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A systems biology approach to multicellular and multi-generational radiation responses
Mutation Research 597 (2006) 32–38
A systems biology approach to multicellular and multi-generational radiation responses Mary Helen Barcellos-Hoff ∗ , Sylvain V. Costes Life Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Building 74-355, Berkeley, CA 94705, United States Received 2 March 2005; received in revised form 30 June 2005; accepted 14 September 2005 Available online 18 January 2006
Abstract Recent studies have highlighted crosstalk between irradiated cells and non-irradiated bystander cells and have uncovered highfrequency phenotypes of genomic instability in the progeny of irradiated cells that cannot be solely explained by radiation-induced mutation. It is difficult to explain these multicellular and multi-generational phenomena using the current paradigm of radiation biology. Radiation-induced bystander effect is a type of multicellular response to radiation that illustrates that the unit of function in multicellular organisms is neither the genome nor the cell. Cell function in complex three-dimensional tissues is coordinated by soluble signaling peptides and by small molecules within the context of insoluble scaffolding provided by the extracellular matrix. Adaptive response and radiation-induced genomic instability could thus result from persistent signaling perturbations following radiation exposures. A model of radiation response based on the systems biology principles of network interconnectivity and spatial organization should reconcile the apparent contradiction of these cellular phenotypes within the higher order structure of tissues and organisms. © 2005 Elsevier B.V. All rights reserved. Keywords: Ionizing radiation; Systems biology; TGF; Extracellular matrix; Genomic instability
1. Introduction The challenge in predicting radiation health effects in humans is to understand how cellular responses occurring in a multicellular context are integrated to produce an organism response. Experimental studies, detailed elsewhere in this volume, show that radiation exposure elicits responses that can produce effects in non-
Abbreviations: ECM, extracellular matrix; MMP, matrix metalloproteinases; TGF1, transforming growth factor 1 ∗ Corresponding author. Tel.: +1 510 486 6371; fax: +1 510 486 2535. E-mail address: [email protected] (M.H. Barcellos-Hoff). 0027-5107/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2005.09.008
irradiated bystander cells or can lead to a high frequency of genomic instability in the progeny of irradiated cells. This has motivated a substantial effort to both describe and quantify these non-targeted responses. One may argue that, more importantly, those data have heightened awareness that many types of cell interactions contribute to long-term radiation effects, and that multicellular responses are poorly integrated into the current paradigms of radiation effects and their consequences in terms of human health. Understanding how cell and molecular responses to ionizing radiation produce individual organism response may be difficult in reductionism models that emphasize components and pathways, rather than on network interconnectivity and tissue context that produce complexity.
M.H. Barcellos-Hoff, S.V. Costes / Mutation Research 597 (2006) 32–38
Even cell function is an elaborately branched signaling network mediated by receptors, ligands and small molecules that can activate DNA repair, alter chromatin organization, switch on cell cycle checkpoints and modulate various cellular metabolic processes [1]. Likewise, tissue function is the culmination of multicellular network coordinated by soluble endocrine, autocrine and paracrine signals and linked through a scaffolding of extracellular matrix that dynamically maintains homeostasis by regulating tissue composition, function, and phenotype. Just as DNA damage elicits a dramatic transition in signaling within a cell, each irradiated tissue has its own set of signals and cell types, distinct from that of the unirradiated tissue and different from that of other irradiated tissue. The sum of these events occurring in different organs, highly modulated by genotype, results in the organism response, but predicting this response for individuals remains an elusive. Thus, one may consider the problem from a systems biology viewpoint, i.e. how is the whole greater than the sum of its parts? Key differences between systems biology and current ‘analytical’ paradigms are that systems biology places emphasis on networks versus components, distributed versus centralized effort, and redundancy versus uniqueness [2]. Systems biology attempts to organize multiscale data obtained following environmental perturbations, e.g. radiation, and use that data to build a descriptive and mechanistic model of the biological phenomena [3]. Top-down analysis of the radiation response of any organism, much less humans, is beyond present capabilities because neither the tools nor detailed, global data are available. Yet, it is feasible to use systems biology concepts to place radiation-induced bystander effect, adaptive response and genomic instability into the context of an irradiated system (i.e. tissue). Radiation-induced bystander effects are a type of multicellular responses to radiation, while adaptive response and multi-generational radiation-induced genomic instability may result from persistent network perturbations following radiation exposures. The goal of systems biology is to analyze the whole, rather than the parts. Since there is a profound prejudice against waste, complexity and redundancy in human enterprise, we tend to impose these same constraints on biological functions. And as humans, we value individuals, uniqueness, and independence, and thus tend to frame biological problems in such terms. Systems biology provides a means to incorporate redundant, multifactorial and contradictory mechanisms into achieving meaningful goals. Three systems biology principles will be discussed as we interpret them to relate to radiation biology: information layering, scaffolding, and robust-
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ness. Neither comprehensive, nor expert, the intent of this commentary is to induce discussion rather than to instruct. 2. Information layering: reiterative, recycled and redundant processes Recasting radiation biology in terms of system biology begins by regarding the tissue, organ or organism as the primary responder rather than the cell or molecular event. This is a fundamental shift from most current models because it de-emphasizes the well-characterized effects of ionizing radiation on a central cellular target, DNA. Most models place DNA damage, in particular double-strand breaks, as the pivotal event that initiates the radiation response and subsequent effects. Although a great deal has been learned about mechanisms of DNA damage and repair machinery, this focus has led to a virtual ‘blind spot’ where cell–cell interactions have no place in modern radiation biology, which leads in turn to a certain skepticism that irradiated cells can produce effects in unirradiated cells. This last is blatantly at odds with modern cell biology and extracellular signaling via ROS, cytokines, peptide hormones, chemokines, matrikines and growth factors and the cross-talk required between cell types to execute tissue, organ and organism functions. We previously postulated the existence of a coordinated multicellular damage response program based on the rapid and dynamic cell biology that occurs in irradiated tissues [4]. Although some events may appear to augment damage, we believe that in most cases tissue damage response programs are directed towards restoring tissue function. A main feature of the program is that individual cell responses are coordinated by extracellular signaling. In normal tissue, a major role of extracellular signaling is to inhibit carcinogenesis by eliminating abnormal cells and suppressing neoplastic behavior. Since oxygen metabolism results in continuous bombardment of DNA and proteins by reactive oxygen species by-products, this program(s) is likely operative at all times, but is co-opted, and possibly corrupted, by the exigencies of acute radiation damage. Tissue pathology and organ failure result when radiation response severely disrupts communication between cells or among different cell types [5,6]. Thus, radiation-induced bystander effects and genomic instability can be seen as, respectively, positive and negative cellular manifestations of multicellular damage responses [5]. Bystander effects are evidence of the extracellular signaling that results from such multicellular programs that attempt to re-establish
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homeostasis and eliminate abnormal cells. This type of effect can be within a tissue, between tissues (e.g. stroma and epithelium) or between organs (i.e. abscopal effects). But if high dose radiation exposure corrupts normal signaling or chronic irradiation persistently alters cellular phenotype, then surveillance of abnormal cells is compromised and aberrant, potentially genomically unstable, cells can accumulate and proliferate. This effect would be compounded in tissues by radiation-induced activation of inflammatory cells that generate additional damage long after exposure [7]. Information layering means that there are multiple primary responses, redundancy and inefficiency, and that the penultimate response is a function of their combination. Whether well-designed, as in the case of humans, or highly evolved in the case of biology, systems respond to stimuli at multiple levels and each critical response has several back-ups. Death due to gastrointestinal syndrome at high doses (>14 Gy) is generally attributed to the denuded intestinal lining due to p53-dependent apoptosis, but Fuks and colleagues have suggested that this is secondary to endothelial cell death mediated via membrane damage that releases ceremide [8]. While this example appears to show redundancy of deleterious consequences, it is significant that neither the damage to epithelial DNA, or the subsequent signaling via p53, appear to dictate the organism response. Furthermore, a network view of radiation biology highlights the potential for radiation effects on target cells, e.g. the epithelial cell in carcinogenesis, to be highly modulated by non-target, i.e. stromal, tissue. Our previous studies have shown that radiation alters the environment in which mammary epithelial cells reside and that these radiation-induced changes in the mammary tissue can under certain circumstances contribute to the known action of radiation as a carcinogen. Radiation chimeric tissue was created by transplanting unirradiated preneoplastic mammary cells to an irradiated mammary gland [9]. The female mammary gland is unique among all glands in that the epithelium develops postnatally from a rudiment that can be removed from the inguinal glands at approximately 3 weeks of age [10]. Surgical removal of the parenchyma results in a gland-free mammary fat pad, referred to as a cleared fat pad, suitable for receiving donor tissue at the time of clearing or later. Transplantation of normal mammary epithelial cells produces ductal outgrowths that fill the fat pad and are nearly indistinguishable in wholemounts or histologically from intact gland. An occasional mouse mammary epithelial cell line, like the COMMA-D, retain the ability to undergo morphogenesis in vivo [11]. COMMA-D cells are non-tumorigenic if injected into the cleared fat
pads of 3-week-old mice or subcutaneously of immature or adult mice, or into nude mice. Although clonal in origin, COMMA-D cells exhibit morphological and phenotypic diversity in culture suggesting a ‘stem’ cell like population [12]. However, the cell line harbors two mutant p53 alleles that confer neoplastic potential [13]. We found that the irradiated stroma dramatically promoted the ability of COMMA-D cells to progress to tumors. COMMA-D tumor incidence was up to four times greater in the cleared fat pads of hosts irradiated with 4 Gy from 1 to 14 days prior to transplantation and the tumors were biologically distinct, i.e. larger, in the irradiated host. Hemibody irradiation resulted in tumors only in the irradiated side, suggesting that local versus systemic effects were dominant. In this model, the carcinogenic target cells were true bystanders to radiation response expressed in stromal cells. Thus, networked, multicellular responses, rather than the damage per se, dictate whether homeostasis is restored or if pathology ensues. In other words, system failure lies less with the components, which are individually dispensable, than with system performance. When radiation-induced bystander effects are placed into this context, then the apparently extraneous events, e.g. response of unirradiated cells to the presence of irradiated cells, are less perturbing, since the system is responding. Furthermore, the observation of bystander effects does not signify ‘more danger’ from radiation any more than the fact that everyone ducks when a gun goes off means that the bullet is more lethal. 3. Scaffolding: distributed information Part of the complexity of radiation effects, particularly when considered from the initial physical damage, is that even when energy is relatively evenly distributed in an organism (e.g. high energy X- or ␥-radiation), not all cells, tissues or organs respond similarly. Some organisms are remarkably resistant to radiation, certain tissues exhibit little to no functional response while others undergo mass suicide, and long-term carcinogenic risks are highly dependent on tissue type. Similarly recent application of gene expression microarray technology for genome-wide analysis of radiation response reveals remarkably distinct features. The same amount of energy elicits vastly different p53 responses in cells from different tissues [14]. Two tissues with essential identical functional radiation response, i.e. p53-dependent apoptosis, initiate very different p53-dependent transcription [15]. Likewise, the same amount of energy deposited in different volumes of the cell elicits different transcriptional events [16].
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Since the initial physical and chemical events due to energy deposition are similar within the genome of any given cell within an organism, the different responses are generally interpreted as differences in cellular response to DNA damage or as a function of DNA repair capacity. Certainly inhibition of various DNA repair function can augment various tissue responses such as apoptosis and cancer induction, but again these are highly tissue-type dependent. For example, deletion of p53 in the mouse genome can block both cell cycle arrest and apoptotic cell responses. However, apoptosis is the primary response in hematopoietic cells, whereas fibroblasts undergo growth arrest, and epithelial cells will go one way or the other depending on tissue source and degree of damage [17]. One might ask how these events are coordinated within a tissue, between organs, and over the course of time? Network information dictating phenotype, function and response to stimuli can reside in the tissue-specific extracellular matrix (ECM). ECM consists of a complex, fibrillar network of large, generally insoluble, proteins that serve as a scaffold for cell adhesion and a reservoir of many peptide growth factors. In contrast to cytokines, whose transient actions are frequently a function of release from cells, or growth factors that are activated to both maintain homeostasis and mediate response to stimuli, ECM is both constant and dynamic. Thus the extracellular space provides an adhesion scaffold that contains solid state information (i.e. ECM), reserve messengers (i.e. growth factors), transit conduit (e.g. cytokines), and dynamic modifiers (e.g. matrix metalloproteinases (MMPs)). ECM remodeling, similar to that elicited by wounding, is generated within hours of radiation exposure [19] and can persist for months after a single exposure [20,21]. MMPs regulate the composition and release of cryptic information from the ECM that profoundly influence cellular behavior [18]. Likewise the pattern of soluble peptide factors activity and production is rapidly altered by radiation exposure, creating a rapid and wide spread signal to recruit cells (even bystanders) and modulate cell phenotype [4,22]. Fundamental changes in scaffolding may persist from radiation exposure that modify subsequent behaviors. Wound healing and tissue composition can be compromised for decades following therapeutic radiation treatment [23]. In experimental models, irradiated stroma can promote neoplastic behavior even in unirradiated cells [9,24]. Thus, both adaptive response and radiationinduced bystander effects, such as those evident by transfer of medium conditioned by irradiated cells, may be due to alterations in the scaffolding that cells reside in. Restoration of the ECM to a pristine state could register the degree and success of tissue response to radiation
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while persistent modification of the ECM would act as a record of radiation exposure. A primary limitation to our current understanding of this biology is the tendency to model these as hierarchical, linear responses, rather than combinatorial networks. At the biological level, new tools and technologies, and new experimental strategies, will be needed. At the modeling level, new programming approaches will be required to represent the non-linear aspect and adaptive behavior of system biology. Thinking of these responses as assets of a network, rather than programmed responses of cells, points to the dynamic flexibility of the network to use resources conditionally. This capability implies that part of the variation between cells, organs and organisms in response to radiation can reside in the ECM scaffolding of a particular tissue, the history that is etched by MMPs in its architecture, and the reserves of growth factors that afford instruction upon damage. 4. Robustness: the system resists change To return to the initial premise that the coordinated cellular response of tissues and organs is based on supracellular signaling through the scaffold of the ECM, one may wonder why are individual cells apparently so wellequipped to respond to energy deposition in DNA? Can reductionism experiments lead to complete understanding of organism response if we continue to identify molecular mechanisms, signaling pathways and cell fate decision points? Would the organism work as well if each cell acted autonomously using a set of well-defined checkpoints as have been described for virtually every critical function? Is the fact that we cannot predict how an individual will respond to radiation just a matter of time and resources, or is the paradigm at fault? In the context of multicellular organisms, an orchestrated response to DNA damage by ionizing radiation is important for rapid restoration of homeostasis and longterm prevention of cancer but it is unclear how factors outside the cell interact with DNA damage responses. The complexity of multicellular, multi-tissue, multiorgan response is the fundamental strength of the system. Rather than relying on a given cell to ‘do the right thing’, the system imposes directives, acts as a register for damage and records response, and provides democratic coordination without a ‘leader’. Indeed several extracellular factors have been implicated as critical determinants of DNA damage responses. Our laboratory showed that transforming growth factor 1 (TGF1) is rapidly and persistently activated in tissues following IR [25]. TGF1 signals via ubiquitously expressed cell surface receptors that in turn
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phosphorylates Smad transcription proteins and activate other signaling pathways that regulate differentiation, function, proliferation and apoptosis depending on cell type and context (reviewed in [26]). Mice deficient for TGF1 show increased tumor progression [27] and haploid insufficiency for carcinogenesis [28], which are commonly thought to be due to loss of growth control. However, TGF1 is secreted in a latent complex [29], which is widely distributed in the microenvironment where the extracellular matrix serves as a reservoir [30]. A protein redox switch leading to its extracellular activation endows latent TGF1 with the ability to serve as an extracellular sensor of oxidative stress [31]. Surprisingly, radiation-induced apoptosis is significantly decreased in Tgfβ1 heterozygote mouse mammary gland while embryonic Tgfβ1 null skin and liver fail to undergo both apoptosis and inhibition of cell cycle in response to IR [32]. Furthermore, keratinocytes from Tgfβ1 null mice exhibit 100–1000-fold greater genomic instability than wildtype cells [33]. Together, these data suggest that TGF1, in addition to its role in homoeostatic growth control, plays a more complex role in regulating tissue response to damage, the failure of which could contribute to the development of cancer. Recent results from our experiments indicate a surprising and unexpected TGF dependence of cellular DNA damage programs that dictate epithelial cell fate decision in response to radiation damage (Jobling et al., unpublished data). We found that activation of ATM, a nuclear sensor of DNA damage that initiates, recruits and activates a complex program of checkpoints for cell cycle, apoptosis and genomic integrity (see reviews [34,35]), is severely compromised in irradiated cells depleted of TGF1. TGF is a vital extracellular signal that initiates and orchestrates multiple cell types and behaviors in development (reviewed in [36]), in a variety of diseases, including cancer (reviewed in [37]), and in response to insults including radiation [4]. The failure of the ATM genotoxic stress program in Tgfβ1 null cells illustrates that the microenvironment is a critical determinant of the cellular stress programs. Importantly, the failure of the extracellular network, as exemplified by TGF, is just as deleterious as the intracellular damage response since it would lead to compromised damage signaling, persistence of abnormal cells, and increased proliferation. This is one link in a network that would ensure that cell fate decisions functionally interact in response to tissue damage and thus enables orchestrated restoration of homeostasis. When operating at multiple levels redundancy produces a robust system to ensure that appropriate steps for repair and recovery are executed.
5. Conclusions Biology, and its application in medicine, has traditionally been an individual pursuit at the observational level, as well as at the conceptual level, that has often led to a reductionism view of complex processes. This has served well to identify the important components of biological systems and to learn great detail about the mechanics of processes. However integration of these details into a truly dynamic view of the processes that maintain normal cells and, conversely, how they are disturbed by ionizing radiation, is elusive. Rather than trying to explain observable phenomena by reducing them to interplay between elementary units that can be investigated independently of each other, systems biology conceives of problems of organization and tackles phenomena not resolvable into local events [38]. The most significant challenge of the post-genomic era is the application of systems biology to functional genomics, i.e., understanding how the genome is expressed to produce myriad cell phenotypes. A phenotype is the result of selective expression of the genome in response to the microenvironment; thus the phenome is the sum of biological components, responses and signaling pathways of cells studied in context, i.e., within a proper tissue structure. The current technology that permits evaluation of many concomitant events means it is now a feasible, if difficult, task to monitor global cell DNA translational events, metabolic by-products, protein abundance and modifications. New models for integrative biology will use tools that bring diverse disciplines such as biology and computational sciences together. The challenge is coming to terms with abundance of information and in putting it into meaningful models. Using systems biology to first address the multiscale nature of phenotype, it can then be applied to understand how the behavior of multiple cell types is coordinated physiologically or in response to an external stimulus like radiation. Acknowledgments The authors wish to acknowledge funding from NASA Specialized Center for Research in Radiation Health Effects and the Low Dose Radiation Program of the Office of Biological and Environmental Research, United States Department of Energy DE AC03 76SF00098. References [1] Y. Shiloh, A.R. Lehmann, Maintaining integrity, Nat. Cell Biol. 6 (2004) 923–928.
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A systems biology approach to multicellular and multi-generational radiation responses Mary Helen Barcellos-Hoff ∗ , Sylvain V. Costes Life Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Building 74-355, Berkeley, CA 94705, United States Received 2 March 2005; received in revised form 30 June 2005; accepted 14 September 2005 Available online 18 January 2006
Abstract Recent studies have highlighted crosstalk between irradiated cells and non-irradiated bystander cells and have uncovered highfrequency phenotypes of genomic instability in the progeny of irradiated cells that cannot be solely explained by radiation-induced mutation. It is difficult to explain these multicellular and multi-generational phenomena using the current paradigm of radiation biology. Radiation-induced bystander effect is a type of multicellular response to radiation that illustrates that the unit of function in multicellular organisms is neither the genome nor the cell. Cell function in complex three-dimensional tissues is coordinated by soluble signaling peptides and by small molecules within the context of insoluble scaffolding provided by the extracellular matrix. Adaptive response and radiation-induced genomic instability could thus result from persistent signaling perturbations following radiation exposures. A model of radiation response based on the systems biology principles of network interconnectivity and spatial organization should reconcile the apparent contradiction of these cellular phenotypes within the higher order structure of tissues and organisms. © 2005 Elsevier B.V. All rights reserved. Keywords: Ionizing radiation; Systems biology; TGF; Extracellular matrix; Genomic instability
1. Introduction The challenge in predicting radiation health effects in humans is to understand how cellular responses occurring in a multicellular context are integrated to produce an organism response. Experimental studies, detailed elsewhere in this volume, show that radiation exposure elicits responses that can produce effects in non-
Abbreviations: ECM, extracellular matrix; MMP, matrix metalloproteinases; TGF1, transforming growth factor 1 ∗ Corresponding author. Tel.: +1 510 486 6371; fax: +1 510 486 2535. E-mail address: [email protected] (M.H. Barcellos-Hoff). 0027-5107/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2005.09.008
irradiated bystander cells or can lead to a high frequency of genomic instability in the progeny of irradiated cells. This has motivated a substantial effort to both describe and quantify these non-targeted responses. One may argue that, more importantly, those data have heightened awareness that many types of cell interactions contribute to long-term radiation effects, and that multicellular responses are poorly integrated into the current paradigms of radiation effects and their consequences in terms of human health. Understanding how cell and molecular responses to ionizing radiation produce individual organism response may be difficult in reductionism models that emphasize components and pathways, rather than on network interconnectivity and tissue context that produce complexity.
M.H. Barcellos-Hoff, S.V. Costes / Mutation Research 597 (2006) 32–38
Even cell function is an elaborately branched signaling network mediated by receptors, ligands and small molecules that can activate DNA repair, alter chromatin organization, switch on cell cycle checkpoints and modulate various cellular metabolic processes [1]. Likewise, tissue function is the culmination of multicellular network coordinated by soluble endocrine, autocrine and paracrine signals and linked through a scaffolding of extracellular matrix that dynamically maintains homeostasis by regulating tissue composition, function, and phenotype. Just as DNA damage elicits a dramatic transition in signaling within a cell, each irradiated tissue has its own set of signals and cell types, distinct from that of the unirradiated tissue and different from that of other irradiated tissue. The sum of these events occurring in different organs, highly modulated by genotype, results in the organism response, but predicting this response for individuals remains an elusive. Thus, one may consider the problem from a systems biology viewpoint, i.e. how is the whole greater than the sum of its parts? Key differences between systems biology and current ‘analytical’ paradigms are that systems biology places emphasis on networks versus components, distributed versus centralized effort, and redundancy versus uniqueness [2]. Systems biology attempts to organize multiscale data obtained following environmental perturbations, e.g. radiation, and use that data to build a descriptive and mechanistic model of the biological phenomena [3]. Top-down analysis of the radiation response of any organism, much less humans, is beyond present capabilities because neither the tools nor detailed, global data are available. Yet, it is feasible to use systems biology concepts to place radiation-induced bystander effect, adaptive response and genomic instability into the context of an irradiated system (i.e. tissue). Radiation-induced bystander effects are a type of multicellular responses to radiation, while adaptive response and multi-generational radiation-induced genomic instability may result from persistent network perturbations following radiation exposures. The goal of systems biology is to analyze the whole, rather than the parts. Since there is a profound prejudice against waste, complexity and redundancy in human enterprise, we tend to impose these same constraints on biological functions. And as humans, we value individuals, uniqueness, and independence, and thus tend to frame biological problems in such terms. Systems biology provides a means to incorporate redundant, multifactorial and contradictory mechanisms into achieving meaningful goals. Three systems biology principles will be discussed as we interpret them to relate to radiation biology: information layering, scaffolding, and robust-
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ness. Neither comprehensive, nor expert, the intent of this commentary is to induce discussion rather than to instruct. 2. Information layering: reiterative, recycled and redundant processes Recasting radiation biology in terms of system biology begins by regarding the tissue, organ or organism as the primary responder rather than the cell or molecular event. This is a fundamental shift from most current models because it de-emphasizes the well-characterized effects of ionizing radiation on a central cellular target, DNA. Most models place DNA damage, in particular double-strand breaks, as the pivotal event that initiates the radiation response and subsequent effects. Although a great deal has been learned about mechanisms of DNA damage and repair machinery, this focus has led to a virtual ‘blind spot’ where cell–cell interactions have no place in modern radiation biology, which leads in turn to a certain skepticism that irradiated cells can produce effects in unirradiated cells. This last is blatantly at odds with modern cell biology and extracellular signaling via ROS, cytokines, peptide hormones, chemokines, matrikines and growth factors and the cross-talk required between cell types to execute tissue, organ and organism functions. We previously postulated the existence of a coordinated multicellular damage response program based on the rapid and dynamic cell biology that occurs in irradiated tissues [4]. Although some events may appear to augment damage, we believe that in most cases tissue damage response programs are directed towards restoring tissue function. A main feature of the program is that individual cell responses are coordinated by extracellular signaling. In normal tissue, a major role of extracellular signaling is to inhibit carcinogenesis by eliminating abnormal cells and suppressing neoplastic behavior. Since oxygen metabolism results in continuous bombardment of DNA and proteins by reactive oxygen species by-products, this program(s) is likely operative at all times, but is co-opted, and possibly corrupted, by the exigencies of acute radiation damage. Tissue pathology and organ failure result when radiation response severely disrupts communication between cells or among different cell types [5,6]. Thus, radiation-induced bystander effects and genomic instability can be seen as, respectively, positive and negative cellular manifestations of multicellular damage responses [5]. Bystander effects are evidence of the extracellular signaling that results from such multicellular programs that attempt to re-establish
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homeostasis and eliminate abnormal cells. This type of effect can be within a tissue, between tissues (e.g. stroma and epithelium) or between organs (i.e. abscopal effects). But if high dose radiation exposure corrupts normal signaling or chronic irradiation persistently alters cellular phenotype, then surveillance of abnormal cells is compromised and aberrant, potentially genomically unstable, cells can accumulate and proliferate. This effect would be compounded in tissues by radiation-induced activation of inflammatory cells that generate additional damage long after exposure [7]. Information layering means that there are multiple primary responses, redundancy and inefficiency, and that the penultimate response is a function of their combination. Whether well-designed, as in the case of humans, or highly evolved in the case of biology, systems respond to stimuli at multiple levels and each critical response has several back-ups. Death due to gastrointestinal syndrome at high doses (>14 Gy) is generally attributed to the denuded intestinal lining due to p53-dependent apoptosis, but Fuks and colleagues have suggested that this is secondary to endothelial cell death mediated via membrane damage that releases ceremide [8]. While this example appears to show redundancy of deleterious consequences, it is significant that neither the damage to epithelial DNA, or the subsequent signaling via p53, appear to dictate the organism response. Furthermore, a network view of radiation biology highlights the potential for radiation effects on target cells, e.g. the epithelial cell in carcinogenesis, to be highly modulated by non-target, i.e. stromal, tissue. Our previous studies have shown that radiation alters the environment in which mammary epithelial cells reside and that these radiation-induced changes in the mammary tissue can under certain circumstances contribute to the known action of radiation as a carcinogen. Radiation chimeric tissue was created by transplanting unirradiated preneoplastic mammary cells to an irradiated mammary gland [9]. The female mammary gland is unique among all glands in that the epithelium develops postnatally from a rudiment that can be removed from the inguinal glands at approximately 3 weeks of age [10]. Surgical removal of the parenchyma results in a gland-free mammary fat pad, referred to as a cleared fat pad, suitable for receiving donor tissue at the time of clearing or later. Transplantation of normal mammary epithelial cells produces ductal outgrowths that fill the fat pad and are nearly indistinguishable in wholemounts or histologically from intact gland. An occasional mouse mammary epithelial cell line, like the COMMA-D, retain the ability to undergo morphogenesis in vivo [11]. COMMA-D cells are non-tumorigenic if injected into the cleared fat
pads of 3-week-old mice or subcutaneously of immature or adult mice, or into nude mice. Although clonal in origin, COMMA-D cells exhibit morphological and phenotypic diversity in culture suggesting a ‘stem’ cell like population [12]. However, the cell line harbors two mutant p53 alleles that confer neoplastic potential [13]. We found that the irradiated stroma dramatically promoted the ability of COMMA-D cells to progress to tumors. COMMA-D tumor incidence was up to four times greater in the cleared fat pads of hosts irradiated with 4 Gy from 1 to 14 days prior to transplantation and the tumors were biologically distinct, i.e. larger, in the irradiated host. Hemibody irradiation resulted in tumors only in the irradiated side, suggesting that local versus systemic effects were dominant. In this model, the carcinogenic target cells were true bystanders to radiation response expressed in stromal cells. Thus, networked, multicellular responses, rather than the damage per se, dictate whether homeostasis is restored or if pathology ensues. In other words, system failure lies less with the components, which are individually dispensable, than with system performance. When radiation-induced bystander effects are placed into this context, then the apparently extraneous events, e.g. response of unirradiated cells to the presence of irradiated cells, are less perturbing, since the system is responding. Furthermore, the observation of bystander effects does not signify ‘more danger’ from radiation any more than the fact that everyone ducks when a gun goes off means that the bullet is more lethal. 3. Scaffolding: distributed information Part of the complexity of radiation effects, particularly when considered from the initial physical damage, is that even when energy is relatively evenly distributed in an organism (e.g. high energy X- or ␥-radiation), not all cells, tissues or organs respond similarly. Some organisms are remarkably resistant to radiation, certain tissues exhibit little to no functional response while others undergo mass suicide, and long-term carcinogenic risks are highly dependent on tissue type. Similarly recent application of gene expression microarray technology for genome-wide analysis of radiation response reveals remarkably distinct features. The same amount of energy elicits vastly different p53 responses in cells from different tissues [14]. Two tissues with essential identical functional radiation response, i.e. p53-dependent apoptosis, initiate very different p53-dependent transcription [15]. Likewise, the same amount of energy deposited in different volumes of the cell elicits different transcriptional events [16].
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Since the initial physical and chemical events due to energy deposition are similar within the genome of any given cell within an organism, the different responses are generally interpreted as differences in cellular response to DNA damage or as a function of DNA repair capacity. Certainly inhibition of various DNA repair function can augment various tissue responses such as apoptosis and cancer induction, but again these are highly tissue-type dependent. For example, deletion of p53 in the mouse genome can block both cell cycle arrest and apoptotic cell responses. However, apoptosis is the primary response in hematopoietic cells, whereas fibroblasts undergo growth arrest, and epithelial cells will go one way or the other depending on tissue source and degree of damage [17]. One might ask how these events are coordinated within a tissue, between organs, and over the course of time? Network information dictating phenotype, function and response to stimuli can reside in the tissue-specific extracellular matrix (ECM). ECM consists of a complex, fibrillar network of large, generally insoluble, proteins that serve as a scaffold for cell adhesion and a reservoir of many peptide growth factors. In contrast to cytokines, whose transient actions are frequently a function of release from cells, or growth factors that are activated to both maintain homeostasis and mediate response to stimuli, ECM is both constant and dynamic. Thus the extracellular space provides an adhesion scaffold that contains solid state information (i.e. ECM), reserve messengers (i.e. growth factors), transit conduit (e.g. cytokines), and dynamic modifiers (e.g. matrix metalloproteinases (MMPs)). ECM remodeling, similar to that elicited by wounding, is generated within hours of radiation exposure [19] and can persist for months after a single exposure [20,21]. MMPs regulate the composition and release of cryptic information from the ECM that profoundly influence cellular behavior [18]. Likewise the pattern of soluble peptide factors activity and production is rapidly altered by radiation exposure, creating a rapid and wide spread signal to recruit cells (even bystanders) and modulate cell phenotype [4,22]. Fundamental changes in scaffolding may persist from radiation exposure that modify subsequent behaviors. Wound healing and tissue composition can be compromised for decades following therapeutic radiation treatment [23]. In experimental models, irradiated stroma can promote neoplastic behavior even in unirradiated cells [9,24]. Thus, both adaptive response and radiationinduced bystander effects, such as those evident by transfer of medium conditioned by irradiated cells, may be due to alterations in the scaffolding that cells reside in. Restoration of the ECM to a pristine state could register the degree and success of tissue response to radiation
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while persistent modification of the ECM would act as a record of radiation exposure. A primary limitation to our current understanding of this biology is the tendency to model these as hierarchical, linear responses, rather than combinatorial networks. At the biological level, new tools and technologies, and new experimental strategies, will be needed. At the modeling level, new programming approaches will be required to represent the non-linear aspect and adaptive behavior of system biology. Thinking of these responses as assets of a network, rather than programmed responses of cells, points to the dynamic flexibility of the network to use resources conditionally. This capability implies that part of the variation between cells, organs and organisms in response to radiation can reside in the ECM scaffolding of a particular tissue, the history that is etched by MMPs in its architecture, and the reserves of growth factors that afford instruction upon damage. 4. Robustness: the system resists change To return to the initial premise that the coordinated cellular response of tissues and organs is based on supracellular signaling through the scaffold of the ECM, one may wonder why are individual cells apparently so wellequipped to respond to energy deposition in DNA? Can reductionism experiments lead to complete understanding of organism response if we continue to identify molecular mechanisms, signaling pathways and cell fate decision points? Would the organism work as well if each cell acted autonomously using a set of well-defined checkpoints as have been described for virtually every critical function? Is the fact that we cannot predict how an individual will respond to radiation just a matter of time and resources, or is the paradigm at fault? In the context of multicellular organisms, an orchestrated response to DNA damage by ionizing radiation is important for rapid restoration of homeostasis and longterm prevention of cancer but it is unclear how factors outside the cell interact with DNA damage responses. The complexity of multicellular, multi-tissue, multiorgan response is the fundamental strength of the system. Rather than relying on a given cell to ‘do the right thing’, the system imposes directives, acts as a register for damage and records response, and provides democratic coordination without a ‘leader’. Indeed several extracellular factors have been implicated as critical determinants of DNA damage responses. Our laboratory showed that transforming growth factor 1 (TGF1) is rapidly and persistently activated in tissues following IR [25]. TGF1 signals via ubiquitously expressed cell surface receptors that in turn
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phosphorylates Smad transcription proteins and activate other signaling pathways that regulate differentiation, function, proliferation and apoptosis depending on cell type and context (reviewed in [26]). Mice deficient for TGF1 show increased tumor progression [27] and haploid insufficiency for carcinogenesis [28], which are commonly thought to be due to loss of growth control. However, TGF1 is secreted in a latent complex [29], which is widely distributed in the microenvironment where the extracellular matrix serves as a reservoir [30]. A protein redox switch leading to its extracellular activation endows latent TGF1 with the ability to serve as an extracellular sensor of oxidative stress [31]. Surprisingly, radiation-induced apoptosis is significantly decreased in Tgfβ1 heterozygote mouse mammary gland while embryonic Tgfβ1 null skin and liver fail to undergo both apoptosis and inhibition of cell cycle in response to IR [32]. Furthermore, keratinocytes from Tgfβ1 null mice exhibit 100–1000-fold greater genomic instability than wildtype cells [33]. Together, these data suggest that TGF1, in addition to its role in homoeostatic growth control, plays a more complex role in regulating tissue response to damage, the failure of which could contribute to the development of cancer. Recent results from our experiments indicate a surprising and unexpected TGF dependence of cellular DNA damage programs that dictate epithelial cell fate decision in response to radiation damage (Jobling et al., unpublished data). We found that activation of ATM, a nuclear sensor of DNA damage that initiates, recruits and activates a complex program of checkpoints for cell cycle, apoptosis and genomic integrity (see reviews [34,35]), is severely compromised in irradiated cells depleted of TGF1. TGF is a vital extracellular signal that initiates and orchestrates multiple cell types and behaviors in development (reviewed in [36]), in a variety of diseases, including cancer (reviewed in [37]), and in response to insults including radiation [4]. The failure of the ATM genotoxic stress program in Tgfβ1 null cells illustrates that the microenvironment is a critical determinant of the cellular stress programs. Importantly, the failure of the extracellular network, as exemplified by TGF, is just as deleterious as the intracellular damage response since it would lead to compromised damage signaling, persistence of abnormal cells, and increased proliferation. This is one link in a network that would ensure that cell fate decisions functionally interact in response to tissue damage and thus enables orchestrated restoration of homeostasis. When operating at multiple levels redundancy produces a robust system to ensure that appropriate steps for repair and recovery are executed.
5. Conclusions Biology, and its application in medicine, has traditionally been an individual pursuit at the observational level, as well as at the conceptual level, that has often led to a reductionism view of complex processes. This has served well to identify the important components of biological systems and to learn great detail about the mechanics of processes. However integration of these details into a truly dynamic view of the processes that maintain normal cells and, conversely, how they are disturbed by ionizing radiation, is elusive. Rather than trying to explain observable phenomena by reducing them to interplay between elementary units that can be investigated independently of each other, systems biology conceives of problems of organization and tackles phenomena not resolvable into local events [38]. The most significant challenge of the post-genomic era is the application of systems biology to functional genomics, i.e., understanding how the genome is expressed to produce myriad cell phenotypes. A phenotype is the result of selective expression of the genome in response to the microenvironment; thus the phenome is the sum of biological components, responses and signaling pathways of cells studied in context, i.e., within a proper tissue structure. The current technology that permits evaluation of many concomitant events means it is now a feasible, if difficult, task to monitor global cell DNA translational events, metabolic by-products, protein abundance and modifications. New models for integrative biology will use tools that bring diverse disciplines such as biology and computational sciences together. The challenge is coming to terms with abundance of information and in putting it into meaningful models. Using systems biology to first address the multiscale nature of phenotype, it can then be applied to understand how the behavior of multiple cell types is coordinated physiologically or in response to an external stimulus like radiation. Acknowledgments The authors wish to acknowledge funding from NASA Specialized Center for Research in Radiation Health Effects and the Low Dose Radiation Program of the Office of Biological and Environmental Research, United States Department of Energy DE AC03 76SF00098. References [1] Y. Shiloh, A.R. Lehmann, Maintaining integrity, Nat. Cell Biol. 6 (2004) 923–928.
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