- Email: [email protected]
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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
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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
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provide a molecular framework for developing future treatments for colorectal cancer.
Colorectal cancer and chemoprevention Colorectal cancer is a leading cause of cancer death in the United States and many western nations [1]. The adoption of screening colonoscopy has helped reduce mortality caused by colorectal cancer. In addition, the development of refined surgical techniques, more effective chemotherapy regimens and targeted agents has improved treatment of the disease. Nevertheless, colorectal cancer remains an important cause of cancer-related morbidity and mortality. Given the high incidence of colorectal cancer, chemoprevention – the administration of a pharmacological compound to reduce cancer risk – represents an attractive strategy to tackle this disease. Substantial clinical data show that non-steroidal antiinflammatory drugs (NSAIDs) have anti-tumorigenic properties and reduce the risk of colorectal cancer development. Some epidemiological studies have shown that aspirin or NSAID use results in an impressive reduction in the risk of developing colorectal cancer [2]. However, how these drugs exert their effects is not well defined. The recent reports by Castellone et al. [3] and Holla et al. [4] greatly improve our understanding of the anti-tumorigenic properties of NSAIDs. Several randomized trials have now substantiated the ability of NSAIDs to reduce the risk of colorectal cancer or the development of adenomas, which are precursors to invasive disease. In two randomized, placebo-controlled trials, aspirin decreased the risk of polyp recurrence [5,6]. Other trials have shown that NSAIDs such as sulindac and celecoxib decrease the frequency of colorectal adenomas in patients with familial adenomatous polyposis, a dominantly inherited colon cancer syndrome that is caused by the inactivation of the tumor suppressor gene adenomatous polyposis coli (APC) [7]. How do NSAIDs exert their anti-tumorigenic effects? NSAIDs inhibit cyclooxygenase (COX) enzymes and prostaglandin synthases, which are key components in the arachidonic acid metabolism pathway. A substantial amount of clinical and experimental evidence suggests that this inhibition is responsible, at least in part, for the anti-cancer effects of NSAIDs. Colon cancers overexpress COX-2, the inducible isoform of the COX enzyme. COX-2 overexpression promotes tumor progression [8,9]. Treatment of cancer cells leads to inhibition of COX enzymes and consequent reduction of the levels of prostaglandin E2 (PGE2), a major product of the COX pathway. The inhibition of PGE2 probably suppresses tumorigenesis by either preventing growth or inducing apoptosis in tumor epithelia [10,11]. However, how the inhibition of PGE2 production leads to reduced tumor growth at the molecular level is unclear. Considerable controversy exists on whether inhibition of COX and PGE2 production has a key role in the antitumorigenic effects of NSAIDs. Because COX inhibition is the main mechanism by which NSAIDs inhibit the inflammatory response, investigators hypothesized that www.sciencedirect.com
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the inhibition of COX blocked the growth of cancer cells. Several lines of evidence suggest that COX inhibition is responsible, at least in part, for the anti-tumorigenic effects. First, when cancer cells are treated with NSAIDs such as aspirin, piroxicam, flurbiprofen, indomethacin or sulindac, growth is inhibited [12,13]. These compounds have different chemical structures and seem to share only their ability to inhibit COX. Second, Oshima et al. [9] provided direct genetic evidence for the importance of COX inhibition in preventing colorectal tumorigenesis. These authors observed that, although ApcD716 mice with one mutant Apc allele develop many tumors in their gastrointestinal tracts, ApcD716 mice with both COX-2 alleles deleted had an 86% reduction in polyp number [9]. This result shows that COX-2 is a genetic modifier of colorectal tumorigenesis, and that inhibition of the enzyme suppresses tumorigenesis. Other investigators have shown that COX-independent mechanisms are also involved. Stoner et al. [14] showed that sulindac sulfone, a compound that does not inhibit COX activity, is also effective at preventing tumor growth. NSAIDs suppress proliferation and cause apoptosis in cells that do not express COX enzymes [15]. Other groups have shown that NSAIDs also act via prostaglandin-independent pathways involving ceramide, nuclear factor-kB (NF-kB) or peroxisome proliferator-activated receptors (PPARs) [10,16,17]. Castellone et al. [3] and Holla et al. [4] showed that COXand prostaglandin-dependent mechanisms have important roles in tumorigenesis and provide a mechanistic understanding of the anti-cancer effects of NSAIDs.
Colorectal cancer biology The development of colorectal cancer results from mutations in several oncogenes and tumor suppressors. The multistep process that underlies the transformation of normal colonic epithelium into carcinomas requires mutation of the genes APC, b-catenin, Kirsten rat sarcoma 2 viral oncogene homolog (K-RAS) and p53. APC is of prime importance in the molecular pathogenesis of colorectal cancer because it is likely to act as a ‘gatekeeper’ gene for normal colon epithelial cells. Mutations in gatekeeper genes such as APC initiate the process of neoplastic transformation. In normal cells, APC modulates the b-catenin–T-cellfactor (TCF) signaling pathway. APC is normally present in a complex that contains other proteins: axin, b-catenin and glycogen synthase kinase 3b (GSK-3b). In the absence of growth signals, the APC–axin–GSK-3b complex inhibits b-catenin function and promotes its ubiquitin-dependent degradation. In the presence of growth signals, b-catenin is released and binds to the transcription factor TCF-4, leading to transactivation of genes that are required for growth [18]. In their recent report, Castellone et al. [3] demonstrated that PGE2 directly regulates APC–bcatenin signaling via axin, and provides a rationale for understanding how COX overexpression and inhibition modulates a pathway that is key to colorectal tumorigenesis [3].
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Prostaglandin E2 modulates b-catenin-mediated transcription Castellone et al. [3] showed that PGE2 is a potent mitogen that stimulates growth of colon cancer cells through b-catenin. The investigators treated colon cancer cells with PGE2 and measured b-catenin–TCF-4-dependent transcription with the TOP/FOP reporter assay. The TOP/ FOP assay uses a luciferase reporter gene that is placed under the transcriptional control of TCF-4 response elements. This assay enables the quantification of the level of b-catenin–TCF-4-dependent transcription [19]. Upon treatment with PGE2, b-catenin-dependent transcription increased dramatically. This increase occurred together with a reduction in b-catenin phosphorylation and an increase in cell proliferation, as measured using bromodeoxyuridine (BrdU) incorporation. These proliferative effects depended on b-catenin because smallinterfering-RNA (siRNA)-mediated knockdown of b-catenin blocked PGE2-induced BrdU incorporation. These results indicate that PGE2 can directly regulate the activity of the APC–b-catenin signaling axis. This regulation can change the proliferative status of colon cancer cells. The findings also show that PGE2 can modulate the activity of b-catenin and regulate proliferation independently of apoptosis. The importance of axin Several studies have shown that PGE2 binds to transmembrane, G-protein-coupled receptors such as EP2. Upon binding, EP2 activates Gs, which, in turn, modulates downstream signaling molecules [20]. Using an elegant series of in vitro experiments, Castellone et al. [3] showed that EP2 and PGE2 activate b-catenin–TCF transcription via the Gs protein but independently of cyclic adenosine monophosphate (cAMP) or protein kinase A (PKA). How is the PGE2 growth signal transduced to the b-catenin–TCF-4 complex? The authors observed that axin contains a regulatory domain of G-protein signaling (RGS); therefore, axin seemed to be a good candidate for linking EP2-activated Gas activity with regulation of b-catenin-dependent transcription. Axin binds to the APC–b-catenin–GSK-3b complex and inhibits b-catenin– TCF activity [21]. Using immunoprecipitation experiments, Castellone et al. [3] showed that, upon binding of PGE2 to EP2, Gas associates with axin via its RGS domain. This, in turn, stimulates the dissociation of axin from GSK-3b and relieves b-catenin from the inhibitory phosphorylation of GSK-3b. Thus, by promoting the release of GSK-3b from axin-containing complexes, PGE2 and Gas activate b-catenin–TCF activity, leading to the transcription of growth-promoting genes. Convergent pathways GSK-3b is a key component of both the signaling pathways of b-catenin and TCF and of phosphoinositide 3 kinase (PI3K) and AKT and, thus, is important in the regulation of cell growth and survival [22]. Activated PI3K and AKT lead to phosphorylation and inactivation of GSK-3b. Castellone et al. [3] tested whether the PI3K–AKT pathway also had a role in PGE2 signaling. They observed that PGE2 stimulated PI3K and AKT activity, resulting in www.sciencedirect.com
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the phosphorylation and inactivation of GSK-3b. Inactivation of GSK-3b resulted in the stabilization of b-catenin and the activation of b-catenin–TCF transcription. These effects of PGE2 on AKT and GSK-3b were mediated by PI3K-dependent signaling. Thus, the observations of Castellone et al. [3] suggest that the effects of PGE2 on b-catenin signaling occurs through at least two coordinated mechanisms, one that is initiated by Gas, leading to the dissociation of axin and GSK-3b, and another that act through Gbg and AKT, leading to the phosphorylation and inactivation of GSK-3b. Although intriguing, G-protein-coupled signaling through axin and GSK-3b might not be the only way PGE2 exerts its action. As reported in the paper by Castellone et al. [3], binding of PGE2 to its receptor results in the production of cAMP and the activation of PKA. Holla et al. [4] showed that this part of the signaling cascade might also have important functional consequences. The authors demonstrated that PGE2 induces a nuclear receptor called nuclear receptor subfamily 4, group A, member 2 (NR4A2). Originally identified as an important dopaminergic receptor in neurons, NR4A2 is rapidly and transiently induced by PGE2 in a cAMP- and PKA-dependent manner in colorectal carcinoma cells. The authors also showed that NR4A2 acts downstream of PGE2 and might be important in enabling PGE2 to prevent apoptosis after serum starvation. Moreover, Holla et al. [4] showed that NR4A2 is induced in the intestines of mice that are treated with PGE2 and is overexpressed in human colorectal carcinomas. These results demonstrate that the mechanism of action of PGE2 is complex and involves multiple signaling pathways (Figure 1). They also demonstrate that COX inhibition can regulate some nuclear receptors in a PGE2-dependent manner (e.g. NR4A2) and others in a prostaglandinindependent manner (e.g. PPARd). Concluding remarks The recent observations reported by Castellone et al. [3] and Holla et al. [4] greatly increased the knowledge of the molecular mechanisms that underlie PGE2 function. Castellone et al. [3] showed that PGE2 regulates cell proliferation by modulating the b-catenin–axin signaling pathway, which is essential for the development of colorectal cancer. Holla et al. [4] showed that PGE2 can also regulate apoptosis by transactivating the nuclear receptor NR4A2. These findings are relevant for both the understanding of the mechanisms of NSAIDmediated chemoprevention and of the molecular pathogenesis of colorectal cancer. However, several important questions remain to be answered. First, it will be important to understand the role of APC in the pathway described in the study by Castellone et al. [3]. APC is a key player in wnt signaling and also regulates b-catenin-dependent transcription. It is likely that APC modulates the effects of PGE2. It would be interesting to determine the effect that overexpression of APC has on the interaction between Gas and axin. Second, do the mechanisms described above have a direct role in NSAID-mediated chemoprevention? It will be important to determine whether NSAID treatment of
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† What role does APC have in the pathway proposed by Castellone et al. [3]? † Does treatment of colorectal cancer cells with NSAIDs modulate Gas–axin signaling directly? † Which of the many proposed mechanisms of NSAID-mediated chemoprevention are the most important clinically? † Can knowledge of prostaglandin–axin signaling be used to develop more effective therapies for chemoprevention?
NSAIDs COX
Answers to these important questions will help develop more effective agents for the prevention of colorectal and other tumors.
PGE2
EP receptor
+ cAMP–PKA
+ Gβγ
+ Gαs Axin + PI3K–AkT
NR4A2
β-catenin (active)
both alleles of axin have been replaced with altered axin alleles that have mutations in the RGS domain can be generated. These mice can then be crossed with multiple-intestinal-neoplasia mice (Min) to determine whether disruption of axin–Gas interaction affects tumor development. Additional work will be needed to answer these questions and identify which of the many proposed mechanisms are the most important (Box 1). Nevertheless, the findings of Castellone et al. [3] and Holla et al. [4] will help build a molecular framework for understanding NSAID-mediated chemoprevention. Such information can be used to help design better chemopreventive agents and targeted therapies.
GSK-3β
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Figure 1. The signaling pathway of prostaglandin E2. COX enzyme produces PGE2. PGE2 binds to and activates EP receptors. Upon ligand binding, EP receptor activates the cAMP–PKA signaling pathway, which leads to increased NR4A2 production [15]. Receptor activation also induces Gas to bind axin and displace GSK-3b. This occurs together with receptor activation of the PI3K–AKT pathway, leading to the phosphorylation and inactivation of GSK-3b. Together, these events result in the stabilization of b-catenin, enabling it to couple with TCF and transactivate genes that are required for growth and survival of cancer cells [11]. Abbreviations: AA, arachidonic acid; Gbg, G-protein bg subunits.
colorectal cancer cells directly modulates the pathways described by Castellone et al. [3] and Holla et al. [4], and whether regulation of these pathways is necessary for the chemopreventive effects of NSAIDs. If so, manipulation of the proteins that are involved in signaling through Gas and axin should alter the chemopreventive effects of NSAIDs. Studies that use transgenic mice and human somatic cell knockouts, in which various combinations of these genes are disrupted, might be useful in helping answer these questions. Indeed, it is known that NSAIDs can act on cancer cells via COX-dependent and COX-independent mechanisms, but the relative importance of these modes of action is unclear [17,23]. Third, which of the many mechanisms described in the literature about NSAID function are the most important for inhibition of tumorigenesis in vivo? Detailed examination of expression patterns in human tumors and dissection of the various pathways suggested that using genetic mouse models will help determine whether the models that were proposed by these two studies [3,4] prove to be relevant in tumors. For example, a mouse in which www.sciencedirect.com
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AA
Other eicosanoids
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We thank members of the Vogelstein-Kinzler laboratory for helpful discussions. We also thank the Department of Radiation Oncology and Molecular Radiation Sciences for support.
References 1 Jemal, A. et al. (2005) Cancer statistics, 2005. CA Cancer J. Clin. 55, 10–30 2 Janne, P.A. and Mayer, R.J. (2000) Chemoprevention of colorectal cancer. N. Engl. J. Med. 342, 1960–1968 3 Castellone, M.D. et al. (2005) Prostaglandin E2 promotes colon cancer cell growth through a Gs–axin–b-catenin signaling axis. Science 310, 1504–1510 4 Holla, V.R. et al. (2006) Prostaglandin E2 regulates the nuclear receptor NR4A2 in colorectal cancer. J. Biol. Chem. 281, 2676–2682 5 Baron, J.A. et al. (2003) A randomized trial of aspirin to prevent colorectal adenomas. N. Engl. J. Med. 348, 891–899 6 Sandler, R.S. et al. (2003) A randomized trial of aspirin to prevent colorectal adenomas in patients with previous colorectal cancer. N. Engl. J. Med. 348, 883–890 7 Jalving, M. et al. (2005) Review article: the potential of combinational regimen with non-steroidal anti-inflammatory drugs in the chemoprevention of colorectal cancer. Aliment. Pharmacol. Ther. 21, 321–339 8 Eberhart, C.E. et al. (1994) Up-regulation of cyclooxygenase 2 gene expression in human colorectal adenomas and adenocarcinomas. Gastroenterology 107, 1183–1188 9 Oshima, M. et al. (1996) Suppression of intestinal polyposis in ApcD716 knockout mice by inhibition of cyclooxygenase 2 (COX-2). Cell 87, 803–809 10 Chan, T.A. et al. (1998) Mechanisms underlying nonsteroidal antiinflammatory drug-mediated apoptosis. Proc. Natl. Acad. Sci. U. S. A. 95, 681–686 11 Tsujii, M. et al. (1998) Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell 93, 705–716 12 Mahmoud, N. et al. (1998) Aspirin prevents tumors in a murine model of familial adenomatous polyposis. Surgery 124, 225–231 13 Beazer-Barclay, Y. et al. (1996) Sulindac suppresses tumorigenesis in the Min mouse. Carcinogenesis 17, 1757–1760
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14 Stoner, G.D. et al. (1999) Sulindac sulfone induced regression of rectal polyps in patients with familial adenomatous polyposis. Adv. Exp. Med. Biol. 470, 45–53 15 Hanif, R. et al. (1996) Effects of nonsteroidal anti-inflammatory drugs on proliferation and on induction of apoptosis in colon cancer cells by a prostaglandin-independent pathway. Biochem. Pharmacol. 52, 237–245 16 Yamamoto, Y. et al. (1999) Sulindac inhibits activation of the NF-kB pathway. J. Biol. Chem. 274, 27307–27314 17 He, T.C. et al. (1999) PPARd is an APC-regulated target of nonsteroidal anti-inflammatory drugs. Cell 99, 335–345 18 Vogelstein, B. and Kinzler, K.W. (2004) Cancer genes and the pathways they control. Nat. Med. 10, 789–799 19 Korinek, V. et al. (1997) Constitutive transcriptional activation by a b-catenin–Tcf complex in APCK/K colon carcinoma. Science 275, 1784–1787
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20 Breyer, R.M. et al. (2001) Prostanoid receptors: subtypes and signaling. Annu. Rev. Pharmacol. Toxicol. 41, 661–690 21 Hart, M.J. et al. (1998) Downregulation of b-catenin by human axin and its association with the APC tumor suppressor, b-catenin and GSK3 b. Curr. Biol. 8, 573–581 22 Kim, L. and Kimmel, A.R. (2000) GSK3, a master switch regulating cell-fate specification and tumorigenesis. Curr. Opin. Genet. Dev. 10, 508–514 23 Chan, T.A. (2003) Cyclooxygenase inhibition and mechanisms of colorectal cancer prevention. Curr. Cancer Drug Targets 3, 455–463
1471-4914/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.molmed.2006.04.006
Reviving exhausted T lymphocytes during chronic virus infection by B7-H1 blockade Sheng Yao and Lieping Chen Department of Dermatology, Department of Oncology and the Institute for Cell Engineering, The Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA
Cytotoxic T lymphocytes (CTLs) are killer cells that are crucial in the control of viral pathogens and cancers. They can become exhausted during chronic viral infection, a phenomenon that consists of a reduction in both number and functionality of CTLs. Recently, Barber and colleagues demonstrated that B7-H1 (also called PD-L1), a cell-surface molecule that is widely distributed in tissues, was necessary for the maintenance of T-cell exhaustion in a chronic-infection mouse model of lymphocytic choriomeningitis virus (LCMV). PD-1, the receptor of B7-H1, was greatly upregulated on CTLs in response to LCMV, and its expression was maintained during chronic infection. Blockade of the B7H1–PD-1 pathway by a monoclonal antibody restored CTL function and reduced viral burden. These results suggest a new strategy for the treatment of chronic viral infection.
T cell and chronic viral infection In the first week of viral infection, a vigorous CD8C cytotoxic-T-lymphocyte (CTL) response is mounted. This acute response is virus-specific and includes proliferation and maturation of CTL effector function, leading to virus clearance and containment. Effector CTLs can either directly lyse infected cells through perforin- and/or Fas– FasL-dependent pathways, or inhibit virus replication by secreting cytokines such as interferon-g (IFN-g) and tumor necrosis factor (TNF). CTLs subsequently enter a contraction phase where most cells undergo apoptosis. Some of the remaining cells become memory T cells, which Corresponding author: Chen, L. ([email protected]). Available online 2 May 2006 www.sciencedirect.com
persist by a poorly understood homeostatic mechanism for a long period of time [1]. Memory CD8C T cells can quickly proliferate and regain effector function upon antigen reexposure. If initial CTL response during the acute phase of infection fails to eradicate viral pathogens, a chronic viral infection is established. Persistent infections of human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV) are world-wide health problems [2,3].
Impairment of CTL function in chronic viral infection During chronic infection, viral-antigen-specific CTLs lose function by three major mechanisms: deletion, exhaustion and peripheral tolerance (Box 1). In a mouse model of chronic infection of lymphocytic choriomeningitis virus (LCMV) [4,5], LCMV is cleared by healthy wild-type mice during acute-infection phase, whereas several highly invasive laboratory-derived LCMV clones cause longterm infection in blood and multiple organs. Barber et al. [5] demonstrated that, when compared with viruscleared mice 60 days post-infection, mice that are chronically infected with an invasive LCMV clone had only half of the antigen-specific memory CD8C T-cell population and their remaining memory T cells were severely impaired in effector function, which is a typical case of CTL exhaustion. To identify genes responsible for this functional suppression, the authors compared gene expression profiles of memory CD8C T cells derived from virus-cleared mice with those of chronically infected mice. Programmed death-1 (PD-1) emerged as a candidate that is highly upregulated on chronically infected memory T cells at both mRNA and protein level. In chronically infected mice, Barber et al. [5] also observed persistent
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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
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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
Update
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provide a molecular framework for developing future treatments for colorectal cancer.
Colorectal cancer and chemoprevention Colorectal cancer is a leading cause of cancer death in the United States and many western nations [1]. The adoption of screening colonoscopy has helped reduce mortality caused by colorectal cancer. In addition, the development of refined surgical techniques, more effective chemotherapy regimens and targeted agents has improved treatment of the disease. Nevertheless, colorectal cancer remains an important cause of cancer-related morbidity and mortality. Given the high incidence of colorectal cancer, chemoprevention – the administration of a pharmacological compound to reduce cancer risk – represents an attractive strategy to tackle this disease. Substantial clinical data show that non-steroidal antiinflammatory drugs (NSAIDs) have anti-tumorigenic properties and reduce the risk of colorectal cancer development. Some epidemiological studies have shown that aspirin or NSAID use results in an impressive reduction in the risk of developing colorectal cancer [2]. However, how these drugs exert their effects is not well defined. The recent reports by Castellone et al. [3] and Holla et al. [4] greatly improve our understanding of the anti-tumorigenic properties of NSAIDs. Several randomized trials have now substantiated the ability of NSAIDs to reduce the risk of colorectal cancer or the development of adenomas, which are precursors to invasive disease. In two randomized, placebo-controlled trials, aspirin decreased the risk of polyp recurrence [5,6]. Other trials have shown that NSAIDs such as sulindac and celecoxib decrease the frequency of colorectal adenomas in patients with familial adenomatous polyposis, a dominantly inherited colon cancer syndrome that is caused by the inactivation of the tumor suppressor gene adenomatous polyposis coli (APC) [7]. How do NSAIDs exert their anti-tumorigenic effects? NSAIDs inhibit cyclooxygenase (COX) enzymes and prostaglandin synthases, which are key components in the arachidonic acid metabolism pathway. A substantial amount of clinical and experimental evidence suggests that this inhibition is responsible, at least in part, for the anti-cancer effects of NSAIDs. Colon cancers overexpress COX-2, the inducible isoform of the COX enzyme. COX-2 overexpression promotes tumor progression [8,9]. Treatment of cancer cells leads to inhibition of COX enzymes and consequent reduction of the levels of prostaglandin E2 (PGE2), a major product of the COX pathway. The inhibition of PGE2 probably suppresses tumorigenesis by either preventing growth or inducing apoptosis in tumor epithelia [10,11]. However, how the inhibition of PGE2 production leads to reduced tumor growth at the molecular level is unclear. Considerable controversy exists on whether inhibition of COX and PGE2 production has a key role in the antitumorigenic effects of NSAIDs. Because COX inhibition is the main mechanism by which NSAIDs inhibit the inflammatory response, investigators hypothesized that www.sciencedirect.com
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the inhibition of COX blocked the growth of cancer cells. Several lines of evidence suggest that COX inhibition is responsible, at least in part, for the anti-tumorigenic effects. First, when cancer cells are treated with NSAIDs such as aspirin, piroxicam, flurbiprofen, indomethacin or sulindac, growth is inhibited [12,13]. These compounds have different chemical structures and seem to share only their ability to inhibit COX. Second, Oshima et al. [9] provided direct genetic evidence for the importance of COX inhibition in preventing colorectal tumorigenesis. These authors observed that, although ApcD716 mice with one mutant Apc allele develop many tumors in their gastrointestinal tracts, ApcD716 mice with both COX-2 alleles deleted had an 86% reduction in polyp number [9]. This result shows that COX-2 is a genetic modifier of colorectal tumorigenesis, and that inhibition of the enzyme suppresses tumorigenesis. Other investigators have shown that COX-independent mechanisms are also involved. Stoner et al. [14] showed that sulindac sulfone, a compound that does not inhibit COX activity, is also effective at preventing tumor growth. NSAIDs suppress proliferation and cause apoptosis in cells that do not express COX enzymes [15]. Other groups have shown that NSAIDs also act via prostaglandin-independent pathways involving ceramide, nuclear factor-kB (NF-kB) or peroxisome proliferator-activated receptors (PPARs) [10,16,17]. Castellone et al. [3] and Holla et al. [4] showed that COXand prostaglandin-dependent mechanisms have important roles in tumorigenesis and provide a mechanistic understanding of the anti-cancer effects of NSAIDs.
Colorectal cancer biology The development of colorectal cancer results from mutations in several oncogenes and tumor suppressors. The multistep process that underlies the transformation of normal colonic epithelium into carcinomas requires mutation of the genes APC, b-catenin, Kirsten rat sarcoma 2 viral oncogene homolog (K-RAS) and p53. APC is of prime importance in the molecular pathogenesis of colorectal cancer because it is likely to act as a ‘gatekeeper’ gene for normal colon epithelial cells. Mutations in gatekeeper genes such as APC initiate the process of neoplastic transformation. In normal cells, APC modulates the b-catenin–T-cellfactor (TCF) signaling pathway. APC is normally present in a complex that contains other proteins: axin, b-catenin and glycogen synthase kinase 3b (GSK-3b). In the absence of growth signals, the APC–axin–GSK-3b complex inhibits b-catenin function and promotes its ubiquitin-dependent degradation. In the presence of growth signals, b-catenin is released and binds to the transcription factor TCF-4, leading to transactivation of genes that are required for growth [18]. In their recent report, Castellone et al. [3] demonstrated that PGE2 directly regulates APC–bcatenin signaling via axin, and provides a rationale for understanding how COX overexpression and inhibition modulates a pathway that is key to colorectal tumorigenesis [3].
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Prostaglandin E2 modulates b-catenin-mediated transcription Castellone et al. [3] showed that PGE2 is a potent mitogen that stimulates growth of colon cancer cells through b-catenin. The investigators treated colon cancer cells with PGE2 and measured b-catenin–TCF-4-dependent transcription with the TOP/FOP reporter assay. The TOP/ FOP assay uses a luciferase reporter gene that is placed under the transcriptional control of TCF-4 response elements. This assay enables the quantification of the level of b-catenin–TCF-4-dependent transcription [19]. Upon treatment with PGE2, b-catenin-dependent transcription increased dramatically. This increase occurred together with a reduction in b-catenin phosphorylation and an increase in cell proliferation, as measured using bromodeoxyuridine (BrdU) incorporation. These proliferative effects depended on b-catenin because smallinterfering-RNA (siRNA)-mediated knockdown of b-catenin blocked PGE2-induced BrdU incorporation. These results indicate that PGE2 can directly regulate the activity of the APC–b-catenin signaling axis. This regulation can change the proliferative status of colon cancer cells. The findings also show that PGE2 can modulate the activity of b-catenin and regulate proliferation independently of apoptosis. The importance of axin Several studies have shown that PGE2 binds to transmembrane, G-protein-coupled receptors such as EP2. Upon binding, EP2 activates Gs, which, in turn, modulates downstream signaling molecules [20]. Using an elegant series of in vitro experiments, Castellone et al. [3] showed that EP2 and PGE2 activate b-catenin–TCF transcription via the Gs protein but independently of cyclic adenosine monophosphate (cAMP) or protein kinase A (PKA). How is the PGE2 growth signal transduced to the b-catenin–TCF-4 complex? The authors observed that axin contains a regulatory domain of G-protein signaling (RGS); therefore, axin seemed to be a good candidate for linking EP2-activated Gas activity with regulation of b-catenin-dependent transcription. Axin binds to the APC–b-catenin–GSK-3b complex and inhibits b-catenin– TCF activity [21]. Using immunoprecipitation experiments, Castellone et al. [3] showed that, upon binding of PGE2 to EP2, Gas associates with axin via its RGS domain. This, in turn, stimulates the dissociation of axin from GSK-3b and relieves b-catenin from the inhibitory phosphorylation of GSK-3b. Thus, by promoting the release of GSK-3b from axin-containing complexes, PGE2 and Gas activate b-catenin–TCF activity, leading to the transcription of growth-promoting genes. Convergent pathways GSK-3b is a key component of both the signaling pathways of b-catenin and TCF and of phosphoinositide 3 kinase (PI3K) and AKT and, thus, is important in the regulation of cell growth and survival [22]. Activated PI3K and AKT lead to phosphorylation and inactivation of GSK-3b. Castellone et al. [3] tested whether the PI3K–AKT pathway also had a role in PGE2 signaling. They observed that PGE2 stimulated PI3K and AKT activity, resulting in www.sciencedirect.com
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the phosphorylation and inactivation of GSK-3b. Inactivation of GSK-3b resulted in the stabilization of b-catenin and the activation of b-catenin–TCF transcription. These effects of PGE2 on AKT and GSK-3b were mediated by PI3K-dependent signaling. Thus, the observations of Castellone et al. [3] suggest that the effects of PGE2 on b-catenin signaling occurs through at least two coordinated mechanisms, one that is initiated by Gas, leading to the dissociation of axin and GSK-3b, and another that act through Gbg and AKT, leading to the phosphorylation and inactivation of GSK-3b. Although intriguing, G-protein-coupled signaling through axin and GSK-3b might not be the only way PGE2 exerts its action. As reported in the paper by Castellone et al. [3], binding of PGE2 to its receptor results in the production of cAMP and the activation of PKA. Holla et al. [4] showed that this part of the signaling cascade might also have important functional consequences. The authors demonstrated that PGE2 induces a nuclear receptor called nuclear receptor subfamily 4, group A, member 2 (NR4A2). Originally identified as an important dopaminergic receptor in neurons, NR4A2 is rapidly and transiently induced by PGE2 in a cAMP- and PKA-dependent manner in colorectal carcinoma cells. The authors also showed that NR4A2 acts downstream of PGE2 and might be important in enabling PGE2 to prevent apoptosis after serum starvation. Moreover, Holla et al. [4] showed that NR4A2 is induced in the intestines of mice that are treated with PGE2 and is overexpressed in human colorectal carcinomas. These results demonstrate that the mechanism of action of PGE2 is complex and involves multiple signaling pathways (Figure 1). They also demonstrate that COX inhibition can regulate some nuclear receptors in a PGE2-dependent manner (e.g. NR4A2) and others in a prostaglandinindependent manner (e.g. PPARd). Concluding remarks The recent observations reported by Castellone et al. [3] and Holla et al. [4] greatly increased the knowledge of the molecular mechanisms that underlie PGE2 function. Castellone et al. [3] showed that PGE2 regulates cell proliferation by modulating the b-catenin–axin signaling pathway, which is essential for the development of colorectal cancer. Holla et al. [4] showed that PGE2 can also regulate apoptosis by transactivating the nuclear receptor NR4A2. These findings are relevant for both the understanding of the mechanisms of NSAIDmediated chemoprevention and of the molecular pathogenesis of colorectal cancer. However, several important questions remain to be answered. First, it will be important to understand the role of APC in the pathway described in the study by Castellone et al. [3]. APC is a key player in wnt signaling and also regulates b-catenin-dependent transcription. It is likely that APC modulates the effects of PGE2. It would be interesting to determine the effect that overexpression of APC has on the interaction between Gas and axin. Second, do the mechanisms described above have a direct role in NSAID-mediated chemoprevention? It will be important to determine whether NSAID treatment of
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† What role does APC have in the pathway proposed by Castellone et al. [3]? † Does treatment of colorectal cancer cells with NSAIDs modulate Gas–axin signaling directly? † Which of the many proposed mechanisms of NSAID-mediated chemoprevention are the most important clinically? † Can knowledge of prostaglandin–axin signaling be used to develop more effective therapies for chemoprevention?
NSAIDs COX
Answers to these important questions will help develop more effective agents for the prevention of colorectal and other tumors.
PGE2
EP receptor
+ cAMP–PKA
+ Gβγ
+ Gαs Axin + PI3K–AkT
NR4A2
β-catenin (active)
both alleles of axin have been replaced with altered axin alleles that have mutations in the RGS domain can be generated. These mice can then be crossed with multiple-intestinal-neoplasia mice (Min) to determine whether disruption of axin–Gas interaction affects tumor development. Additional work will be needed to answer these questions and identify which of the many proposed mechanisms are the most important (Box 1). Nevertheless, the findings of Castellone et al. [3] and Holla et al. [4] will help build a molecular framework for understanding NSAID-mediated chemoprevention. Such information can be used to help design better chemopreventive agents and targeted therapies.
GSK-3β
Acknowledgements TRENDS in Molecular Medicine
Figure 1. The signaling pathway of prostaglandin E2. COX enzyme produces PGE2. PGE2 binds to and activates EP receptors. Upon ligand binding, EP receptor activates the cAMP–PKA signaling pathway, which leads to increased NR4A2 production [15]. Receptor activation also induces Gas to bind axin and displace GSK-3b. This occurs together with receptor activation of the PI3K–AKT pathway, leading to the phosphorylation and inactivation of GSK-3b. Together, these events result in the stabilization of b-catenin, enabling it to couple with TCF and transactivate genes that are required for growth and survival of cancer cells [11]. Abbreviations: AA, arachidonic acid; Gbg, G-protein bg subunits.
colorectal cancer cells directly modulates the pathways described by Castellone et al. [3] and Holla et al. [4], and whether regulation of these pathways is necessary for the chemopreventive effects of NSAIDs. If so, manipulation of the proteins that are involved in signaling through Gas and axin should alter the chemopreventive effects of NSAIDs. Studies that use transgenic mice and human somatic cell knockouts, in which various combinations of these genes are disrupted, might be useful in helping answer these questions. Indeed, it is known that NSAIDs can act on cancer cells via COX-dependent and COX-independent mechanisms, but the relative importance of these modes of action is unclear [17,23]. Third, which of the many mechanisms described in the literature about NSAID function are the most important for inhibition of tumorigenesis in vivo? Detailed examination of expression patterns in human tumors and dissection of the various pathways suggested that using genetic mouse models will help determine whether the models that were proposed by these two studies [3,4] prove to be relevant in tumors. For example, a mouse in which www.sciencedirect.com
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Box 1. Outstanding questions
AA
Other eicosanoids
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We thank members of the Vogelstein-Kinzler laboratory for helpful discussions. We also thank the Department of Radiation Oncology and Molecular Radiation Sciences for support.
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14 Stoner, G.D. et al. (1999) Sulindac sulfone induced regression of rectal polyps in patients with familial adenomatous polyposis. Adv. Exp. Med. Biol. 470, 45–53 15 Hanif, R. et al. (1996) Effects of nonsteroidal anti-inflammatory drugs on proliferation and on induction of apoptosis in colon cancer cells by a prostaglandin-independent pathway. Biochem. Pharmacol. 52, 237–245 16 Yamamoto, Y. et al. (1999) Sulindac inhibits activation of the NF-kB pathway. J. Biol. Chem. 274, 27307–27314 17 He, T.C. et al. (1999) PPARd is an APC-regulated target of nonsteroidal anti-inflammatory drugs. Cell 99, 335–345 18 Vogelstein, B. and Kinzler, K.W. (2004) Cancer genes and the pathways they control. Nat. Med. 10, 789–799 19 Korinek, V. et al. (1997) Constitutive transcriptional activation by a b-catenin–Tcf complex in APCK/K colon carcinoma. Science 275, 1784–1787
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20 Breyer, R.M. et al. (2001) Prostanoid receptors: subtypes and signaling. Annu. Rev. Pharmacol. Toxicol. 41, 661–690 21 Hart, M.J. et al. (1998) Downregulation of b-catenin by human axin and its association with the APC tumor suppressor, b-catenin and GSK3 b. Curr. Biol. 8, 573–581 22 Kim, L. and Kimmel, A.R. (2000) GSK3, a master switch regulating cell-fate specification and tumorigenesis. Curr. Opin. Genet. Dev. 10, 508–514 23 Chan, T.A. (2003) Cyclooxygenase inhibition and mechanisms of colorectal cancer prevention. Curr. Cancer Drug Targets 3, 455–463
1471-4914/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.molmed.2006.04.006
Reviving exhausted T lymphocytes during chronic virus infection by B7-H1 blockade Sheng Yao and Lieping Chen Department of Dermatology, Department of Oncology and the Institute for Cell Engineering, The Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA
Cytotoxic T lymphocytes (CTLs) are killer cells that are crucial in the control of viral pathogens and cancers. They can become exhausted during chronic viral infection, a phenomenon that consists of a reduction in both number and functionality of CTLs. Recently, Barber and colleagues demonstrated that B7-H1 (also called PD-L1), a cell-surface molecule that is widely distributed in tissues, was necessary for the maintenance of T-cell exhaustion in a chronic-infection mouse model of lymphocytic choriomeningitis virus (LCMV). PD-1, the receptor of B7-H1, was greatly upregulated on CTLs in response to LCMV, and its expression was maintained during chronic infection. Blockade of the B7H1–PD-1 pathway by a monoclonal antibody restored CTL function and reduced viral burden. These results suggest a new strategy for the treatment of chronic viral infection.
T cell and chronic viral infection In the first week of viral infection, a vigorous CD8C cytotoxic-T-lymphocyte (CTL) response is mounted. This acute response is virus-specific and includes proliferation and maturation of CTL effector function, leading to virus clearance and containment. Effector CTLs can either directly lyse infected cells through perforin- and/or Fas– FasL-dependent pathways, or inhibit virus replication by secreting cytokines such as interferon-g (IFN-g) and tumor necrosis factor (TNF). CTLs subsequently enter a contraction phase where most cells undergo apoptosis. Some of the remaining cells become memory T cells, which Corresponding author: Chen, L. ([email protected]). Available online 2 May 2006 www.sciencedirect.com
persist by a poorly understood homeostatic mechanism for a long period of time [1]. Memory CD8C T cells can quickly proliferate and regain effector function upon antigen reexposure. If initial CTL response during the acute phase of infection fails to eradicate viral pathogens, a chronic viral infection is established. Persistent infections of human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV) are world-wide health problems [2,3].
Impairment of CTL function in chronic viral infection During chronic infection, viral-antigen-specific CTLs lose function by three major mechanisms: deletion, exhaustion and peripheral tolerance (Box 1). In a mouse model of chronic infection of lymphocytic choriomeningitis virus (LCMV) [4,5], LCMV is cleared by healthy wild-type mice during acute-infection phase, whereas several highly invasive laboratory-derived LCMV clones cause longterm infection in blood and multiple organs. Barber et al. [5] demonstrated that, when compared with viruscleared mice 60 days post-infection, mice that are chronically infected with an invasive LCMV clone had only half of the antigen-specific memory CD8C T-cell population and their remaining memory T cells were severely impaired in effector function, which is a typical case of CTL exhaustion. To identify genes responsible for this functional suppression, the authors compared gene expression profiles of memory CD8C T cells derived from virus-cleared mice with those of chronically infected mice. Programmed death-1 (PD-1) emerged as a candidate that is highly upregulated on chronically infected memory T cells at both mRNA and protein level. In chronically infected mice, Barber et al. [5] also observed persistent