broad but insensitive approach for the
Hsa21 (247) Mmu16 (165)
Mmu10 (57) Mmu17 (24)
N.r. (29) Ts65Dn (136) Ts1Cje (83) Ts1Rhr (41)
TRENDS in Molecular Medicine
Figure 1. Gene content of human chromosome 21 (Hsa21) and orthologous regions of mouse chromosome (Mmu) 16, Mmu17 and Mmu10, and widely used mouse models. Hsa21 contains 247 genes. Ts65Dn mice are trisomic for a region of Hsa21 that contains 136 genes. Ts1Cje mice contain a segment with 83 genes and Ts1Rhr mice have three copies of 41 genes from a region of Hsa21 previously known as the ‘critical region’ [2]. Mouse models with segmental trisomy for Hsa21-orthologous regions of Mmu17 and Mmu10 are under development in several laboratories. Abbreviation: n.r., not represented. Gene numbers were adapted from Ref. [16]. www.sciencedirect.com
Update
TRENDS in Molecular Medicine
identification of small changes in gene expression levels that are expected in trisomy. In Tc1 mice, approximately one third (39 in 131) of the Hsa21 genes on the chip gave a significantly elevated signal compared with euploid control RNA. It will be important to analyze gene expression levels with more sensitive methods to determine which human genes, if any, do not respond properly to their human regulatory signals in the developing mouse. Quantitative analysis of protein expression and activity remains to be completed.
DS phenotypes are recapitulated in Tc1 mice It will require much work to determine whether Tc1 mice display the many phenotypes that have been characterized in other mouse models of DS and, thus, assess the applicability of the Tc1 model to DS research, but initial observations are encouraging. Hippocampal function is especially affected in DS, and in Ts65Dn and Ts1Cje mouse models [8–10]. Similar to these mouse models, Tc1 mice display abnormal LTP. Tc1 mice also show decreased performance in a novel hippocampal-based object-recognition test, although no effects have been observed in several other tests related to hippocampal function. Decreased density of cerebellar granule cells occurs in Ts65Dn mice and in the brains of individuals with DS [11], and this has been also observed in Tc1 mice. Similar to Ts65Dn mice, Tc1 mice fail to recapitulate the histopathology of Alzheimer’s disease, which is present in all individuals with DS by the fourth decade. This might indicate that the biochemistry of neuritic plaque and tangle formation requires an absolute passage of time, not just ageing within the lifespan of the mouse. Tc1 mice have small mandibles, which is characteristic of individuals with DS and is observed in other mouse models [12]. An integrated morphometric assessment of the entire skull is still missing for Tc1 mice, but it is reasonable to assume that additional skeletal effects that are analogous to those in individuals with DS and other mouse models occur in these mice. For all phenotypes, individual variability might contribute to different outcomes in Tc1 mice. Further direct comparisons between Tc1 mice and other DS mouse models will be required in additional paradigms. However, these initial results indicate several parallels between important features of DS, which are recapitulated in other DS mouse models, and those observed in the Tc1 model. An important advantage of Tc1 mice over other DS mouse models is the occurrence of heart-septation defects. Trisomy 21 is the leading cause of congenital heart disease [13]. Although rare in Ts65Dn and other mouse models, heart defects occur in most Tc1 mice (a surprising 20% of control mice also showed ventricular septal defects). Two important conclusions from this observation are that: (i) trisomic mice display heart defects similar to those seen in DS; and (ii) at least one dosage-sensitive gene that affects heart septation resides in regions of the genome that are not triplicated in Ts65Dn mice. New mouse models with segmental trisomy in the regions of Mmu17 and Mmu10 that are orthologous to Hsa21 will enable further www.sciencedirect.com
Vol.12 No.6 June 2006
239
understanding of the genetic contributions that produce this effect in Tc1 mice. Limitations of the Tc1 model The Tc1 model is not a perfect model for DS (Box 1). In addition to the caveats mentioned, an important aim of DS research is to develop models that are easily propagated, to encourage more researchers to apply methods from their own fields to DS research. Like Ts65Dn mice, male Tc1 mice are usually sterile. Viable Tc1 mice are propagated at sub-Mendelian frequencies through trisomic females, which provide a trisomic intra-uterine environment and frequently show poor nurturing skills towards newborn pups. Tc1 mice display a high degree of mosaicism – that is, the human chromosome is lost from a subset of cells so that up to 50% of the cells in adult tissues of Tc1 mice no longer contain Hsa21. Previous experiments with chimeric mice that were generated using euploid embryos and mouse embryonic stem cells that contain Hsa21 demonstrated that trisomic cells were present in different Box 1. Outstanding questions † When and where does mosaicism occur during development? Is there a single event with subsequent selection of euploid versus trisomic cells, or is the human chromosome lost from multiple cell lineages at different times in development? † What additional phenotype assessments of Tc1 mice will be needed to compare them with phenotypes that have already been measured in other mouse models? Some assessments include: (i) Morris water maze, which is used to test hippocampal function. Ts65Dn mice show a robust deficit in this test; other models have milder effects that are correlated with the amount of genetic information from Hsa21 (or its mouse homologs) that is present. (ii) Cerebellar structure and function. Cerebellum is small and depauperate of neurons in individuals with DS and mouse models [11], including Tc1 mice. Sensitive tests are needed to assess outcomes of interventions to ameliorate this structural deficit [17]. (iii) Integrated morphometric assessment of the skull [12], which demonstrates direct similarities between DS and several other mouse models [2] should be undertaken in Tc1 mice. The prediction is that Tc1 mice should be more affected than Ts65Dn mice, with the caveat that mosaicism is predicted a priori to attenuate the effect of trisomy. If the frequency of mosaicism can be correlated with the degree of dysmorphology, this assessment might provide important insights into the pathogenesis of this phenotype. (iv) Assessment of LTP and LTD with the same methods that were used for other models [9]. The use of different paradigms for the same measurements in multiple electrophysiological (and behavioral) studies of trisomic mouse models has sometimes led to conflicting results that might reflect differences in methodology as much as or more than real differences in the models. † Which additional DS phenotypes can be studied in Tc1 mice and other models? Reduced tumor frequency in DS has been shown in several epidemiological studies [18]. Along with observations that suggest resistance to atherosclerosis in DS, these should be assessed in Tc1 and other DS models to identify contributing genes and mechanisms. † How are DS phenotypes caused (i.e. why does a slight elevation in the level of a normal gene or genes disrupt development)? Which genes contribute to which phenotypes? How can the effects of trisomy be ameliorated?
Update
240
TRENDS in Molecular Medicine
proportions in different tissues of the same mouse [14]. This was presumed to result from selection against trisomic cells, which differed in different tissues, as proposed earlier for chimeric mice in which one donor embryo contained a complete extra copy of Mmu16 due to a Robertsonian translocation [15]. However, mosaicism in Tc1 mice can only result from loss of Hsa21 that has been inherited from the mother, which was initially present in all cells. This is probably a mechanism by which ‘mosaic DS’ occurs in humans. The timing of Hsa21 loss in Tc1 mice, and whether it occurs once or in multiple cell lineages through development, is unknown. The impact in a given individual is not identical to that in any other individual, even if they have the same percentage of trisomic cells. This variability represents a substantial source of phenotypic variation. There is reasonable inferential evidence that this kind of mosaicism does not normally occur in mouse models and in humans with trisomy 21, but this finding in Tc1 mice suggests that the possibility of mosaicism should be examined more thoroughly in trisomic humans and mice. Concluding remarks The derivation of Tc1 mice has been an epic undertaking spanning O12 years. The effort has led to a great advance in DS research because this compelling model expands the range of phenotypes recapitulated between species with the analogous aneuploid gene set. The Tc1 model also provides an excellent system for the assessment of comparative gene-regulation effects in development. Acknowledgements We thank R. Roper, T. Sussan and C. Epstein for thoughtful suggestions about the manuscript and figures.
References 1 Epstein, C.J. (2001) Down syndrome (trisomy 21). In The Metabolic and Molecular Bases of Inherited Disease (Vol. 1) (Scriver, C.R. et al., eds), pp. 1223–1256, McGraw-Hill 2 Olson, L.E. et al. (2004) A chromosome 21 critical region does not cause specific down syndrome phenotypes. Science 306, 687–690
Vol.12 No.6 June 2006
3 Deutsch, S. et al. (2005) Gene expression variation and expression quantitative trait mapping of human chromosome 21 genes. Hum. Mol. Genet. 14, 3741–3749 4 Goodbourn, S. et al. (2000) Interferons: cell signalling, immune modulation, antiviral response and virus countermeasures. J. Gen. Virol. 81, 2341–2364 5 O’Doherty, A. et al. (2005) An aneuploid mouse strain carrying human chromosome 21 with down syndrome phenotypes. Science 309, 2033–2037 6 Kahlem, P. et al. (2004) Transcript level alterations reflect gene dosage effects across multiple tissues in a mouse model of down syndrome. Genome Res. 14, 1258–1267 7 Lyle, R. et al. (2004) Gene expression from the aneuploid chromosome in a trisomy mouse model of down syndrome. Genome Res. 14, 1268–1274 8 Pennington, B.F. et al. (2003) The neuropsychology of Down syndrome: evidence for hippocampal dysfunction. Child Dev. 74, 75–93 9 Kleschevnikov, A.M. et al. (2004) Hippocampal long-term potentiation suppressed by increased inhibition in the Ts65Dn mouse, a genetic model of Down syndrome. J. Neurosci. 24, 8153–8160 10 Siarey, R.J. et al. (1999) Increased synaptic depression in the Ts65Dn mouse, a model for mental retardation in Down syndrome. Neuropharmacology 38, 1917–1920 11 Baxter, L.L. et al. (2000) Discovery and genetic localization of Down syndrome cerebellar phenotypes using the Ts65Dn mouse. Hum. Mol. Genet. 9, 195–202 12 Richtsmeier, J. et al. (2000) Parallels of craniofacial maldevelopment in Down syndrome and Ts65Dn mice. Dev. Dyn. 217, 137–145 13 Ferencz, C. et al. (1989) Congenital cardiovascular malformations associated with chromosome abnormalities: an epidemiologic study. J. Pediatr. 114, 79–86 14 Shinohara, T. et al. (2001) Mice containing a human chromosome 21 model behavioral impairment and cardiac anomalies of Down’s syndrome. Hum. Mol. Genet. 10, 1163–1175 15 Gearhart, J. et al. (1986) Mouse chimeras composed of trisomy 16 and normal (2N) cells: preliminary studies. Brain Res. Bull. 16, 815–824 16 Antonarakis, S.E. et al. (2004) Chromosome 21 and down syndrome: from genomics to pathophysiology. Nat. Rev. Genet. 5, 725–738 17 Roper, R.J. et al. (2006) Defective cerebellar response to mitogenic Hedgehog signaling in Down syndrome mice. Proc. Natl. Acad. Sci. U. S. A. 103, 1452–1456 18 Yang, Q. et al. (2002) Mortality associated with Down’s syndrome in the USA from 1983 to 1997: a population-based study. Lancet 359, 1019–1025
1471-4914/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.molmed.2006.04.005
Prostaglandins and the colon cancer connection Timothy A. Chan Department of Radiation Oncology and Molecular Radiation Sciences, Sidney Kimmel Cancer Center, Johns Hopkins Hospital, 401 N. Broadway Street, Suite 1440, Baltimore, MD 21231, USA
Colorectal cancer is a leading cause of cancer-related deaths throughout the world. Non-steroidal anti-inflammatory drugs (NSAIDs) are among the few agents that are known to inhibit colorectal tumorigenesis. The mechanisms that underlie this effect are poorly Corresponding author: Chan, T.A. ([email protected]). Available online 2 May 2006 www.sciencedirect.com
understood. Two recent studies have provided some significant insight. Castellone and colleagues showed that prostaglandin E2 modulates the b-catenin signaling axis, a key pathway for colorectal tumorigenesis. Holla and colleagues showed that prostaglandin E2 might act via a nuclear receptor. These findings shed light on the mechanisms that underlie prostaglandin action, and