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The Rho-activating CNF1 toxin from pathogenic E. coli: A risk factor for human cancer development?

The Rho-activating CNF1 toxin from pathogenic E. coli: A risk factor for human cancer development? Nowadays, there is increasing evidence that some pathogenic bacteria can contribute to specific stages of cancer development. The concept that bacterial infection could be involved in carcinogenesis acquired a widespread interest with the discovery that H. pylori is able to establish chronic infections in the stomach and that this infection is associated with an increased risk of gastric adenocarcinoma and mucosa associated lymphoid tissue lymphoma. Chronic infections triggered by bacteria can facilitate tumor initiation or progression since, during the course of infection, normal cell functions can come under the control of pathogen factors that directly manipulate the host regulatory pathways and the inflammatory reactions. Renowned publications have recently corroborated the molecular mechanisms that link bacterial infections, inflammation and cancer, indicating certain strains of Escherichia coli as a risk factor for patients with colon cancer. E. coli is a normal inhabitant of the human intestine that becomes highly pathogenic following the acquisition of virulence factors, including a protein toxin named cytotoxic necrotizing factor 1 (CNF1). This toxin permanently activates the small GTP-binding proteins belonging to the Rho family, thus promoting a prominent polymerization of the actin cytoskeleton as well as a number of cellular responses, including changes in protein expression and functional modification of the cell physiology. CNF1 is receiving an increasing attention as a putative factor involved in transformation because of its ability to: (i) induce COX2 expression, an immediate-early gene over-expressed in some type of cancers; (ii) induce a long-lasting activation of the transcription factor NF-kB, a largely accepted marker of tumor cells; (iii) protect epithelial cells from apoptosis; (iv) ensue the release of pro-inflammatory cytokines in epithelial and endothelial cells; and (v) promote cellular motility. As cancer may arise through dysfunction of the same regulatory systems, it seems likely that CNF1-producing E. coli infections can contribute to tumor development. This review focuses on the aspects of CNF1 activity linked to cell transformation with the aim of contributing to the identification of a possible carcinogenic agent from the microbial world. Page 1 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:4 http://www.infectagentscancer.com/content/3/1/4 pathway is triggered by bacterial and viral infections, as Introduction Bacterial infection and cancer well as by pro-inflammatory cytokines, such as TNF-α and In the last century, cancer research thoroughly established IL-1, all of which activate the IKK complex [17]. This com- the role of major carcinogenic agents of different nature, plex phosphorilates the NF-kB inhibitors IkBs, thereby including infectious agents. However, although there is a targeting them for proteosomal degradation and freeing general agreement that some viruses, such as hepatitis B NF-kB to enter the nucleus and mediate transcription of virus (HBV), Epstein-Barr virus (EBV) and human papil- target genes. It is worth noting that many of the genes able loma virus (HPV) can cause cancer, the involvement of to mediate alterations characterizing a tumor cell are bacteria in carcinogenesis remains controversial. The role under the transcriptional control of NF-kB (reviewed in of viral infections in tumor onset is widely accepted [18,19]). For example, the activity and expression of cyc- because of the direct action of single viral genes (onco- lin D1, CDK2 kinase, c-myc, p21, p53 and pRb, which are genes) that result in cell transformation [1]. By contrast, involved in the control of cell cycle and are altered in sev- the molecular mechanism(s) by which bacteria might eral types of cancer, are NF-kB-dependent. The expression promote tumorigenesis are still poorly characterized. of numerous cytokines, that are growth factors for tumor Hence, one of the main challenges, nowadays, is to define cells (IL-1β, TNF, IL-6, EGF) are also regulated by NF-kB. the impact of bacterial infections as a cause of cancer and Tissue invasion and metastasis, two crucial events of eventually design strategies for their prevention and con- tumor progression, are regulated by NF-kB-dependent trol. genes, including metalloproteases (MMPs), urokinase type of plasminogen activator (uPA), IL-8, the adhesion Bacterial infections are usually believed to cause acute dis- molecules VCAM-1, ICAM-1 and ELAM-1. NF-kB is also eases, but it is now becoming clear that some bacteria can involved in the regulation of angiogenesis, the process by contribute to the establishment of chronic diseases, which tumor cells promote neo-vascularization. Finally, including cancer [2]. The concept that bacterial infection altered expression of genes involved in suppression of could be involved in carcinogenesis was first proposed in apoptosis (i.e. Bcl-2 family members and IAP proteins), a the late nineteenth and early twentieth centuries, based key feature of cancer cells, is often due to deregulated NF- on the discovery of bacteria at the sites of tumors, kB activity. although there was no proof that the bacteria were in any way causative [3]. Since then, the putative link between Concerning this last point, several pathogenic bacteria, chronic infection and cancer acquired a widespread inter- particularly those that can establish a persistent intracellu- est with the discovery that Helicobacter pylori is able to lar infection, activate NF-kB in the host cell and suppress establish chronic infections in the stomach and that this cell death, thus creating a niche in which the bacterium infection is associated with an increased risk of gastric can survive, in spite of the attempts of the host immune adenocarcinoma [4] and mucosa associated lymphoid tis- system to destroy the infected cell [20]. As a consequence, sue (MALT) lymphoma [5]. In this context, it is worth not- the suppression of apoptosis by a pathogen might also ing that H. pylori is classified as a class I carcinogenic factor allow a partially transformed cell to evade the self-destruc- [6]. Other chronic bacterial infections have been linked to tive process and to progress to a higher level of transfor- human carcinogenesis although the underlying mecha- mation. nisms remain to be defined (reviewed in [2]). The strong- est epidemiological case is for Salmonella enterica serovar Another important feature of inflammation-associated typhi (S. typhi), the agent of typhoid, which can also lead cancer is the production of reactive oxygen species (ROS) to chronic bacterial carriage in the gallbladder [7-11]. Sur- and nitric oxide (NO) by inflammatory and epithelial veys of typhoid outbreaks have shown that those who cells. This leads to increased mutations and altered func- become carriers have an increased risk of developing tions of important enzymes and proteins in inflamed tis- hepatobiliary carcinoma compared with people who have sue, thus contributing to the multistage carcinogenetic had acute typhoid and have cleared the infection [9]. process [21]. For example, during a chronic H. pylori infec- tion, production of ROS and nitroxides and the associated A recurring theme in the link between bacterial infection inflammatory response are assumed to contribute to the and carcinogenesis is that of chronic inflammation, which induction of a gastric carcinogenic process [22,23]. These is often a common feature of persistent infection [2,12]. chemical species are mainly produced by inflammatory One of the key molecules that link chronic inflammation cells to fight infection and are source of oxidative DNA and cancer is represented by the NF-kB family of transcrip- damage, thus contributing to carcinogenesis [22,23]. In tion factors [12,13]. In particular, different mouse studies particular, it has been demonstrated that within 6 hours, provide strong and direct genetic evidence that the classi- H. pylori infection had a mutagenic effect on gastric epi- cal, IKK-β dependent NF-kB activation pathway is indeed thelial cells [24]. The major form of oxidative DNA dam- a crucial mediator of tumor promotion [14-16]. This age is the formation of 8-oxoG lesions, specifically Page 2 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:4 http://www.infectagentscancer.com/content/3/1/4 repaired by the OGG1 DNA glycosylase. The inactivation and cell matrix interactions may serve as an early event in of this enzyme inhibits the level of inflammatory lesions H. pylori-induced carcinogenesis [30]. Very recently, in and abolishes the mutagenic effect induced by the infec- experimental studies dealing with H. pylori strains carrying tion at the gastric level, thus strengthening a close relation or not CagA, it has been demonstrated that this factor acti- between chronic inflammation and genotoxicity [24]. vates host cell survival and anti-apoptotic pathways to overcome self-renewal of the gastric epithelium, thus Bacterial toxins and cancer enhancing bacterial colonization of the stomach and As stated above, there is increasing evidence that some helping sustained H. pylori infection. In this context, it is pathogenic bacteria can contribute to specific stages of interesting to note that, patients infected with H. pylori cancer development. In particular, chronic infections trig- encoding the cag pathogeniticy island (PAI) are associated gered by bacteria can facilitate tumor initiation or progres- with an increased risk of gastric cancer [31]. sion since, during the course of infection, normal cell functions can undergo the control of factors released by In addition to the link between H. pylori and stomach can- the pathogen [2]. These bacterial factors, namely virulence cers, very recent studies evidenced that some other toxins factors, can directly manipulate the host regulatory path- may contribute to bowel and urogenital tract cancers [3]. ways and the inflammatory reaction [3]. In this context, it has been reported that adherent and invasive strains of Escherichia coli are a risk factor for Bacteria express a wide range of virulence factors, includ- patients with pre-cancerous and cancerous colon diseases ing protein toxins that have evolved to interact with [32,33]. Interestingly, in one of these studies, 3 out of 8 eukaryotic cellular machinery in a precise way. These tox- cancer-associated E. coli were reported to possess the cyto- ins interfere with key eukaryotic processes, such as cellular toxic necrotizing factor 1 (cnf1) gene [32], the gene coding signaling components, and some directly attack the for the protein toxin CNF1. The aim of this review is to genome [25-27]. These last can damage DNA via different highlight the main cell responses to CNF1, particularly mechanisms: i) directly by enzymatic attack, ii) indirectly those related to signaling pathways linked to inflamma- by provoking an inflammatory reaction that produces free tion and to cell transformation. radicals, or even iii) by affecting DNA repair mechanisms. Nougayrède and colleagues [28] have recently identified a CNF1 from E. coli: a protein toxin that activates novel hybrid peptide-polyketide compound from the Rho GTPases Escherichia coli that leads to DNA damage. This novel com- Although belonging to the normal human intestinal flora, pound is produced by pathogenic and, most interestingly, E. coli becomes highly pathogenic following the acquisi- commensal isolates. Although it is not yet clear how the tion of genes coding for virulence factors, one of which peptide-polyketide compound functions at the molecular being CNF1 [34]. CNF1-producing E. coli strains are occa- level, it is possible that it contributes to bacterial patho- sionally detected in isolates from feces of children with genesis and bacterially-induced carcinogenesis. diarrhea [35-37], but, more frequently, are responsible of extraintestinal infections, particularly in the urinary tract Any bacterial product that interferes with signaling, result- (UTIs) [38-40]. Also, these strains can be detected in cases ing in the disruption of the normal balance of growth, cell of bacteraemia [41] and of meningitis in neonates [42]. division and apoptosis, could facilitate tumor promotion. CNF1, first described in 1983 by Caprioli and coworkers Similarly, the ability to promote anchorage-independent as a toxin capable of causing multinucleation ("cyto- growth could favor metastatic potential and lead to cancer toxic") in cultured cells (Fig. 1a, b) and necrosis in rabbit progression. So far, the best example of potentially carci- skin ("necrotizing") [43,44], is a single-chain multido- nogenic toxin is H. pylori CagA that interferes with cellular main protein toxin, containing a binding domain to a cell signaling mechanisms in a way that is characteristic of receptor (N-terminal domain), a translocation domain tumor promoters. Indeed, CagA intracellularly interacts, (middle domain) and an enzymatic one (C-terminal in a phosphorylation-dependent and independent way, domain) [45,46], which modifies a specific cellular target with many host proteins that regulate cell growth, motil- in the host cell cytosol. CNF1 binds to the surface of cul- ity and polarity, thus leading to gastric epithelial prolifer- tured epithelial cells with high affinity [47], and the 67- ation, cell-cell dissociation and increased cell scattering kDa laminin receptor has been suggested (67LR) as a and motility (for a review [29]). In particular, Bagnoli and putative receptor for CNF1 [48]. After binding to its recep- coworkers [30] showed that CagA is sufficient to disrupt tor, CNF1 is endocytosed and routed to an endosomal the mechanisms that maintain normal epithelial differen- compartment [47], from where the toxin injects its cata- tiation, including cell adhesion, cell polarity, and the inhi- lytic activity into the cytosol [47], by using two hydropho- bition of migration. Since the cellular behavior induced bic structures present in the middle part of the CNF1 by CagA is reminiscent of oncogenes that disrupt cytoskel- molecule [49]. etal signaling, the authors proposed that altered cell-cell Page 3 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:4 http://www.infectagentscancer.com/content/3/1/4 Differen Figure 1 t aspects of CNF1 activity on epithelial cells Different aspects of CNF1 activity on epithelial cells. (a-b) Transmission electron micrographs showing: a control mononucleated cell (a), and a CNF1-treated cell (b), bearing four nuclei in the cytoplasm. (c-d) Fluorescence micrographs of control (c) or CNF1-treated (d) cells stained for F-actin detection. CNF1-treated cells display polymerization of actin into stress fibers (asterisks) and prominent ruffles (arrows). (e-f) Scanning (e) and transmission (f) electron micrographs showing different stages of the internalization process of non-invasive bacteria by CNF1-treated cells. After being contacted by mem- brane ruffles (e), bacteria are internalized within vacuoles (f). (g-h) Fluorescence micrographs of cells stained with an antibody that recognizes tubulin, the main component of microtubules. Note in h, an example of multipolar mitosis induced by CNF1. (i- l) Fluoresce micrographs of control (i) and CNF1-treated (l) cells transfected with the Ds-Red plasmid to visualize mitochon- drial organization. CNF1 induces the formation of elongated and interconnected mitochondria. Page 4 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:4 http://www.infectagentscancer.com/content/3/1/4 The cytoplasmic target of CNF1 is represented by the Rho intracellular bacterium Salmonella [60], for which a link GTPases, important molecular switches belonging to the with cancer has been evidenced [9]. This bacterium first Ras superfamily, that cycle between an inactive GDP- activates the Rho GTPases, by the GEF-like toxin SopE, to bound state and an active GTP-bound one, under the strict promote macropinocytosis that allows its entry into cells control of activators (guanine nucleotide exchange fac- and soon after, once inside, deactivates the GTPases via a tors, GEFs) and inactivators (GTPase-activating proteins, GAP-mimicking protein (SptP), thus allowing a moderate GAPs) [50]. The conformational changes, induced by the threshold of Rho protein activation for a high invasion binding of GTP or GDP, occur inside two molecular efficiency [61]. domains of the Rho proteins called switches that are responsible of the coupling of the G-proteins with their By regulating the actin cytoskeleton, the Rho GTPases also downstream effectors (switch 1) and allow the interaction play a crucial role in certain aspects of the malignant phe- of the activated GTPase with GAPs (switch 2). The enzy- notype, such as tumor cell motility, invasiveness and matic activity of CNF1 consists in the deamidation of a metastasis [62]. Indeed, RhoC has been implicated as a specific glutamine residue located in the switch 2 domain marker for highly angiogenic and aggressive breast cancer of the G proteins (glutamine 63 of Rho [51,52] or with a high metastatic ability [63]. An additional clue in glutamine 61 of Rac and Cdc42 [53]). This glutamine res- favor of our hypothesis comes from the observation that idue is essential for the GTPase activity of Rho proteins, CNF1 provokes cell junctions disruption and strongly either intrinsic or GAP-mediated [54]. By modifying enhances cellular motility in uroepithelial 804G cells glutamine into glutamic acid, CNF1 impairs the role of [59]. The augmented motility of epithelial cells highlights Rho GAP, allowing Rho proteins to be permanently another characteristic that somehow links this toxin to locked in their activated GTP-bound state and, thus, cancer. enhancing the activity of these proteins on their effectors. CNF1 hinders apoptosis via the pro- It is worth noting that the modification on Rho proteins inflammatory Akt/IKK/NF-kB pathway induced by CNF1 corresponds to the modification found It is also known that Rho proteins are crucially involved in on Ras proteins in many tumors [55]. In fact, Rho the development of inflammatory processes and that a glutamine 63 corresponds to Ras glutamine 61, a well- key player in the link existing between Rho, chronic known residue that, after mutagenization, leads to a per- inflammation and cancer is the Nuclear Factor-kB (NF- manent activation of the molecule and to tumor onset. kB) [64]. As mentioned in the Introduction, NF-kB is rep- resented by a group of structurally related and evolution- CNF1 and Rho GTPases: actin remodeling and arily conserved transcription factors involved in regulating the expression of genes that control different host cell invasion The Rho GTPases are pivotal in controlling the actin aspects of the tumor cell biology, including inflamma- cytoskeleton architecture [50]. In fact, one of the first tion, cell growth, and suppression of apoptosis (reviewed described cell responses to CNF1 is a remarkable reorgan- in [65]). Tumor cells can show elevated NF-κB-regulated ization of the actin cytoskeleton in epithelial cells that transcription, which can inhibit TNF-α-induced apoptosis consists in the assembly of F-actin in prominent stress fib- through the up-regulation of the anti-apoptotic proteins ers, membrane ruffles and filopodia [56] (Fig. 1c, d). The of the Bcl-2 family. prominent ruffling activity promoted by CNF1 permits epithelial cells to behave as phagocytes, developing a mac- In this context, we previously reported that CNF1 can acti- ropinocytotic activity that allows the capture and engulf- vate the nuclear factor-kB (NF-kB) [66] in epithelial cells. ment of different types of particles, including bacteria Such an activation, is responsible for the ability of the [57,58]. This aspect is of particular relevance for the bac- toxin to stimulate the expression of pro-inflammatory fac- terial pathogenicity since the macropinocytic activity tors and to protect host cell from apoptotic stimuli. As ensued by CNF1 in epithelial cells may possibly represent concerns the pro-survival activity, we have shown that the route of entry of CNF1-producing E. coli, similarly to CNF1 can increase the expression of proteins related to what occurs with other intestinal pathogens (Fig. 1e). Fur- cell adhesion (integrins, Focal Adhesion Kinase, cadher- thermore, upon activation by CNF1, the Rho GTPases ins, catenins), thus improving cell spreading and the abil- undergo sensitization to ubiquitylation and subsequent ity of cells to adhere to each other and to the extracellular proteosomal degradation [59], a process that would turn matrix [67]. In fact, prolonged cell survival, together with off the ruffling process, thus allowing an efficient internal- increased adhesion to matrix components might have sig- ization of bacteria inside the cells (Fig. 1f). It is worth not- nificant biological consequences and affect the tumori- ing that the activity of CNF1, with its ability to switch on genic potential of epithelial cells. Moreover, CNF1 the Rho GTPases and then coerce their degradation in the protects epithelial cells against the drop of the mitochon- proteasome, is somehow similar to the activity of the drial membrane potential provoked by UVB radiation and Page 5 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:4 http://www.infectagentscancer.com/content/3/1/4 increases the expression of the anti-apoptotic members of the Bcl-2 family, Bcl-2 and Bcl-X [68]. Although the causal relationship between Bcl-2 and mitochondrial membrane potential has not yet been clarified, we have very recently demonstrated that the up-regulation of the anti-apoptotic protein Bcl-2 somehow controls the mito- chondrial morphology, and that this depends on the acti- vation of the pro-inflammatory Rac1/PI3K/Akt/IKK/NF- kB pathway [69]. In fact, besides blocking the activity of the pro-apoptotic members of Bcl-2 family, Bcl-2 is involved in the regulation of mitochondrial morphology, since Bcl-2-over-expressing mitochondria present both increased volume and structural complexity [70]. In living cells, mitochondria continuously divide (fission) and fuse (fusion) with one another [71] and Bcl-2 family members are involved in these processes, the pro-apoptotic mem- bers regulating fission whereas the anti-apoptotic regulat- ing fusion of mitochondria [72]. In this context, we very recently demonstrated that CNF1 can induce, in epithelial cells, the formation of a complex network of elongated and interconnected mitochondria with an increased aver- age length [[69]; Fig. 1i, l]. Importantly, Bcl-2 silencing reduces the ability of CNF1 to protect cells against apop- Hypothetic model on how the ity Figure 2 can be connected to cancerpro-inflammatory CNF1 activ- tosis and also prevents the CNF1-induced mitochondrial Hypothetic model on how the pro-inflammatory changes. Therefore, since the mitochondrial remodeling is CNF1 activity can be connected to cancer. CNF1- of direct relevance for the role of these organelles in cell dependent Rho activation stimulates NF-κB nuclear translo- physiology and the mitochondrial dysfunction can con- cation and trans-activation, through the classical Akt/IKK- tribute to a number of human disorders, including cancer mediated pathway. NF-κB induces the transcription of genes [73], the role of CNF1 as a factor favoring transformation coding for proteins involved in inflammation and apoptosis, that is the pro-inflammatory molecules IL-6, IL-8, TNF-α and can be further supported by this novel finding. Cox-2, and anti-apoptotic factors, such as Bcl-2. This last also causes elongation and enrichment of the mitochondrial net- As concerns inflammation, CNF1 ensues the transcription work, an aspect somehow linked to transformation. On the and release of pro-inflammatory cytokines, such as IL-6, other hand, by mean of its activity on the actin cytoskeleton IL-8 and TNF-α in uroepithelial [74] and endothelial [75] organization, CNF1 is able to provoke cell junctions disrup- cells, probably contributing to the establishing of the tion and to strongly enhance cellular motility, a phenomenon inflammatory process due to CNF1-producing E. coli. The strictly linked to invasiveness and metastasis. pro-inflammatory role of CNF1 is also supported by the demonstration that the toxin strongly up-regulate the transcription of cyclooxygenase- 2 (COX-2) [76], an and modulates autophagy [81], cellular phenomena that immediate-early gene induced in response to pro-inflam- are frequently observed in different types of cancer cells, matory cytokines, tumor promoters, and growth factors and blocks the cell cycle G2/M transition in epithelial cells and over-expressed in cancers of the lung, colon, stomach, [82]. The ability of CNF1 to block the cell cycle progres- and breast [77-79]. sion suggests a strategy that permits to contain the host damage, and to induce specific cellular responses rather than rapid cell death. This could in turn facilitate the bac- Conclusion On the whole, it appears that CNF1 touches some of the terial invasion of underlying tissues. The activity of CNF1, signaling pathways that are engaged by carcinogens and with its ability to switch on the Rho GTPases and then tumor promoters, as schematized in Fig. 2. Particularly coerce their degradation in the proteasome, probably relevant are the activation of the transcription factor NF- renders the CNF1-producing E. coli intracellular parasites, κB, the pro-survival activity, the increased expression of hence permitting their potentially harmful and presuma- RNA messengers for COX-2 and pro-inflammatory bly transforming activity inside the cell, where they can cytokines as well as the augmented cell motility. In addi- escape the host immune system attack. Finally, it is worth tion, CNF1 impairs the cytokinesis, thus leading to multi- noting that CNF1 activity on cells shares several properties nucleation [44,56], induces nuclear segmentation, with CagA from the carcinogenic bacterium H. pylori, amitotic cell division, multipolar mitosis (Fig. 1g, h) [80], which can be indicated as the best example of a poten- Page 6 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:4 http://www.infectagentscancer.com/content/3/1/4 Table 1: Comparison between the cellular effects of E. coli CNF1 and those of the major virulence factors (VacA and CagA) of H. pylori CNF1 CagA VacA Effects on cytoskeleton and • Actin polymerization • Actin rearrangement • Actin disruption cell morphology • Spreading • "hummingbird" phenotype • Vacuolization • Multinucleation • loss of cell polarity • cell scattering Apoptosis regulation Anti-apoptotic effect Anti-apoptotic effect Pro-apoptotic effect Production of inflammatory TNF-α IL-8 TNF-α mediators IL-6 ROS IL-8 IL-8 IL-1β ROS IL-6 Activation of transcription NF-kB NF-kB NF-kB factors NFAT ATF-2 SRF NFAT AP1 /M Inhibition of G /S progression Induction of G /S progression Effect on cell cycle Block in G 2 1 1 Effects on mitochondria Inhibition of UVB-induced Reduction of mitochondrial mitochondrial membrane membrane potential, cytochrome c depolarization release 3. 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"BioMed Central will be the most significant development for coli cytotoxin: autophagy as a double-edged sword for the disseminating the results of biomedical researc h in our lifetime." host. Autophagy 2006, 2:310-311. Sir Paul Nurse, Cancer Research UK 82. Falzano L, Filippini P, Travaglione S, Miraglia AG, Fabbri A, Fiorentini C: Escherichia coli cytotoxic necrotizing factor 1 blocks cell Your research papers will be: cycle G2/M transition in uroepithelial cells. Infect Immun 2006, available free of charge to the entire biomedical community 74:3765-3772. peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 9 of 9 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Infectious Agents and Cancer Springer Journals

The Rho-activating CNF1 toxin from pathogenic E. coli: A risk factor for human cancer development?

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Copyright © 2008 by Travaglione et al; licensee BioMed Central Ltd.
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Biomedicine; Cancer Research; Infectious Diseases; Oncology
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Abstract

Nowadays, there is increasing evidence that some pathogenic bacteria can contribute to specific stages of cancer development. The concept that bacterial infection could be involved in carcinogenesis acquired a widespread interest with the discovery that H. pylori is able to establish chronic infections in the stomach and that this infection is associated with an increased risk of gastric adenocarcinoma and mucosa associated lymphoid tissue lymphoma. Chronic infections triggered by bacteria can facilitate tumor initiation or progression since, during the course of infection, normal cell functions can come under the control of pathogen factors that directly manipulate the host regulatory pathways and the inflammatory reactions. Renowned publications have recently corroborated the molecular mechanisms that link bacterial infections, inflammation and cancer, indicating certain strains of Escherichia coli as a risk factor for patients with colon cancer. E. coli is a normal inhabitant of the human intestine that becomes highly pathogenic following the acquisition of virulence factors, including a protein toxin named cytotoxic necrotizing factor 1 (CNF1). This toxin permanently activates the small GTP-binding proteins belonging to the Rho family, thus promoting a prominent polymerization of the actin cytoskeleton as well as a number of cellular responses, including changes in protein expression and functional modification of the cell physiology. CNF1 is receiving an increasing attention as a putative factor involved in transformation because of its ability to: (i) induce COX2 expression, an immediate-early gene over-expressed in some type of cancers; (ii) induce a long-lasting activation of the transcription factor NF-kB, a largely accepted marker of tumor cells; (iii) protect epithelial cells from apoptosis; (iv) ensue the release of pro-inflammatory cytokines in epithelial and endothelial cells; and (v) promote cellular motility. As cancer may arise through dysfunction of the same regulatory systems, it seems likely that CNF1-producing E. coli infections can contribute to tumor development. This review focuses on the aspects of CNF1 activity linked to cell transformation with the aim of contributing to the identification of a possible carcinogenic agent from the microbial world. Page 1 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:4 http://www.infectagentscancer.com/content/3/1/4 pathway is triggered by bacterial and viral infections, as Introduction Bacterial infection and cancer well as by pro-inflammatory cytokines, such as TNF-α and In the last century, cancer research thoroughly established IL-1, all of which activate the IKK complex [17]. This com- the role of major carcinogenic agents of different nature, plex phosphorilates the NF-kB inhibitors IkBs, thereby including infectious agents. However, although there is a targeting them for proteosomal degradation and freeing general agreement that some viruses, such as hepatitis B NF-kB to enter the nucleus and mediate transcription of virus (HBV), Epstein-Barr virus (EBV) and human papil- target genes. It is worth noting that many of the genes able loma virus (HPV) can cause cancer, the involvement of to mediate alterations characterizing a tumor cell are bacteria in carcinogenesis remains controversial. The role under the transcriptional control of NF-kB (reviewed in of viral infections in tumor onset is widely accepted [18,19]). For example, the activity and expression of cyc- because of the direct action of single viral genes (onco- lin D1, CDK2 kinase, c-myc, p21, p53 and pRb, which are genes) that result in cell transformation [1]. By contrast, involved in the control of cell cycle and are altered in sev- the molecular mechanism(s) by which bacteria might eral types of cancer, are NF-kB-dependent. The expression promote tumorigenesis are still poorly characterized. of numerous cytokines, that are growth factors for tumor Hence, one of the main challenges, nowadays, is to define cells (IL-1β, TNF, IL-6, EGF) are also regulated by NF-kB. the impact of bacterial infections as a cause of cancer and Tissue invasion and metastasis, two crucial events of eventually design strategies for their prevention and con- tumor progression, are regulated by NF-kB-dependent trol. genes, including metalloproteases (MMPs), urokinase type of plasminogen activator (uPA), IL-8, the adhesion Bacterial infections are usually believed to cause acute dis- molecules VCAM-1, ICAM-1 and ELAM-1. NF-kB is also eases, but it is now becoming clear that some bacteria can involved in the regulation of angiogenesis, the process by contribute to the establishment of chronic diseases, which tumor cells promote neo-vascularization. Finally, including cancer [2]. The concept that bacterial infection altered expression of genes involved in suppression of could be involved in carcinogenesis was first proposed in apoptosis (i.e. Bcl-2 family members and IAP proteins), a the late nineteenth and early twentieth centuries, based key feature of cancer cells, is often due to deregulated NF- on the discovery of bacteria at the sites of tumors, kB activity. although there was no proof that the bacteria were in any way causative [3]. Since then, the putative link between Concerning this last point, several pathogenic bacteria, chronic infection and cancer acquired a widespread inter- particularly those that can establish a persistent intracellu- est with the discovery that Helicobacter pylori is able to lar infection, activate NF-kB in the host cell and suppress establish chronic infections in the stomach and that this cell death, thus creating a niche in which the bacterium infection is associated with an increased risk of gastric can survive, in spite of the attempts of the host immune adenocarcinoma [4] and mucosa associated lymphoid tis- system to destroy the infected cell [20]. As a consequence, sue (MALT) lymphoma [5]. In this context, it is worth not- the suppression of apoptosis by a pathogen might also ing that H. pylori is classified as a class I carcinogenic factor allow a partially transformed cell to evade the self-destruc- [6]. Other chronic bacterial infections have been linked to tive process and to progress to a higher level of transfor- human carcinogenesis although the underlying mecha- mation. nisms remain to be defined (reviewed in [2]). The strong- est epidemiological case is for Salmonella enterica serovar Another important feature of inflammation-associated typhi (S. typhi), the agent of typhoid, which can also lead cancer is the production of reactive oxygen species (ROS) to chronic bacterial carriage in the gallbladder [7-11]. Sur- and nitric oxide (NO) by inflammatory and epithelial veys of typhoid outbreaks have shown that those who cells. This leads to increased mutations and altered func- become carriers have an increased risk of developing tions of important enzymes and proteins in inflamed tis- hepatobiliary carcinoma compared with people who have sue, thus contributing to the multistage carcinogenetic had acute typhoid and have cleared the infection [9]. process [21]. For example, during a chronic H. pylori infec- tion, production of ROS and nitroxides and the associated A recurring theme in the link between bacterial infection inflammatory response are assumed to contribute to the and carcinogenesis is that of chronic inflammation, which induction of a gastric carcinogenic process [22,23]. These is often a common feature of persistent infection [2,12]. chemical species are mainly produced by inflammatory One of the key molecules that link chronic inflammation cells to fight infection and are source of oxidative DNA and cancer is represented by the NF-kB family of transcrip- damage, thus contributing to carcinogenesis [22,23]. In tion factors [12,13]. In particular, different mouse studies particular, it has been demonstrated that within 6 hours, provide strong and direct genetic evidence that the classi- H. pylori infection had a mutagenic effect on gastric epi- cal, IKK-β dependent NF-kB activation pathway is indeed thelial cells [24]. The major form of oxidative DNA dam- a crucial mediator of tumor promotion [14-16]. This age is the formation of 8-oxoG lesions, specifically Page 2 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:4 http://www.infectagentscancer.com/content/3/1/4 repaired by the OGG1 DNA glycosylase. The inactivation and cell matrix interactions may serve as an early event in of this enzyme inhibits the level of inflammatory lesions H. pylori-induced carcinogenesis [30]. Very recently, in and abolishes the mutagenic effect induced by the infec- experimental studies dealing with H. pylori strains carrying tion at the gastric level, thus strengthening a close relation or not CagA, it has been demonstrated that this factor acti- between chronic inflammation and genotoxicity [24]. vates host cell survival and anti-apoptotic pathways to overcome self-renewal of the gastric epithelium, thus Bacterial toxins and cancer enhancing bacterial colonization of the stomach and As stated above, there is increasing evidence that some helping sustained H. pylori infection. In this context, it is pathogenic bacteria can contribute to specific stages of interesting to note that, patients infected with H. pylori cancer development. In particular, chronic infections trig- encoding the cag pathogeniticy island (PAI) are associated gered by bacteria can facilitate tumor initiation or progres- with an increased risk of gastric cancer [31]. sion since, during the course of infection, normal cell functions can undergo the control of factors released by In addition to the link between H. pylori and stomach can- the pathogen [2]. These bacterial factors, namely virulence cers, very recent studies evidenced that some other toxins factors, can directly manipulate the host regulatory path- may contribute to bowel and urogenital tract cancers [3]. ways and the inflammatory reaction [3]. In this context, it has been reported that adherent and invasive strains of Escherichia coli are a risk factor for Bacteria express a wide range of virulence factors, includ- patients with pre-cancerous and cancerous colon diseases ing protein toxins that have evolved to interact with [32,33]. Interestingly, in one of these studies, 3 out of 8 eukaryotic cellular machinery in a precise way. These tox- cancer-associated E. coli were reported to possess the cyto- ins interfere with key eukaryotic processes, such as cellular toxic necrotizing factor 1 (cnf1) gene [32], the gene coding signaling components, and some directly attack the for the protein toxin CNF1. The aim of this review is to genome [25-27]. These last can damage DNA via different highlight the main cell responses to CNF1, particularly mechanisms: i) directly by enzymatic attack, ii) indirectly those related to signaling pathways linked to inflamma- by provoking an inflammatory reaction that produces free tion and to cell transformation. radicals, or even iii) by affecting DNA repair mechanisms. Nougayrède and colleagues [28] have recently identified a CNF1 from E. coli: a protein toxin that activates novel hybrid peptide-polyketide compound from the Rho GTPases Escherichia coli that leads to DNA damage. This novel com- Although belonging to the normal human intestinal flora, pound is produced by pathogenic and, most interestingly, E. coli becomes highly pathogenic following the acquisi- commensal isolates. Although it is not yet clear how the tion of genes coding for virulence factors, one of which peptide-polyketide compound functions at the molecular being CNF1 [34]. CNF1-producing E. coli strains are occa- level, it is possible that it contributes to bacterial patho- sionally detected in isolates from feces of children with genesis and bacterially-induced carcinogenesis. diarrhea [35-37], but, more frequently, are responsible of extraintestinal infections, particularly in the urinary tract Any bacterial product that interferes with signaling, result- (UTIs) [38-40]. Also, these strains can be detected in cases ing in the disruption of the normal balance of growth, cell of bacteraemia [41] and of meningitis in neonates [42]. division and apoptosis, could facilitate tumor promotion. CNF1, first described in 1983 by Caprioli and coworkers Similarly, the ability to promote anchorage-independent as a toxin capable of causing multinucleation ("cyto- growth could favor metastatic potential and lead to cancer toxic") in cultured cells (Fig. 1a, b) and necrosis in rabbit progression. So far, the best example of potentially carci- skin ("necrotizing") [43,44], is a single-chain multido- nogenic toxin is H. pylori CagA that interferes with cellular main protein toxin, containing a binding domain to a cell signaling mechanisms in a way that is characteristic of receptor (N-terminal domain), a translocation domain tumor promoters. Indeed, CagA intracellularly interacts, (middle domain) and an enzymatic one (C-terminal in a phosphorylation-dependent and independent way, domain) [45,46], which modifies a specific cellular target with many host proteins that regulate cell growth, motil- in the host cell cytosol. CNF1 binds to the surface of cul- ity and polarity, thus leading to gastric epithelial prolifer- tured epithelial cells with high affinity [47], and the 67- ation, cell-cell dissociation and increased cell scattering kDa laminin receptor has been suggested (67LR) as a and motility (for a review [29]). In particular, Bagnoli and putative receptor for CNF1 [48]. After binding to its recep- coworkers [30] showed that CagA is sufficient to disrupt tor, CNF1 is endocytosed and routed to an endosomal the mechanisms that maintain normal epithelial differen- compartment [47], from where the toxin injects its cata- tiation, including cell adhesion, cell polarity, and the inhi- lytic activity into the cytosol [47], by using two hydropho- bition of migration. Since the cellular behavior induced bic structures present in the middle part of the CNF1 by CagA is reminiscent of oncogenes that disrupt cytoskel- molecule [49]. etal signaling, the authors proposed that altered cell-cell Page 3 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:4 http://www.infectagentscancer.com/content/3/1/4 Differen Figure 1 t aspects of CNF1 activity on epithelial cells Different aspects of CNF1 activity on epithelial cells. (a-b) Transmission electron micrographs showing: a control mononucleated cell (a), and a CNF1-treated cell (b), bearing four nuclei in the cytoplasm. (c-d) Fluorescence micrographs of control (c) or CNF1-treated (d) cells stained for F-actin detection. CNF1-treated cells display polymerization of actin into stress fibers (asterisks) and prominent ruffles (arrows). (e-f) Scanning (e) and transmission (f) electron micrographs showing different stages of the internalization process of non-invasive bacteria by CNF1-treated cells. After being contacted by mem- brane ruffles (e), bacteria are internalized within vacuoles (f). (g-h) Fluorescence micrographs of cells stained with an antibody that recognizes tubulin, the main component of microtubules. Note in h, an example of multipolar mitosis induced by CNF1. (i- l) Fluoresce micrographs of control (i) and CNF1-treated (l) cells transfected with the Ds-Red plasmid to visualize mitochon- drial organization. CNF1 induces the formation of elongated and interconnected mitochondria. Page 4 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:4 http://www.infectagentscancer.com/content/3/1/4 The cytoplasmic target of CNF1 is represented by the Rho intracellular bacterium Salmonella [60], for which a link GTPases, important molecular switches belonging to the with cancer has been evidenced [9]. This bacterium first Ras superfamily, that cycle between an inactive GDP- activates the Rho GTPases, by the GEF-like toxin SopE, to bound state and an active GTP-bound one, under the strict promote macropinocytosis that allows its entry into cells control of activators (guanine nucleotide exchange fac- and soon after, once inside, deactivates the GTPases via a tors, GEFs) and inactivators (GTPase-activating proteins, GAP-mimicking protein (SptP), thus allowing a moderate GAPs) [50]. The conformational changes, induced by the threshold of Rho protein activation for a high invasion binding of GTP or GDP, occur inside two molecular efficiency [61]. domains of the Rho proteins called switches that are responsible of the coupling of the G-proteins with their By regulating the actin cytoskeleton, the Rho GTPases also downstream effectors (switch 1) and allow the interaction play a crucial role in certain aspects of the malignant phe- of the activated GTPase with GAPs (switch 2). The enzy- notype, such as tumor cell motility, invasiveness and matic activity of CNF1 consists in the deamidation of a metastasis [62]. Indeed, RhoC has been implicated as a specific glutamine residue located in the switch 2 domain marker for highly angiogenic and aggressive breast cancer of the G proteins (glutamine 63 of Rho [51,52] or with a high metastatic ability [63]. An additional clue in glutamine 61 of Rac and Cdc42 [53]). This glutamine res- favor of our hypothesis comes from the observation that idue is essential for the GTPase activity of Rho proteins, CNF1 provokes cell junctions disruption and strongly either intrinsic or GAP-mediated [54]. By modifying enhances cellular motility in uroepithelial 804G cells glutamine into glutamic acid, CNF1 impairs the role of [59]. The augmented motility of epithelial cells highlights Rho GAP, allowing Rho proteins to be permanently another characteristic that somehow links this toxin to locked in their activated GTP-bound state and, thus, cancer. enhancing the activity of these proteins on their effectors. CNF1 hinders apoptosis via the pro- It is worth noting that the modification on Rho proteins inflammatory Akt/IKK/NF-kB pathway induced by CNF1 corresponds to the modification found It is also known that Rho proteins are crucially involved in on Ras proteins in many tumors [55]. In fact, Rho the development of inflammatory processes and that a glutamine 63 corresponds to Ras glutamine 61, a well- key player in the link existing between Rho, chronic known residue that, after mutagenization, leads to a per- inflammation and cancer is the Nuclear Factor-kB (NF- manent activation of the molecule and to tumor onset. kB) [64]. As mentioned in the Introduction, NF-kB is rep- resented by a group of structurally related and evolution- CNF1 and Rho GTPases: actin remodeling and arily conserved transcription factors involved in regulating the expression of genes that control different host cell invasion The Rho GTPases are pivotal in controlling the actin aspects of the tumor cell biology, including inflamma- cytoskeleton architecture [50]. In fact, one of the first tion, cell growth, and suppression of apoptosis (reviewed described cell responses to CNF1 is a remarkable reorgan- in [65]). Tumor cells can show elevated NF-κB-regulated ization of the actin cytoskeleton in epithelial cells that transcription, which can inhibit TNF-α-induced apoptosis consists in the assembly of F-actin in prominent stress fib- through the up-regulation of the anti-apoptotic proteins ers, membrane ruffles and filopodia [56] (Fig. 1c, d). The of the Bcl-2 family. prominent ruffling activity promoted by CNF1 permits epithelial cells to behave as phagocytes, developing a mac- In this context, we previously reported that CNF1 can acti- ropinocytotic activity that allows the capture and engulf- vate the nuclear factor-kB (NF-kB) [66] in epithelial cells. ment of different types of particles, including bacteria Such an activation, is responsible for the ability of the [57,58]. This aspect is of particular relevance for the bac- toxin to stimulate the expression of pro-inflammatory fac- terial pathogenicity since the macropinocytic activity tors and to protect host cell from apoptotic stimuli. As ensued by CNF1 in epithelial cells may possibly represent concerns the pro-survival activity, we have shown that the route of entry of CNF1-producing E. coli, similarly to CNF1 can increase the expression of proteins related to what occurs with other intestinal pathogens (Fig. 1e). Fur- cell adhesion (integrins, Focal Adhesion Kinase, cadher- thermore, upon activation by CNF1, the Rho GTPases ins, catenins), thus improving cell spreading and the abil- undergo sensitization to ubiquitylation and subsequent ity of cells to adhere to each other and to the extracellular proteosomal degradation [59], a process that would turn matrix [67]. In fact, prolonged cell survival, together with off the ruffling process, thus allowing an efficient internal- increased adhesion to matrix components might have sig- ization of bacteria inside the cells (Fig. 1f). It is worth not- nificant biological consequences and affect the tumori- ing that the activity of CNF1, with its ability to switch on genic potential of epithelial cells. Moreover, CNF1 the Rho GTPases and then coerce their degradation in the protects epithelial cells against the drop of the mitochon- proteasome, is somehow similar to the activity of the drial membrane potential provoked by UVB radiation and Page 5 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:4 http://www.infectagentscancer.com/content/3/1/4 increases the expression of the anti-apoptotic members of the Bcl-2 family, Bcl-2 and Bcl-X [68]. Although the causal relationship between Bcl-2 and mitochondrial membrane potential has not yet been clarified, we have very recently demonstrated that the up-regulation of the anti-apoptotic protein Bcl-2 somehow controls the mito- chondrial morphology, and that this depends on the acti- vation of the pro-inflammatory Rac1/PI3K/Akt/IKK/NF- kB pathway [69]. In fact, besides blocking the activity of the pro-apoptotic members of Bcl-2 family, Bcl-2 is involved in the regulation of mitochondrial morphology, since Bcl-2-over-expressing mitochondria present both increased volume and structural complexity [70]. In living cells, mitochondria continuously divide (fission) and fuse (fusion) with one another [71] and Bcl-2 family members are involved in these processes, the pro-apoptotic mem- bers regulating fission whereas the anti-apoptotic regulat- ing fusion of mitochondria [72]. In this context, we very recently demonstrated that CNF1 can induce, in epithelial cells, the formation of a complex network of elongated and interconnected mitochondria with an increased aver- age length [[69]; Fig. 1i, l]. Importantly, Bcl-2 silencing reduces the ability of CNF1 to protect cells against apop- Hypothetic model on how the ity Figure 2 can be connected to cancerpro-inflammatory CNF1 activ- tosis and also prevents the CNF1-induced mitochondrial Hypothetic model on how the pro-inflammatory changes. Therefore, since the mitochondrial remodeling is CNF1 activity can be connected to cancer. CNF1- of direct relevance for the role of these organelles in cell dependent Rho activation stimulates NF-κB nuclear translo- physiology and the mitochondrial dysfunction can con- cation and trans-activation, through the classical Akt/IKK- tribute to a number of human disorders, including cancer mediated pathway. NF-κB induces the transcription of genes [73], the role of CNF1 as a factor favoring transformation coding for proteins involved in inflammation and apoptosis, that is the pro-inflammatory molecules IL-6, IL-8, TNF-α and can be further supported by this novel finding. Cox-2, and anti-apoptotic factors, such as Bcl-2. This last also causes elongation and enrichment of the mitochondrial net- As concerns inflammation, CNF1 ensues the transcription work, an aspect somehow linked to transformation. On the and release of pro-inflammatory cytokines, such as IL-6, other hand, by mean of its activity on the actin cytoskeleton IL-8 and TNF-α in uroepithelial [74] and endothelial [75] organization, CNF1 is able to provoke cell junctions disrup- cells, probably contributing to the establishing of the tion and to strongly enhance cellular motility, a phenomenon inflammatory process due to CNF1-producing E. coli. The strictly linked to invasiveness and metastasis. pro-inflammatory role of CNF1 is also supported by the demonstration that the toxin strongly up-regulate the transcription of cyclooxygenase- 2 (COX-2) [76], an and modulates autophagy [81], cellular phenomena that immediate-early gene induced in response to pro-inflam- are frequently observed in different types of cancer cells, matory cytokines, tumor promoters, and growth factors and blocks the cell cycle G2/M transition in epithelial cells and over-expressed in cancers of the lung, colon, stomach, [82]. The ability of CNF1 to block the cell cycle progres- and breast [77-79]. sion suggests a strategy that permits to contain the host damage, and to induce specific cellular responses rather than rapid cell death. This could in turn facilitate the bac- Conclusion On the whole, it appears that CNF1 touches some of the terial invasion of underlying tissues. The activity of CNF1, signaling pathways that are engaged by carcinogens and with its ability to switch on the Rho GTPases and then tumor promoters, as schematized in Fig. 2. Particularly coerce their degradation in the proteasome, probably relevant are the activation of the transcription factor NF- renders the CNF1-producing E. coli intracellular parasites, κB, the pro-survival activity, the increased expression of hence permitting their potentially harmful and presuma- RNA messengers for COX-2 and pro-inflammatory bly transforming activity inside the cell, where they can cytokines as well as the augmented cell motility. In addi- escape the host immune system attack. Finally, it is worth tion, CNF1 impairs the cytokinesis, thus leading to multi- noting that CNF1 activity on cells shares several properties nucleation [44,56], induces nuclear segmentation, with CagA from the carcinogenic bacterium H. pylori, amitotic cell division, multipolar mitosis (Fig. 1g, h) [80], which can be indicated as the best example of a poten- Page 6 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:4 http://www.infectagentscancer.com/content/3/1/4 Table 1: Comparison between the cellular effects of E. coli CNF1 and those of the major virulence factors (VacA and CagA) of H. pylori CNF1 CagA VacA Effects on cytoskeleton and • Actin polymerization • Actin rearrangement • Actin disruption cell morphology • Spreading • "hummingbird" phenotype • Vacuolization • Multinucleation • loss of cell polarity • cell scattering Apoptosis regulation Anti-apoptotic effect Anti-apoptotic effect Pro-apoptotic effect Production of inflammatory TNF-α IL-8 TNF-α mediators IL-6 ROS IL-8 IL-8 IL-1β ROS IL-6 Activation of transcription NF-kB NF-kB NF-kB factors NFAT ATF-2 SRF NFAT AP1 /M Inhibition of G /S progression Induction of G /S progression Effect on cell cycle Block in G 2 1 1 Effects on mitochondria Inhibition of UVB-induced Reduction of mitochondrial mitochondrial membrane membrane potential, cytochrome c depolarization release 3. 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"BioMed Central will be the most significant development for coli cytotoxin: autophagy as a double-edged sword for the disseminating the results of biomedical researc h in our lifetime." host. Autophagy 2006, 2:310-311. Sir Paul Nurse, Cancer Research UK 82. Falzano L, Filippini P, Travaglione S, Miraglia AG, Fabbri A, Fiorentini C: Escherichia coli cytotoxic necrotizing factor 1 blocks cell Your research papers will be: cycle G2/M transition in uroepithelial cells. Infect Immun 2006, available free of charge to the entire biomedical community 74:3765-3772. peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 9 of 9 (page number not for citation purposes)

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