- Email: [email protected]
Aberrant extracellular signaling induced by ionizing radiation and its role in carcinogenesis
International Congress Series 1236 (2002) 399 – 405
Aberrant extracellular signaling induced by ionizing radiation and its role in carcinogenesis Rhonda L. Henshall-Powell a, Catherine C. Park b, Mary Helen Barcellos-Hoff a,* a
Life Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA b University of California, San Francisco, CA 94143, USA
Abstract Multicellular organisms orchestrate the behavior of individual cells via extracellular signaling through the microenvironment. The lines of communication are diverse, ranging from the insoluble scaffold of the extracellular matrix, permeated with small, diffusible molecules of cytokines to the cell surface that is structured by adhesion receptors and gap junctions forming communication channels between cells. Ionizing radiation elicits rapid and persistent changes in extracellular signaling, as exemplified in our studies of the irradiated mammary gland by the rapid and persistent activation of transforming growth factor-h1. We have shown that such events can contribute to radiation’s carcinogenic action in experiments in which nonirradiated, preneoplastic mammary epithelial cells are transplanted to an irradiated stroma, in which significantly larger tumors arose more frequently. In recent studies, we analyzed the effect of radiation on extracellular signaling in human mammary cells using a three-dimensional culture model. Preliminary data indicate that the progeny of irradiated cells, i.e., survivors, display aberrant morphogenesis and cell – cell interactions, resulting in behaviors characteristic of malignancy in this model. We hypothesized that under certain conditions, radiation exposure prevents normal cell interactions, which in turn could predispose susceptible cells to genomic instability. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Transforming growth factor h; Mammary gland; Breast cancer; Carcinogenesis; Ionizing radiation; Microenvironment; Genomic instability
Abbreviations: TGF-h, transforming growth factor-h1; ECM, extracellular matrix; HMEC, Human mammary epithelial cells. * Corresponding author. Tel.: +1-510-486-6371; fax: +1-510-486-4545. E-mail address: [email protected] (M.H. Barcellos-Hoff ). 0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 1 ) 0 0 7 7 0 - 1
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1. The role of the microenvironment The behavior of individual cells is dictated by their interactions with each other via the microenvironment. Indeed, coordinated multicellular behavior is necessary for tissue function, which is orchestrated by extracellular signaling through the microenvironment [1]. The microenvironment, which consists of insoluble proteins in the extracellular matrix (ECM), soluble proteins like cytokines, and cell adhesion molecules (CAM) that link cells to the ECM and to each other, is essential for tissue-specific organization and differentiation [2,3]. In normal tissues, extracellular signaling is also critical for eliminating abnormal cells and suppressing neoplastic behavior (reviewed in Ref. [4]). Extracellular signaling controls cell migration, proliferation, and morphogenesis, all of which are disturbed during carcinogenesis. The disruption of cell adhesion has been postulated to contribute a rate-limiting step in neoplastic progression. Whether referred to as ‘soil’ or ‘landscapers’ for cancer, an intact, normal microenvironment is a critical barrier to neoplastic behavior. Disruption of ECM integrity, and thereby cell adhesion using transgenic manipulations can promote tumorigenesis [5]. Loss of E-cadherin, a cell-adhesion molecule crucial for epithelial cell interactions, leads to invasive cell behavior while restitution of E-cadherin to tumor cells impedes malignant behavior [6,7]. Nonmalignant human mammary epithelial cells (HMEC) treated with antibodies that alter ECM receptors known as integrins, exhibit disorganized growth characteristic of tumors. Conversely, manipulating integrins can revert breast cancer cell into normal mammary specific acinar organization. This normalization of cell interactions also reduces tumor formation in vivo [8]. These experiments suggest that appropriate microenvironment signaling can override malignant behavior even in tumor cells with an unstable genome. The importance of understanding extracellular control is further underscored by interferon-g treatment for chronic myelogenous leukemia, which induces integrin expression that force cancer cells to reattach to the stroma [9]. We have previously proposed that the cell biology of irradiated tissues is indicative of a coordinated multicellular damage response program in which individual cell contributions are directed towards repair of the tissue [10]. However, as a function of dose or radiation quality, radiation-induced extracellular signaling can also disrupt multicellular communication. Our data, using murine and human models of breast cancer, suggest that such radiation-induced microenvironment remodeling can promote malignant progression in susceptible cell populations.
2. Radiation exposure leads to rapid and dynamic microenvironment remodeling Over the last decade, we have demonstrated that radiation elicits a rapid and dynamic program of ECM remodeling. By comparing the composition ECM in irradiated mammary gland to that of liver and skin following high and low LET radiation exposures, we have concluded that the composition of irradiated microenvironment is a function of the tissue type, the dose, and the radiation quality [11,12]. In the mammary gland, ECM remodeling is mediated by the activation of a potent cytokine, transforming growth factor h1 (TGF-h).
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TGF-h is produced as a latent complex that is secreted and requires extracellular activation that permits TGF-h to bind to ubiquitous receptors [13]. We defined antibodies that reveal activation in situ and showed that it is evidenced by the loss of the latent complex and unmasking of TGF-h [14]. Following radiation exposure, TGF-h activation is evident within an hour, persistent for at least a week, and detected following whole body doses of as little as 0.1 Gy [15]. As a consequence of its rapid activation, we demonstrated a novel mechanism of TGF-h activation via exposure to reactive oxygen species, which endows TGF-h with a redox sensor capability. As such, we proposed that TGF-h acts as an extracellular lynch pin released by radiation and other oxidative stressors, to orchestrate multicellular response to damage [16].
3. The irradiated microenvironment promotes neoplastic potential To test the contribution of such alterations to the process of carcinogenesis, we created radiation chimeric mammary glands by taking advantage of the postnatal development of the mammary gland to surgically create a stroma without epithelium [17]. We then transplanted unirradiated mammary epithelial cells to an irradiated mammary stroma. The mammary cells we used were nontumorigenic by several criteria but harbor mutation in both alleles of the p53 gene [18]. Infrequent small tumors that regress occur in shamirradiated adult stroma. Nevertheless, when these cells were transplanted to irradiated (4 Gy) stroma, tumors formed in three-quarters of the transplants and the tumors were significantly larger. Furthermore, the effect of the irradiated stroma persisted up to 14 days after exposure. Since hemi-body irradiation resulted in tumors only on the irradiated side, we concluded that this effect was primarily due to altered stromal microenvironment. Radiation-induced microenvironments constitute a new class of its carcinogenic action that affects the ability of cells to maintain appropriate communication between each other. As a consequence of disrupted communication, we hypothesized that the cells harboring neoplastic mutations are released from extracellular signals that suppress their proliferation in normal tissue [19].
4. Radiation induces a heritable HMEC phenotype that is characteristic of malignancy To test whether radiation exposure alters cell communication, we used a model of nonmalignant HMEC cultured within an artificial ECM. Under these culture conditions, single cells proliferate as clones that organize into a structure typical of glandular epithelium, i.e., an acinus consisting of a single layer of polarized epithelial cells organized into a hollow sphere. Morphogenesis in these colonies is accompanied by appropriately localized proteins necessary for tissue structure and polarity. For example, E-cadherin is localized at the interface between cells, h1-integrin is baso-lateral and a6-integrin is basal [8]. Importantly, tumorigenic and nontumorigenic mammary epithelial cells, which are nearly indistinguishable when cultured as monolayers, are readily classified by their morphogenesis in three-dimensional ECM [20]. Tumor cells proliferate, fail to establish
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appropriate cell– cell and cell– ECM connections, and thus, form clumps. In contrast, nonmalignant mammary epithelial cells growth arrest after a short period of growth and form acini similar to those found in situ. We used this tissue-specific morphogenesis to ask whether irradiated HMEC maintained appropriate microenvironment interactions. Cells were irradiated shortly after plating and TGF-h was added to some cultures to mimic the presence of an irradiated stroma. We observed that most colonies arising from cells treated with radiation and TGFh showed pronounced morphological disorganization in comparison to colonies from sham controls or following single treatments (Barcellos-Hoff et al., unpublished data). Surprisingly, we also found that the number of cells per colony was increased in double treated specimens. Furthermore, using confocal microscopy and immunofluorescence, we observed that colonies from irradiated cells cultured in the presence of TGF-h showed a dramatic loss of E-cadherin, significantly increased h1-integrin, and decreased a6integrin. A distinct collagen IV containing basement membrane was observed in all treatment groups, suggesting that the altered integrin expression was not due to the lack of appropriate ligand. Importantly, the phenotype occurred in almost all cells that survive irradiation, which clearly limits the role of mutational mechanisms. Together, these data suggested that colonies arising from irradiated cells exhibit a consistent phenotype consisting of inappropriate intercellular adhesion, deranged extracellular adhesion molecules, loss of gap junction proteins, and disorganized tissue-specific organization. These observations indicate that radiation exposure of individual cells leads to a persistently altered phenotype in daughter cells that is characterized by the loss of critical controls imposed by the microenvironment to maintain tissue integrity. The resulting behavior is consistent with malignant progression. We have argued that microenvironment perturbations induced by irradiation are means of fostering neoplastic progression in susceptible (i.e., premalignant) cells [4]. Tlsty [21] has recently proposed that disturbances in cell adhesion can modulate pathways that control genomic stability. We predict that if such colonies arising from irradiated cells show increased genomic instability, it is a consequence of the lack of microenvironment control rather than as a direct result of DNA damage. Future studies will test whether the irradiated HMEC phenotype contributes to radiation-induced genomic instability.
5. Potential mechanisms underlying an irradiated phenotype Since the disruption of extracellular interactions occurs in almost all colonies formed by cells that survive irradiation, the role of mutational mechanisms is clearly limited as the mechanism underlying this phenomenon. Phenotypic evolution is a more likely basis for the behavior of irradiated HMEC. Phenotype is driven by biochemical changes, due in part to extracellular signaling and epigenetic modulation of the genome. Both can lead to heritable phenotypes (as evidenced by the differentiation of more than 300 cell types from a human genome) or a reversible phenotype, such as in the ‘activated’ phenotypes that are observed in certain cells during inflammation or wound healing. If the mechanisms by which the irradiated phenotype is perpetuated involve extracellular signaling via soluble molecules or cell contact, irradiated cells will be able to influence unirradiated cells via a
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‘‘bystander’’ mechanism. Radiation ‘‘bystander’’ effects have been demonstrated both in vitro and in vivo [22 –24]. It is unclear whether bystander effects that promote neoplastic behavior are a function of direct cell contact, or mediated by soluble factors, or (more likely) both. The relevance of studying epigenetic mechanisms in irradiated cells lies in their potency as harbingers of change: under certain circumstances, epigenetic modifications occur at high frequency in specific genes known to be players in the carcinogenic process. Epigenetic mechanisms encompass a wide range of events that affect how the genome is expressed but are not as well characterized as genomic sequence changes during cancer progression. DNA cytosine methylation in regions that are CpG-rich (CpG island) recruits methylated-DNA binding proteins that in turn interact with histone deacetylases and alter chromatin structure; each of these processes can act independently or in concert to favor inhibition of gene expression necessary for certain phenotypes. Little is known about mechanisms leading to aberrant methylation in epithelial cells and scarcely any data exist regarding methylation following radiation exposure. In addition to several Russian reports in the 1970s, one study in 1989 showed a dose-dependent decrease in 5-methylcytosine at 24, 48 and 72 h post-exposure to 0.5– 10 Gy [25]. In tumors, aberrant methylation contributes to gene silencing and may contribute to the generation of heterogeneous phenotypes. Recently, the importance of epigenetic changes during carcinogenesis has gained attention [26]. Studies have demonstrated that hypermethylation of 14-3-3 sigma occurs in 91% of primary breast cancers and is strongly associated with the loss of gene expression in these tumors [27]. Also 50% of breast tumors exhibit E-cadherin epigenetic alterations [28]. The p16/INK4A and retinoblastoma are also among the genes that show changes in the methylation pattern during cancer progression. Romanov et al. [29] have shown that HMEC spontaneously bypass senescence and exhibit genomic instability which is accompanied by p16 methylation.
6. Conclusions Our studies of the irradiated mammary gland and the progeny of irradiated human cells suggest that radiation exposure can promote malignant progression by pathways other than that of mutational mechanisms. Since the frequency of chromosome aberrations increases many cell generations after irradiation by an, as yet, unknown mechanism [30 –32], we suggest that disruption of extracellular signaling, adhesion, and communication could precede, and may augment, destablization of the genome. Our hypothesis that genomic instability is as much a matter of the loss of a multicellular controls as the induction of cellular process [33]. Our future studies will concern the molecular mechanisms underlying these events.
Acknowledgements Funding for this research was from the Specialized Center of Research and Training in Radiation Health from the National Aeronautics and Space Administration and from the
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Low Dose Radiation Program in the Office of Biological and Environmental Research, Office of Energy Research of the US Department of Energy at Lawrence Berkeley National Laboratory.
References [1] M.J. Bissell, M.H. Barcellos-Hoff, The influence of extracellular matrix on gene expression: is structure the message? J. Cell Sci. 8 (1987) 327 – 343. [2] M.H. Barcellos-Hoff, M.J. Bissell, Mammary epithelial cells as a model for studies of the regulation of gene expression, in: K.S. Matlin, J.D. Valentich (Eds.), Functional Epithelial Cells in Culture, A.R. Liss, New York, 1989, pp. 399 – 433. [3] M.H. Barcellos-Hoff, J. Aggeler, T.G. Ram, M.J. Bissell, Functional differentiation and alveolar morphogenesis of primary mammary epithelial cell cultures on reconstituted basement membrane, Development 105 (1989) 223 – 235. [4] M.H. Barcellos-Hoff, It takes a tissue to make a tumor: epigenetics, cancer and the microenvironment, J. Mammary Gland Biol. Neoplasia 6 (2001) 213 – 221. [5] C.J. Sympson, M.J. Bissell, Z. Werb, Mammary gland tumor formation in transgenic mice overexpressing stromelysin-1, Sem. Cancer Biol. 6 (1995) 159 – 163. [6] K. Vleminckx, L.J. Vakaet, M. Mareel, W. Fiers, F. van Roy, Genetic manipulation of E-cadherin expression by epithelial tumor cells reveals an invasion suppressor role, Cell 66 (1) (1991) 107 – 119. [7] J. Luo, D.M. Lubaroff, M.J. Hendrix, Suppression of prostate cancer invasive potential and matrix metalloproteinase activity by E-cadherin transfection, Cancer Res. 59 (15) (1999) 3552 – 3556. [8] V.M. Weaver, O.W. Petersen, F. Wang, C.A. Larabell, P. Briand, C. Damsky, et al., Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies, J. Cell Biol. 137 (1) (1997) 231 – 245. [9] R. Bhatia, P.B. McGlave, C.M. Verfaillie, Treatment of marrow stroma with interferon-alpha restores normal beta 1 integrin-dependent adhesion of chronic myelogenous leukemia hematopoietic progenitors. Role of MIP-1 alpha, J. Clin. Invest. 96 (2) (1995) 931 – 939. [10] M.H. Barcellos-Hoff, How do tissues respond to damage at the cellular level? The role of cytokines in irradiated tissues, Radiat. Res. 150 (5) (1998) S109 – S120. [11] E.J. Ernhart, HZE and proton induced microenvironment remodeling mediated by transforming growth factor-h1 [Doctoral]. Ft. Collins: Colorado State University; 1996. [12] S.V. Costes, C.H. Streuli, M.H. Barcellos-Hoff, Quantitative image analysis of laminin immunoreactivity in 1 GeV/amu iron particle irradiated skin basement membrane, Radiat. Res. 154 (2000) 389 – 397. [13] D.A. Lawrence, R. Pircher, P. Jullien, Conversion of a high molecular weight latent beta-TGF from chicken embryo fibroblasts into a low molecular weight active beta-TGF under acidic conditions, Biochem. Biophys. Res. Commun. 133 (1985) 1026 – 1034. [14] M.H. Barcellos-Hoff, E.J. Ehrhart, M. Kalia, R. Jirtle, K. Flanders, M.L.-S. Tsang, Immunohistochemical detection of active TGF-h in situ using engineered tissue, Am. J. Pathol. 147 (1995) 1228 – 1237. [15] E.J. Ehrhart, A. Carroll, P. Segarini, M.L.-S. Tsang, M.H. Barcellos-Hoff, Latent transforming growth factor-h activation in situ: quantitative and functional evidence following low dose irradiation, FASEB J. 11 (1997) 991 – 1002. [16] M.H. Barcellos-Hoff, T.A. Dix, Redox-mediated activation of latent transforming growth factor-h1, Mol. Endocrinol. 10 (1996) 1077 – 1083. [17] M.H. Barcellos-Hoff, S.A. Ravani, Irradiated mammary gland stroma promotes the expression of tumorigenic potential by unirradiated epithelial cells, Cancer Res. 60 (2000) 1254 – 1260. [18] D.J. Jerry, D. Medina, J.S. Butel, p53 mutations in COMMA-D cells, In Vitro Cell. Dev. Biol. 30A (1994) 87 – 89. [19] M.H. Barcellos-Hoff, The potential influence of radiation-induced microenvironments in neoplastic progression, J. Mammary Gland Bio. Neoplasia 3 (1998) 165 – 175. [20] O.W. Petersen, L. Ronnov-Jessen, A.R. Howlett, M.J. Bissell, Interaction with basement membrane serves
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to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells, Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 9064 – 9068. T.D. Tlsty, Cell-adhesion-dependent influences on genomic instability and carcinogenesis, Curr. Opin. Cell Biol. 10 (5) (1998) 647 – 653. J.B. Little, Radiation carcinogenesis, Carcinogenesis 21 (3) (2000) 397 – 404. K.M. Prise, O.V. Belyakov, M. Folkard, B.D. Michael, Studies of bystander effects in human fibroblasts using a charged particle microbeam, Int. J. Radiat. Biol. 74 (6) (1998) 793 – 798. R. Ramesh, A.J. Marrogi, A. Munshi, C.N. Abboud, S.M. Freeman, In vivo analysis of the ‘bystander effect’: a cytokine cascade, Exp. Hematol. 24 (1996) 829 – 838. J.F. Kalinich, G.N. Catravas, S.L. Snyder, The effect of gamma radiation on DNA methylation, Radiat. Res. 117 (2) (1989) 185 – 197. L.M. Mielnicki, H.L. Asch, B.B. Asch, Genes, chromatin, and breast cancer: an epigenetic tale, J. Mammary Gland Biol. Neoplasia 6 (2001) 169 – 182. A.T. Ferguson, E. Evron, C.B. Umbricht, T.K. Pandita, T.A. Chang, H. Hermeking, et al., High frequency of hypermethylation at the 14-3-3 sigma locus leads to gene silencing in breast cancer, Proc. Natl. Acad. Sci. U. S. A. 97 (11) (2000) 6049 – 6054. J.R. Graff, E. Gabrielson, H. Fujii, S.B. Baylin, J.G. Herman, Methylation patterns of the E-cadherin 5V CpG island are unstable and reflect the dynamic, heterogeneous loss of E-cadherin expression during metastatic progression, J. Biol. Chem. 275 (4) (2000) 2727 – 2732. S.R. Romanov, B.K. Kozakiewicz, C.R. Holst, M.R. Stampfer, L.M. Haupt, T.D. Tlsty, Normal human mammary epithelilal cells spontaneously escape senescence and acquire genomic changes, Nature 409 (2001) 633 – 637. M.A. Kadhim, S.A. Lorimore, K.M. Townsend, D.T. Goodhead, V.J. Buckle, E.G. Wright, Radiationinduced genomic instability: delayed cytogenetic aberrations and apoptosis in primary human bone marrow cells, Int. J. Radiat. Biol. 67 (3) (1995) 287 – 293. M.A. Kadhim, D.A. Macdonald, D.T. Goodhead, S.A. Lorimore, S.J. Marsden, E.G. Wright, Transmission of chromosomal instability after plutonium alpha-particle irradiation, [see comments], Nature 355 (6362) (1992) 738 – 740. C. Mothersill, M.A. Kadhim, S. O’Reilly, D. Papworth, S.J. Marsden, C.B. Seymour, et al., Dose- and timeresponse relationships for lethal mutations and chromosomal instability induced by ionizing radiation in an immortalized human keratinocyte cell line, Int. J. Radiat. Biol. 76 (6) (2000) 799 – 806. M.H. Barcellos-Hoff, A.L. Brooks, Extracellular signaling via the microenvironment: a hypothesis relating carcinogenesis, bystander effects and genomic instability, Radiat. Res. 156 (2001) 618 – 627.
Aberrant extracellular signaling induced by ionizing radiation and its role in carcinogenesis Rhonda L. Henshall-Powell a, Catherine C. Park b, Mary Helen Barcellos-Hoff a,* a
Life Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA b University of California, San Francisco, CA 94143, USA
Abstract Multicellular organisms orchestrate the behavior of individual cells via extracellular signaling through the microenvironment. The lines of communication are diverse, ranging from the insoluble scaffold of the extracellular matrix, permeated with small, diffusible molecules of cytokines to the cell surface that is structured by adhesion receptors and gap junctions forming communication channels between cells. Ionizing radiation elicits rapid and persistent changes in extracellular signaling, as exemplified in our studies of the irradiated mammary gland by the rapid and persistent activation of transforming growth factor-h1. We have shown that such events can contribute to radiation’s carcinogenic action in experiments in which nonirradiated, preneoplastic mammary epithelial cells are transplanted to an irradiated stroma, in which significantly larger tumors arose more frequently. In recent studies, we analyzed the effect of radiation on extracellular signaling in human mammary cells using a three-dimensional culture model. Preliminary data indicate that the progeny of irradiated cells, i.e., survivors, display aberrant morphogenesis and cell – cell interactions, resulting in behaviors characteristic of malignancy in this model. We hypothesized that under certain conditions, radiation exposure prevents normal cell interactions, which in turn could predispose susceptible cells to genomic instability. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Transforming growth factor h; Mammary gland; Breast cancer; Carcinogenesis; Ionizing radiation; Microenvironment; Genomic instability
Abbreviations: TGF-h, transforming growth factor-h1; ECM, extracellular matrix; HMEC, Human mammary epithelial cells. * Corresponding author. Tel.: +1-510-486-6371; fax: +1-510-486-4545. E-mail address: [email protected] (M.H. Barcellos-Hoff ). 0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 1 ) 0 0 7 7 0 - 1
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1. The role of the microenvironment The behavior of individual cells is dictated by their interactions with each other via the microenvironment. Indeed, coordinated multicellular behavior is necessary for tissue function, which is orchestrated by extracellular signaling through the microenvironment [1]. The microenvironment, which consists of insoluble proteins in the extracellular matrix (ECM), soluble proteins like cytokines, and cell adhesion molecules (CAM) that link cells to the ECM and to each other, is essential for tissue-specific organization and differentiation [2,3]. In normal tissues, extracellular signaling is also critical for eliminating abnormal cells and suppressing neoplastic behavior (reviewed in Ref. [4]). Extracellular signaling controls cell migration, proliferation, and morphogenesis, all of which are disturbed during carcinogenesis. The disruption of cell adhesion has been postulated to contribute a rate-limiting step in neoplastic progression. Whether referred to as ‘soil’ or ‘landscapers’ for cancer, an intact, normal microenvironment is a critical barrier to neoplastic behavior. Disruption of ECM integrity, and thereby cell adhesion using transgenic manipulations can promote tumorigenesis [5]. Loss of E-cadherin, a cell-adhesion molecule crucial for epithelial cell interactions, leads to invasive cell behavior while restitution of E-cadherin to tumor cells impedes malignant behavior [6,7]. Nonmalignant human mammary epithelial cells (HMEC) treated with antibodies that alter ECM receptors known as integrins, exhibit disorganized growth characteristic of tumors. Conversely, manipulating integrins can revert breast cancer cell into normal mammary specific acinar organization. This normalization of cell interactions also reduces tumor formation in vivo [8]. These experiments suggest that appropriate microenvironment signaling can override malignant behavior even in tumor cells with an unstable genome. The importance of understanding extracellular control is further underscored by interferon-g treatment for chronic myelogenous leukemia, which induces integrin expression that force cancer cells to reattach to the stroma [9]. We have previously proposed that the cell biology of irradiated tissues is indicative of a coordinated multicellular damage response program in which individual cell contributions are directed towards repair of the tissue [10]. However, as a function of dose or radiation quality, radiation-induced extracellular signaling can also disrupt multicellular communication. Our data, using murine and human models of breast cancer, suggest that such radiation-induced microenvironment remodeling can promote malignant progression in susceptible cell populations.
2. Radiation exposure leads to rapid and dynamic microenvironment remodeling Over the last decade, we have demonstrated that radiation elicits a rapid and dynamic program of ECM remodeling. By comparing the composition ECM in irradiated mammary gland to that of liver and skin following high and low LET radiation exposures, we have concluded that the composition of irradiated microenvironment is a function of the tissue type, the dose, and the radiation quality [11,12]. In the mammary gland, ECM remodeling is mediated by the activation of a potent cytokine, transforming growth factor h1 (TGF-h).
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TGF-h is produced as a latent complex that is secreted and requires extracellular activation that permits TGF-h to bind to ubiquitous receptors [13]. We defined antibodies that reveal activation in situ and showed that it is evidenced by the loss of the latent complex and unmasking of TGF-h [14]. Following radiation exposure, TGF-h activation is evident within an hour, persistent for at least a week, and detected following whole body doses of as little as 0.1 Gy [15]. As a consequence of its rapid activation, we demonstrated a novel mechanism of TGF-h activation via exposure to reactive oxygen species, which endows TGF-h with a redox sensor capability. As such, we proposed that TGF-h acts as an extracellular lynch pin released by radiation and other oxidative stressors, to orchestrate multicellular response to damage [16].
3. The irradiated microenvironment promotes neoplastic potential To test the contribution of such alterations to the process of carcinogenesis, we created radiation chimeric mammary glands by taking advantage of the postnatal development of the mammary gland to surgically create a stroma without epithelium [17]. We then transplanted unirradiated mammary epithelial cells to an irradiated mammary stroma. The mammary cells we used were nontumorigenic by several criteria but harbor mutation in both alleles of the p53 gene [18]. Infrequent small tumors that regress occur in shamirradiated adult stroma. Nevertheless, when these cells were transplanted to irradiated (4 Gy) stroma, tumors formed in three-quarters of the transplants and the tumors were significantly larger. Furthermore, the effect of the irradiated stroma persisted up to 14 days after exposure. Since hemi-body irradiation resulted in tumors only on the irradiated side, we concluded that this effect was primarily due to altered stromal microenvironment. Radiation-induced microenvironments constitute a new class of its carcinogenic action that affects the ability of cells to maintain appropriate communication between each other. As a consequence of disrupted communication, we hypothesized that the cells harboring neoplastic mutations are released from extracellular signals that suppress their proliferation in normal tissue [19].
4. Radiation induces a heritable HMEC phenotype that is characteristic of malignancy To test whether radiation exposure alters cell communication, we used a model of nonmalignant HMEC cultured within an artificial ECM. Under these culture conditions, single cells proliferate as clones that organize into a structure typical of glandular epithelium, i.e., an acinus consisting of a single layer of polarized epithelial cells organized into a hollow sphere. Morphogenesis in these colonies is accompanied by appropriately localized proteins necessary for tissue structure and polarity. For example, E-cadherin is localized at the interface between cells, h1-integrin is baso-lateral and a6-integrin is basal [8]. Importantly, tumorigenic and nontumorigenic mammary epithelial cells, which are nearly indistinguishable when cultured as monolayers, are readily classified by their morphogenesis in three-dimensional ECM [20]. Tumor cells proliferate, fail to establish
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appropriate cell– cell and cell– ECM connections, and thus, form clumps. In contrast, nonmalignant mammary epithelial cells growth arrest after a short period of growth and form acini similar to those found in situ. We used this tissue-specific morphogenesis to ask whether irradiated HMEC maintained appropriate microenvironment interactions. Cells were irradiated shortly after plating and TGF-h was added to some cultures to mimic the presence of an irradiated stroma. We observed that most colonies arising from cells treated with radiation and TGFh showed pronounced morphological disorganization in comparison to colonies from sham controls or following single treatments (Barcellos-Hoff et al., unpublished data). Surprisingly, we also found that the number of cells per colony was increased in double treated specimens. Furthermore, using confocal microscopy and immunofluorescence, we observed that colonies from irradiated cells cultured in the presence of TGF-h showed a dramatic loss of E-cadherin, significantly increased h1-integrin, and decreased a6integrin. A distinct collagen IV containing basement membrane was observed in all treatment groups, suggesting that the altered integrin expression was not due to the lack of appropriate ligand. Importantly, the phenotype occurred in almost all cells that survive irradiation, which clearly limits the role of mutational mechanisms. Together, these data suggested that colonies arising from irradiated cells exhibit a consistent phenotype consisting of inappropriate intercellular adhesion, deranged extracellular adhesion molecules, loss of gap junction proteins, and disorganized tissue-specific organization. These observations indicate that radiation exposure of individual cells leads to a persistently altered phenotype in daughter cells that is characterized by the loss of critical controls imposed by the microenvironment to maintain tissue integrity. The resulting behavior is consistent with malignant progression. We have argued that microenvironment perturbations induced by irradiation are means of fostering neoplastic progression in susceptible (i.e., premalignant) cells [4]. Tlsty [21] has recently proposed that disturbances in cell adhesion can modulate pathways that control genomic stability. We predict that if such colonies arising from irradiated cells show increased genomic instability, it is a consequence of the lack of microenvironment control rather than as a direct result of DNA damage. Future studies will test whether the irradiated HMEC phenotype contributes to radiation-induced genomic instability.
5. Potential mechanisms underlying an irradiated phenotype Since the disruption of extracellular interactions occurs in almost all colonies formed by cells that survive irradiation, the role of mutational mechanisms is clearly limited as the mechanism underlying this phenomenon. Phenotypic evolution is a more likely basis for the behavior of irradiated HMEC. Phenotype is driven by biochemical changes, due in part to extracellular signaling and epigenetic modulation of the genome. Both can lead to heritable phenotypes (as evidenced by the differentiation of more than 300 cell types from a human genome) or a reversible phenotype, such as in the ‘activated’ phenotypes that are observed in certain cells during inflammation or wound healing. If the mechanisms by which the irradiated phenotype is perpetuated involve extracellular signaling via soluble molecules or cell contact, irradiated cells will be able to influence unirradiated cells via a
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‘‘bystander’’ mechanism. Radiation ‘‘bystander’’ effects have been demonstrated both in vitro and in vivo [22 –24]. It is unclear whether bystander effects that promote neoplastic behavior are a function of direct cell contact, or mediated by soluble factors, or (more likely) both. The relevance of studying epigenetic mechanisms in irradiated cells lies in their potency as harbingers of change: under certain circumstances, epigenetic modifications occur at high frequency in specific genes known to be players in the carcinogenic process. Epigenetic mechanisms encompass a wide range of events that affect how the genome is expressed but are not as well characterized as genomic sequence changes during cancer progression. DNA cytosine methylation in regions that are CpG-rich (CpG island) recruits methylated-DNA binding proteins that in turn interact with histone deacetylases and alter chromatin structure; each of these processes can act independently or in concert to favor inhibition of gene expression necessary for certain phenotypes. Little is known about mechanisms leading to aberrant methylation in epithelial cells and scarcely any data exist regarding methylation following radiation exposure. In addition to several Russian reports in the 1970s, one study in 1989 showed a dose-dependent decrease in 5-methylcytosine at 24, 48 and 72 h post-exposure to 0.5– 10 Gy [25]. In tumors, aberrant methylation contributes to gene silencing and may contribute to the generation of heterogeneous phenotypes. Recently, the importance of epigenetic changes during carcinogenesis has gained attention [26]. Studies have demonstrated that hypermethylation of 14-3-3 sigma occurs in 91% of primary breast cancers and is strongly associated with the loss of gene expression in these tumors [27]. Also 50% of breast tumors exhibit E-cadherin epigenetic alterations [28]. The p16/INK4A and retinoblastoma are also among the genes that show changes in the methylation pattern during cancer progression. Romanov et al. [29] have shown that HMEC spontaneously bypass senescence and exhibit genomic instability which is accompanied by p16 methylation.
6. Conclusions Our studies of the irradiated mammary gland and the progeny of irradiated human cells suggest that radiation exposure can promote malignant progression by pathways other than that of mutational mechanisms. Since the frequency of chromosome aberrations increases many cell generations after irradiation by an, as yet, unknown mechanism [30 –32], we suggest that disruption of extracellular signaling, adhesion, and communication could precede, and may augment, destablization of the genome. Our hypothesis that genomic instability is as much a matter of the loss of a multicellular controls as the induction of cellular process [33]. Our future studies will concern the molecular mechanisms underlying these events.
Acknowledgements Funding for this research was from the Specialized Center of Research and Training in Radiation Health from the National Aeronautics and Space Administration and from the
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Low Dose Radiation Program in the Office of Biological and Environmental Research, Office of Energy Research of the US Department of Energy at Lawrence Berkeley National Laboratory.
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