JAMES E. TROSKO

The survival of multicellular organisms through evolution in an aerobic environment came about by the adaptive responses to both the endogenous oxidative metabolism in the cells of the organism and all the chemicals and low-level radiation to which they had to be exposed. Included in the defense repertoire were preventive mechanisms against excessive oxidative damage to membranes, proteins, and DNA; built-in redundancies for damaged molecules; tight homeostatic and physically coupled redox systems, pools of reductants, antioxidants, DNA repair mechanisms and sensitive "sensor" molecules such as NF-kB; and signal transduction mechanisms affecting both transcription and post-translational modification of proteins needed to cope with the oxidative stress. The biological consequences of the response of a multicellular organism to the low-level radiation that might exceed the background level of oxidative damage to a cell in a tissue could be apoptosis, cell proliferation, or cell differentiation. If these biological endpoints are not detected at rates above non-irradiated control levels in an organism, it is highly unlikely that low-level radiation would play much of a role in the multistep process of carcinogenesis. Finally, gap-junctional intercellular communication, known to be required for homeostatic regulation of cell proliferation and adaptive functions in a multicellular organism, could, by its ability to suppress cell division in coupled cells of a tissue, provide protection to any one cell receiving track-radiation through the sharing of reductants and by triggering apoptosis.

Solving the long-standing, yet extremely germane problem of predicting the risks to human health after exposure to low-level acute and chronic ionizing radiation has had to rely on the extrapolation and integration of theoretical, experimental molecular, in vitro and in vivo cellular and animal studies, in addition to epidemiological examination of therapeutic, accidental, and atomic bomb exposures to ionizing radiation. A rather narrow analysis will be made because of the complexities arising from the initial quality and quantity of the radiation exposure (e.g., high or low LET; acute, chronic, dose, etc.), as well as from the genetic, developmental, sex, and pre- and post-exogenous factors influencing the radiation effects. Specifically, the question of this brief review will be whether one can determine what potential roles ionizing radiation can play in the induction of human cancers at low-level acute or chronic exposures. Therefore, the assumptions to be made explicit are that (1) all cancers arise from a single stem call ["the stem cell theory" or "oncogeny as partially blocked ontogeny theory" (Nowell, 1976; Potter, 1978; Fialkow, 1979)]; (2) the carcinogenic process involves both multiple steps and multiple mechanisms ["initiation," ''promotion" and "progression" phases (Pitot et al., 1981)]; and (3) the ultimate appearance of a cancer is the result of the breakdown of multiple homeostatic mechanisms at the molecular, cellular, tissue, organ, and organ-system levels in a multicellular organism such as a human being (Potter, 1974; Potter, 1983). While this analysis will focus on cancer, many of the implications will relate to other potential long-term health effects related to low-level radiation exposure, such as birth defects, cataracts, atherogenesis, and other chronic diseases.

The fundamental assumption will be that, while the primary injury to a multicellular organism is the energy deposition (tracks or hits to critical molecules) in terms of ionizations and excitations in localized clusters at the atomic and molecular levels in a cell, and while the cell is considered the unit of life (Feinendegen et al., 1995), it is the intercellular coupling or syncytium of cells that is the functional unit of life. It is this understanding of how a "society of cells" within an organism responds to the immediate and long-term effects of radiation that will give us some insight to understand how cancers, attributed to radiation exposure by epidemiological studies, come about and how molecular effects of radiation at the cell level ultimately contribute to the disruption of all the homeostatic mechanisms at each hierarchical level of a multicellular organism.

The concept of molecular epidemiology was developed in this era of new concepts and molecular findings related to cancer—namely, that oncogenes and tumor-suppressor genes exist, and that in most tumors, physical and chemical carcinogens could induce specific lesions in DNA that could form mutations having a unique "fingerprint" (Jones et al., 1991; Shields and Harris, 1991). While this concept of "molecular epidemiology" has been somewhat supported by observations that sunlight-induced skin tumors had mutations in their p53 genes indicative of the pyrimidine dimers induced in skin cells by the ultraviolet light of the sun, associations with other carcinogenic chemicals have not been as successful in supporting the concept (Brash et al., 1991; Dumaz et al., 1993). If, in fact, ionizing radiation of any suspected exposure could induce unique DNA lesions that might cause unique mutations in oncogenes and tumor-suppressor genes that are distinct from spontaneous or background radiation, then possibly molecular biology can contribute to epidemiology in resolving the specific role that ionizing radiation plays in carcinogenesis. However, as will be discussed below, carcinogenesis is more than mutagenesis, and ionizing radiation can do more than just induce DNA damage.

While there is no dispute about neoplastic transformants or tumors appearing in cells, animals, or humans exposed to radiation, in vitro and in vivo, there is no consensus as to the molecular or biological mechanism by which radiation contributes to the carcinogenic process. In addition, great uncertainty exists as to the risks to humans after either low-dose, acute, or chronic, exposure to ionizing radiation (Upton, 1989; Gerber et al., 1991; Sugahara et al., 1992; Hoel, 1993; Kondo, 1993; Gilbert, 1994; Pollycove, 1994; Sinclair, 1994; Thompson et al., 1994; Fliedner et al., 1995).

The carcinogenic process can be conceptualized as consisting of at least three operational phases: "initiation," "promotion," and "progression" (Pitot et al., 1981). The initiation phase has been defined as an irreversible event occurring in a stem cell that prevents it from terminal differentiation but not from proliferation (Figure 11.1). As long as that single initiated cell is surrounded by and communicates with its normal neighbor, it poses no problem to the organism; a problem only arises if that mitogenic suppressing influence is inhibited. Promotion is the process that allows the clonal amplification of the initiated cells (Trosko et al., 1990) and the prevention of the induced programmed cell death or apoptosis of the initiated cell (Burch et al., 1992). Finally, when during the clonal expansion of the initiated cells additional genetic alterations occur (e.g., mutation, amplification, loss of various oncogenes or tumor-suppressor genes), the initiated cell acquires the phenotype by which the cell becomes autonomous or independent of exogenous growth factors or tumor promoters, then the cell has entered the progression phase.

FIGURE 11.1 The initiation/promotion/progression model of carcinogenesis. β₁ = rate of terminal differentiation and death of stem cell; β₂ = rate of death, but not of terminal differentiation of the initiated cell; α₁ = rate of cell division of stem cells; α₂ = rate of cell division of initiated cells; μ₁ = rate of the molecular event leading to initiation (i.e., possibly mutation); μ₂ = rate at which second event occurs within an initiated cell.

The scientific question to be asked is, Can ionizing radiation induce each phase of carcinogenesis? That is, can ionizing radiation convert a normal stem cell to an initiated cell? Can ionizing radiation contribute to the clonal expansion of a pre-existing initiated cell? Or, can ionizing radiation bring about the neoplastic conversion of an initiated clone of promoted cells? It should be obvious in the context of an acute exposure to low-level radiation, by definition, ionizing radiation cannot be a tumor promoter, since a single exposure would not be sufficient to allow sustained proliferation of an initiated cell or sustained prevention of apoptosis of the initiated cell. It is unknown whether sustained proliferation or prevention of apoptosis can occur with low-level chronic exposure to ionizing radiation.

While in vitro experiments with animal cells suggest answers to these questions, because of limitations in the interpretations and extrapolation to human cells and the human organism, it is not possible to conclude rigorously that ionizing radiation is an initiator and a progressor. The same could also be said of the few animal

experiments designed to test if ionizing radiation could affect these three phases of carcinogenesis (Trosko, 1994). On the other hand, on a theoretical level, based on the molecular concepts of oncogenes and tumor-suppressor genes (Bishop, 1995) and current understanding of the molecular nature of how ionizing radiation can damage DNA and induce various kinds of gene and chromosomal mutations (Thacker, 1986; Lucke-Huhle et al., 1990; Renan, 1992; Felber et al., 1992; Ito et al., 1993; Cox, 1994; Goodhead, 1994), one could speculate where one might expect ionizing radiation to play a role in the multistep, multiple mechanism process of carcinogenesis (Trosko, 1992).

It is well established that ionizing radiation can induce DNA singleand double-strand breaks, directly or indirectly, as well as various DNA base modifications (Cox, 1994; Ward, 1995). In addition, point mutations, gene deletions, and a variety of chromosome aberrations can be induced by exposure to ionizing radiation (Thacker, 1986; Renan, 1992; Lucke-Huhle et al., 1990; Felber et al., 1992; Ho et al., 1993; Goodhead, 1994; Nelson et al., 1994). Molecular analyses of a number of genes used as targets for ionizing radiation appear to indicate that the majority of the mutant cells that survived in order to be analyzed contained deletion mutations with gross chromosomal aberrations (Thacker, 1986). Assuming for the sake of this analysis that ionizing radiation is a better deletion-mutagen/clastogen than a point mutagen, ionizing radiation would be better at deactivating a tumor-suppressor gene than activating an oncogene. While chromosome rearrangements (i.e., possibly the transposition of the bcr/abl gene) or small gene deletions (i.e., the truncation of the ErB2/Neu) by ionizing radiation could activate oncogenes without inducing point mutations within the genes, the likelihood of deleting a tumor suppressor gene by ionizing radiation would seem more probable. If ionizing radiation does leave a unique molecular lesion leading to unique types of mutations in either oncogenes or tumor-suppressor genes found in tumors of those known to have been exposed to ionizing radiation (i.e., "molecular epidemiology"), then one might be able to pinpoint the specific role ionizing radiation plays as a mutagen in the different stages of carcinogenesis.

Until recently, it was well established that one of the consequences of ionizing radiation was cell death caused either by DNA/chromosomal lesions or by some non-DNA-related events within the cell. With the explosion of research on the phenomenon of programmed cell death or apoptosis, it now seems clear that much of what previously was thought to be ionizing-radiation-induced cell necrosis was, in fact, apoptosis (Meyn et al., 1993).

In addition, the fact that apoptosis can be induced by targets in the cell other than DNA implies that at low-dose levels, oxidative stress induction of epigenetic events might be important (Fritch et al., 1994). This new insight is now forcing the integration of knowledge on apoptosis, stem cells, various oncogenes (Bcl-2, ras, etc.), tumor-suppressor genes (i.e., p53), non-DNA damage to cells, activation of various transcription factors (AP-1; NF-kB), and tumor promotion.

Clearly, cell removal via surgery or cell death in tissues containing initiated cells has been shown to act to promote tumors in animals (Argyris, 1985). Recently, it has been observed that many chemical tumor promoters, such as phorbol esters, phenobarbital, and DDT can inhibit apoptosis (Burch et al., 1992). The speculation is that tumor promotion is a process that requires not only the prevention of the loss of initiated cells by the blockage of apoptosis, but also the clonal amplification of these initiated cells (Trosko et al., 1983).

In the context of trying to determine if ionizing radiation can promote tumors by killing cells, several diametrically opposite effects of radiation on cell killing must be examined. On a single-cell level (only relevant in vivo for free-existing cells of the "soft tissues," other single migrating cells, and stem cells), the probability of killing the cells after low-level exposure by DNA or non-DNA mechanisms must be considered (1) for various kinds of cells [stem versus progenitor versus terminally differentiated cells (Potten et al., 1994; Vergouwen et al., 1994)]; (2) at various times in the cell cycle (Little, 1994); and (3) for cells that have effective DNA repair (for DNA lesions causing cell death) or effective quenching of non-DNA lesions [e.g., glutathione levels (Miura and Sasaki, 1991; Saunders et al., 1991; Bergelson et al., 1994; Meister, 1994; Sierra-Rivera et al., 1994)]. This consideration will relate to radiation-induced leukemias. For cells in solid tissues, the phenomenon of gap-junctional intercellular communication, which effectively couples many cells of a tissue into a syncytium, the probability of killing a cell in this matrix will be affected by additional factors to be discussed later.

At a low-dose exposure given either acutely or chronically, if radiation does not induce significant cell killing by necrosis (DNA lesions causing lethal mutations or non-DNA mechanisms not triggering apoptosis), the likelihood of ionizing radiation acting as a tumor promoter is small if not nil. Recent evidence regarding ionizing-radiation-induced apoptosis indicates that, depending on the type of cell (Vergouwen et al., 1994; Potten et al., 1994), p53 tumor-suppressor gene can be induced (Potten et al., 1994). The induction of p53, a molecule which has multiple functions (Cox and Lane 1995), would allow for more DNA repair, thus removing DNA lesions and reducing the chance of certain classes of mutations (Gotz and Montenarh, 1995). If a cell with DNA damage is not removed by either necrosis or apoptosis, then this surviving cell could produce mutations which could then contribute to the carcinogenic process (Gotz and Montenarh, 1995). Ionizing-radiation-induced p53-dependent apoptosis is then considered a mechanism by which potentially initiated cells are removed from tissue rather than promoted.

In fact, one of the current ideas for radiation therapy is to increase apoptosis in existing tumor cells (Kerr and Searle, 1979).

If radiation can induce cell killing by necrosis and apoptosis, the question now is, At low-dose exposures, does ionizing radiation primarily induce apoptosis? Also, as will be discussed later, are all cells equally susceptible to radiation-induced apoptosis? Until experiments are designed to answer these questions, it seems impossible to predict if ionizing radiation is a potential tumor promoter at low doses, given acutely or chronically.

Only in the last few years has the idea been advanced that radiation might bring about some of its biological effects by non-DNA damage-related mechanisms. Clearly, ionizing radiation can damage DNA in cells, and this damage can be the substrate for mutations and/or cell death. Under those conditions where the probability is too low that a track or hit occurs within a given cell of a tissue or at or near DNA (Goodhead, 1994; Feinendegen et al., 1995), it is known that the cell does react epigenetically. That is, the cell responds by induction of gene transcription, modified translation of gene messages, and altered post-transitional modification of existing proteins (definition of an epigenetic response) (Trosko et al., 1992).

Specific gene induction by ionizing radiation and activation of pre-existing proteins have been reported and reviewed several times (Woloschak et al., 1990; Boothman and Lee, 1991; Weichselbaum et al., 1991; Uckum et al., 1992; Herrlick and Rahmsdorf, 1994). While the prevailing paradigm has been that DNA damages or arrested DNA forks were the signal for the initiation of new gene transitional activity (Devary et al., 1992), there is evidence that there exists a non-DNA damage source of a radiation-induced signal transduction pathway, including that which leads to apoptosis (Ramakrishan et al., 1993; Buttke and Sandstrom, 1995).

The single isolated cell or the syncytium of cells within a tissue functions because of the use of oxygen as the terminal electron acceptor in order to produce energy. In order to survive potential cellular damage because of the constant generation of reactive oxygen species (ROS), a delicate homeostatic regulatory system had to evolve with the evolution of living organisms in an aerobic world, which would allow a cell (1) to recognize any change in its external environment; (2) to adapt to this signal by proliferating, differentiating, or if differentiated, adaptively responding to the signal (Gotz and Montenarh, 1995). A balance of pro-oxidants and antioxidants provides elements for a homeostatic mechanism (Cerutti et al., 1994). Tipping that balance within limits allows the cell to perform its necessary survival functions. While the cell and tissue have developed mechanisms to minimize and avoid lethal disruptions of the redox homeostatic state (e.g., various repair systems and redundant targets of oxidative damage), in some cases the fact that

oxidative stress is not handled adaptively by a cell may, in fact, be adaptive to the tissue and organism (Buttke and Sandstrom, 1995). In other words, non-adaptive cells would be eliminated by apoptosis.

Ionizing radiation, as well as ultraviolet light and many chemical toxicants, can induce free radicals, directly or indirectly. In those cases, especially at low doses, where there is little or no genomic DNA damage (Feinendegen et al., 1995), the ROS produced can act as signaling molecules, per se, or by oxidatively modifying cellular components. In order to contribute to the alteration of the redox state of the cell and thereby trigger some epigenetic event, the amount of ROS and/or its intracellular localization must exceed the normal background level of oxidative metabolism-generated ROS (Hill and Treisman, 1995).

While the detailed mechanisms for ionizing, UV, or chemical induction of oxidative stress are not yet known, it has been postulated that plasma- and intracellular unsaturated fatty acid-containing membranes are targets of the ROS (Devary et al., 1992; Buttke and Sandstrom, 1995). Cellular and organelle membranes are probably the sensors enabling the cell to gauge the change in the redox state of the cell. In addition, specific protein molecules (e.g., p53, NF-kB, AP-1) are important cellular monitors of the altered redox state (Angel and Karin, 1991; Devary et al., 1992; Hainaut and Milner, 1993; Baeuerle and Henkel, 1994; Hill and Treisman, 1995; Ueda et al., 1995). Their alteration by oxidated/reductive changes would dramatically affect their numerous and critical functions within a cell. The formation of reactive oxygen species by ionizing radiation, as well as by phorbol esters and H₂O₂, can stimulate mitogen-activated protein kinase activity (Stevenson et al., 1994). Free radicals at high doses can cause toxicity; however, at low doses they are associated with cell growth of certain cells (Nicotera et al., 1994; Stevenson et al., 1994; Timblin et al., 1995).

Before anything could happen after the cell experiences an ionizing track, the cellular oxidative protective mechanisms must be breached. That is, if the intracellular pool of reduced glutathione is insufficient and antioxidant deficiencies exist, then lipid peroxidation could occur, since the track is more likely to be in the vicinity of the plasma or other membranous target than in the nucleus (Feinendegen et al., 1995). Any resulting oxidative stress could trigger activation of protein kinases [i.e., tyrosine kinase (Devary et al., 1992)], inactivation of p53 (Hainaut and Milner, 1993; Baeuerle and Henkel, 1994), or activation of a transcription factor NF-kB (Baeuerle and Henkel, 1994). It has been shown that the regulation of the activity of NF-kB might be responsible for the ionizing radiation sensitivity of the ataxia telangiectasia phenotype (Jung et al., 1995).

To help stabilize the redox state against endogenous and exogenously generated oxidative stress, antioxidants—especially glutathione levels—have been shown to influence radioresistance of certain cell lines under certain conditions (Miura and Sasaki, 1991; Saunders et al., 1991). However, the endpoints measured usually included DNA damage and cell survival, and the various studies used a variety of cell types (mostly cancer cell lines) irradiated under a number of conditions.

FIGURE 11.2 Schematic diagram the complex factors (stem cells; coupled progenitor/differentiated cells; intracellular antioxidant protective mechanism; redox or oxidative-stress-sensing signal transducing elements) which could influence the initial radiation exposure in tissues.

Recent studies have implied the role of glutathione depletion in activation of gene expression (Bergelson et al., 1994; Hu and Cotgreave, 1995) in accordance with the prediction that oxidative stress-sensing-molecules such as NF-kB and AP-1 would be triggered under these conditions. Moreover, since in solid tissues, most cells are coupled by gap junctions, glutathione levels would be stabilized by transfer of glutathione which can traverse the gap junctions (Barhoumi et al., 1995; Nakamura et al., 1995). The relation of drug or radiation sensitivity and the ability of cells to couple via gap junctions have been previously shown (Tofilon et al., 1984; Frankfurt et al., 1991; Pitts, 1994) (Figure 11.2).

The implication of these studies for normal cells is that ionizing radiation-induced oxidative stress would be modified by the metabolically coupled cells. To induce either cell proliferation or apoptosis in individual cells within the coupled syncytium must involve some as yet unknown factors.

The tumor-suppressor gene, p53, has been implicated in multiple functions related to the regulation of cell division, differentiation, DNA repair, and programmed

cell death or apoptosis (Cox and Lane, 1995; Gotz and Montenarh, 1995). Indeed, the association of the mutated p53 gene or loss of heterozygosity of the p53 gene has been linked to a large number of human cancers (Hollstein et al., 1991). However, in those cancers having no p53 mutations or loss of heterozygosity, functional inactivation can occur by other means, including amplification of the MDM2 gene product which binds to p53 (Fakharzadeh et al., 1991). These processes involving the mutation, deletion, or inaction by gene amplification are all considered genotoxic events. In the cell whose redox state might be altered without having a genotoxic event, the altered redox state itself could bring about the reversible, but nevertheless potentially disruptive effect of ionizing radiation. Several studies have implicated the functional conversion of the normal p53 protein to a "mutated" form in cells with an altered redox state (Hainaut and Milner, 1993; Ueda et al., 1995).

Phorbol esters, powerful skin tumor promoters, can bring about the phosphorylation of the p53 protein (Baudier et al., 1992). Depending on the cell type, phorbol esters can induce cell proliferation, block apoptosis, or modulate differentiation via their ability to activate protein kinase C (Yamasaki et al., 1984). Ionizing radiation has been shown to induce protein kinase C at rather high dose levels (2 Gy) (Woloschak et al., 1990; Avila et al., 1993). TPA, by down-regulation of PK-C, has been shown to block the induction of transcription by ionizing radiation, presumably by the lack of post-translational modification of critical transcription factors needed for initiation of gene transcription (Lin et al., 1990). The stimulation of tyrosine kinase activity by ionizing radiation and other inducers of oxidative stress has also been shown to activate phospholipase D, which could then lead to signal transduction pathways needed to initiate early-response gene transcription (Avila et al., 1993).

Ionizing radiation is known to induce p53, and it is implicated in DNA damage recognition and apoptosis (Gotz and Montenarh, 1995). Potten and co-workers (1994) have shown that low levels of ionizing radiation could induce apoptosis in the stem cell pool of the small intestine of the mouse, whereas the stem cells of the large intestine were resistant to radiation induction of apoptosis. The expression of Bcl-2, or lack of it, as well as the induction of p53 by radiation in these tissues, correlated with the ability of ionizing radiation to induce cancers in these two tissues. In the p53 knockout mice, the roles of p53 in radiation-induced apoptosis and carcinogenesis have been examined (Clarke et al., 1994). It turns out that the rapidly appearing apoptosis that occurs after ionizing radiation is absent in the p53 null mouse, but that spontaneous apoptosis occurs at the level found in the normal mouse (Potten et al., 1994). While contradictory results have been obtained when testing whether the functional loss of p53 would affect radiation sensitivity, cell-type specificity might have influenced the differences (Jung et al., 1992; Slichenmyer et al., 1993).

The exact cascade of events might include the oxidation of unsaturated fatty acids in the membranes, and the preferential release of the hydroperoxides by

phospholipase A2 enzyme, with the subsequent increase of intracellular calcium. Increased phosphorylation of proteins such as c-Jun due to activation of kinases such as the src kinase after UV radiation (Devary et al., 1992) could activate AP-1 transcription factor. This, in turn, could activate the genes having AP-1 elements in their DNA. Simultaneously, the phosphorylation of p53, the "guardian" of the cell (Lane, 1992) could convert it to a longer lasting molecule, as well as one with dramatically different functions (Woloschak et al., 1990; Uckum et al., 1992).

The alteration in the redox state by ionizing radiation, as well as by non-genotoxic chemicals and growth regulators, can also affect the nuclear factor-kB (NF-kB) (Baeuerle and Henkel, 1994). NF-kB is normally regulated by the 1kB-a protein in the cytoplasm of the cell. Upon phosphorylation of the 1kB-a, the unbound NF-kB translocates to the nucleus where it activates transcription, while the 1kB-a undergoes proteolysis (Jung et al., 1995).

These examples of the ability of agents such as ionizing radiation to activate pre-existing proteins quickly by the generation of radical oxygen species in cells where there has been no genomic DNA damage should support the idea that ionizing radiation can be an epigenetic agent. The rapid effect on pre-existing proteins via either direct conformational changes due to oxidation/reduction events, or by post-translational modification by phosphorylation/dephosphorylation and the subsequent transcriptional activation of early response genes, are within the definition of epigenetic events. These events could not occur, however, if the radiation-induced reactive oxygen species did not exceed the background levels due to normal oxidative metabolism, for which the cell has provided a variety of protective mechanisms. The availability of the intracellular glutathione levels and antioxidants, together with the number and intracellular localization of the ionization tracks, would determine if there would be a disequilibrium of the redox state. Thresholds must be exceeded for this to occur (e.g., exceeding background levels to cause a change in state and the necessity for producing sufficient redox-induced transcription factors for gene activation) (Hill and Treisman, 1995). Most importantly, in the context of the process of carcinogenesis, several factors must be kept in mind, namely (1) whether single isolated cells or cells coupled by gap junctions in a syncytium are oxidatively stressed; (2) what stages of the cell cycle the cells are in at the time of oxidative stress; (3) the state of differentiation at the time of oxidative stress; (4) whether the cells are in different stages of the carcinogenic process (e.g., initiated, promoted, or neoplastically converted); and (5) the wide background of genetic or environmental factors could modify the initial radiation effect in any given individual.

A multicellular organism such as a human being exists because from the single fertilized egg to the adult of 100 × 10¹² cells, all the cells communicated with

FIGURE 11.3 Schematic diagram characterizing the postulated link between extracellular and intercellular and intracellular communication through various intracellular transmembrane signaling mechanisms. Intracellular communication alters membrane function, activates inactive proteins, modulates gap-junction function, and modulates gene expression. This diagram provides an integrated view of how the neuroendocrine immune system (mind or brain/body connection) and other multisystem coordinations could occur. While not shown here, activation or altered expression of various oncogenes (antioncogenes) could also contribute to the regulation of gap-junction function.

each other via extra-, intra- and intercellular communication processes, as shown in Figure 11.3.

Although cell-matrix and cell-cell adhesion are distinct forms of communication, for the purpose of this discussion they will be viewed as a subclass of intercellular communication. Specifically, intercellular communication is mediated by the membrane-associated protein channel, the gap junction. The gap junction appeared very early in the evolution of the multicellular organism, where a delicate balance of the regulation of cell proliferation, cell differentiation, and adaptive functions of the differentiated cells had to be maintained (Revel, 1988).

The gap-junction structure—which consists of hemi-channels, connexons , in each of the two contiguous cell membranes—allows the passive diffusion of ions and molecules below 1,200 daltons between the coupled cells (Evans, 1988). By being coupled via gap junctions, cells can either equilibrate their ions and regulatory molecules or transiently send or receive signals to upset equilibria (Sheridan, 1987). Synchronization of electronic or metabolic functions occurs in various tissues by gap-junctional intercellular communication (GJIC) (Bennett et al., 1991).

The connexons are composed of a hexameric arrangement of proteins called connexins. These connexins are coded by a highly evolutionary conserved family of genes (Dermietzel et al., 1990). The gap-junction genes express themselves in different cells of different organs (Gimlich et al., 1990; Yancey et al., 1992). The connexin genes are regulated at the transcriptional, translational, and post-translational levels (Saez et al., 1990). Many of the second messages needed for signal transduction (e.g., Ca⁺⁺, nitric oxide, diacylglycerol, c-AMP, phosphorylation by protein kinases) have been shown to either up- or down-regulate GJIC. Hormones, growth factors, neurotransmitters, as well as oncogenes (e.g., src, neu, ras, raf, mos), a tumor-suppressor gene, chemical tumor promoters (e.g., TPA, phenobarbital, DDT, TCDD, etc.), anti-tumor promoters and anti-carcinogens (e.g., retinoids, carotenoids, green tea components, lovastatin), have been shown to either up- or down-regulate GJIC (Trosko et al., 1993; Miyachi and Nishikawa, 1994). Most cancer cells have either defective homologous or heterologous GJIC (Kanno, 1985; Yamasaki et al., 1987).

All this evidence seems to be consistent with the original hypothesis by Loewenstein that the loss of GJIC is the causal basis of a cancer (Loewenstein, 1966). In view of the stem cell theory of cancer (Kondo, 1983) and the facts that cancer cells do not have growth control nor are they able to terminally differentiate [''oncogeny as partially blocked ontogeny" (Potter, 1978)], the recent observations that stem cells do not express gap-junction genes (Chang et al., 1987; Kao et al., 1995) might implicate gap junctions in the carcinogenic process (Holder et al., 1993), since gap junctions have been implicated in the regulation of growth control and differentiation (Loewenstein, 1979; Brissett et al., 1994). With the reports that transfection of non-communicating cancer cells with connexin genes could restore normal growth control and reduce tumorigenicity (Eghbali et al., 1991; Metha et al., 1991; Zhu, et al., 1991; Zhu et al., 1992; Jou et al., 1993), and that the antisense connexin gene could eliminate the suppression of neoplastic transformation (Goldberg et al., 1994), more evidence has been provided to support the hypothesis that GJIC is needed to prevent tumorigenesis.

The role of gap junctions in low-level radiation induction of cancer is relevant in several places in the carcinogenic process, namely, the stem cell (the "target" cell for the carcinogenic process), the promotion phase of carcinogenesis (the phase of carcinogenesis that depends on epigenetic events to trigger cell proliferation of

the single initiated cell) and apoptosis (the potential removal of stem cells or initiated cells). To tie these ideas together with radiation-induced redox changes and carcinogenesis, the role of gap junctions might be seen as an integrating element.

If the stem cell is the "target" cell for carcinogenesis, then the apoptosis-related survival of those stem cells that are sensitive to low-level radiation-induced non-genotoxic or epigenetic changes would be affected. This appears to be the case for the stem cells of the small intestine (Potten et al., 1994). The frequency of small intestine cancers is very low compared to cancers of the large intestine, where the stem cell does not seem to be affected by radiation-induced apoptosis. If a stem cell has been initiated by some other carcinogen, a radiation-induced disequilibrium in the redox state might not be handled as well as in stem cells that are not initiated. Although oxidative stress has been implicated in the apoptotic process (e.g., chemicals that induce oxidative stress appear to block apoptosis), it is also linked to mitogenesis (Devary et al., 1992; Ramaishan et al., 1993; Herrlick and Rahmsdorf, 1994). Therefore, the radiation-induced reactive oxygen species might trigger very different responses, depending on the type of stem cell, which genes are expressed in the stem cell (e.g., Bcl-2, p53), the condition of the stem cell (e.g., whether it been initiated or not), and the availability of glutathione and antioxidants.

One additional characteristic of stem cells is that they might not possess expressed gap-junction genes. The fertilized egg, being a totipotent stem cell, does not have expressed or functional gap junctions at an early stage of development (Lo and Gilula, 1979). Two presumptive human epithelial stem cells of the kidney and breast do not express gap junctions (Chang et al., 1987; Kao et al., 1995). The growth of these cells is regulated by extracellular communication mechanisms that modulate intracellular communication-dependent signal transduction.

Gap junctions seem to be associated with the process of stem cell differentiation. Cells in solid tissues coupled by these gap junctions are able to share ions and small molecules, including Ca⁺⁺ and glutathione, which could help stabilize the redox state of a given cell (Kavanagh et al., 1988; Barhoumi et al, 1993). Many chemical tumor promoters that can cause oxidative stress in cells can (1) activate NF-kB, AP-1, and early response genes, and can stimulate protein kinases, inactivate by phosphorylation p53 and 1kB-a, etc.; (2) block GJIC; and (3) block apoptosis (Angel and Karin, 1991; Hainaut and Milner, 1993; Baeuerle and Henkel, 1994; Cerutti et al., 1994; Buttke and Sandstrom, 1995; Hill and Treisman, 1995). On the other hand, glucocorticoids and retinoids can up-regulate GJIC and induce apoptosis, while having dramatically different effects on the redox state of the cell and signal transduction (Hossain et al., 1989; Ren et al., 1994).

Gap junctions have been shown to be able to transfer glutathione between cells (Barhoumi et al., 1995; Nakamura et al., 1995), and the depletion of glutathione levels has been shown to potentiate TPA's ability to inhibit GJIC, presumably by enhancing oxidative stress in the cells (Hu and Cotgreave, 1995). Gap-junctional intercellular communication has been implicated in several studies on radiation

and chemical-induced toxicity. In other words, many studies have shown that cells grown in spheroids, as opposed to cells grown in sparse or even confluent two-dimensional cell cultures, are more radioresistant (Tofilon et al., 1984; Olive and Durand, 1994). This might imply the micro-environment in the spheroid could induce GJIC which is absent or reduced in non-spheroid conditions. There is evidence that the micro-environment can influence the presence and function of gap junctions in cells not normally having functional GJIC (Sasaki et al., 1984; Confort et al., 1986). The existence of contradictory literature related to radiation-and chemotherapeutic drug-resistance and their correlation with the presence or absence of GJIC might simply be related to whether the conditions in each particular experiment are conducive or not to the up-regulation of gap junctions. This is particularly true in the case where several oncogenes, which are associated with the down-regulation of GJIC (e.g., ras, raf, neu, src), have been associated with conferring radiation resistance to the cell (Sklar, 1988; Pirollo et al., 1993).

Since oxidative stress has been linked to the blockage of apoptosis and the block-age of GJIC, and the blockage of GJIC has been linked to tumor promotion, the question is now, Can low-level radiation exposure of solid tissues induce sufficient oxidative stress to block apoptosis in initiated cells, block GJIC, and help bring about the promotion phase of carcinogenesis? Promotion of an initiated cell can only occur if there is sustained application of the promoting stimulus (Siskin et al., 1982). If the low-level ionization can be sufficient to induce oxidative stress without genotoxicity, it might overcome a threshold (background oxidative metabolism) in the cell to activate early response genes, block apoptosis, and stimulate cell division. The tissue must be exposed chronically in order for this to occur. Since it has been shown that doses of 0.5 Gy can induce permeability of tight junctions and transient down-regulation of gap-junctional intercellular communication (Somosy et al., 1993), there is a possibility that an acute exposure to ionizing radiation could cause some cells to proliferate, others to differentiate, and still others, which are terminally differentiated, to produce adaptive responses. Protracted down-regulation of the tight junctions and gap junctions in epithelial cell layers might be responsible for the lethal effects of high doses of radiation (Somosy et al., 1993). Future studies at lower acute doses must be done to study the possible correlation between radiation-induced oxidative stress and down-regulation of GJIC.

Given in vitro and whole-animal experiments with ionizing radiation as well as several epidemiological studies, one would have to conclude, after extrapolation from high-dose levels to low-dose levels with the assumption of a linear no-threshold model, that radiation does indeed contribute to the carcinogenic process. Recent analyses of the incidence of solid tumors found in the survivors of the atomic bombs in Hiroshima and Nagasaki (Thompson et al., 1994) would lead some to conclude

that even at very low-dose exposures, there is a finite probability of radiation-induced cancers. Caution must be taken in the interpretation of these data. As was stated by Sinclair (1994), "Clearly, the linear fit over a dose range up to 4 Gy or 5 Gy is just that—a fit. It does not imply single-hit kinetics or any other mechanism." Among the known uncertainties in trying to derive risk estimates from the Life Span Studies on the atomic bomb survivors are the epidemiological studies themselves, the dosimetry, the projection to lifetime, the generalization from conclusions about the Japanese population of that time to all populations today, and the extrapolation to low-dose and dose-rate (Trosko, 1995).

From the analysis above—together with the fact that carcinogenesis is well known to involve multiple genetic and epigenetic changes before a normal diploid stem cell becomes invasive, malignant, and metastatic—the likelihood that an acute and even chronic low-level ionizing radiation exposure can affect each and all the steps necessary is extremely remote. If it can be shown that frequencies of induced gene mutations and chromosomal aberrations from apoptotic cells versus those from cells that survive radiation are the same or different, then the role of radiation-induced apoptosis of stem-like cells in carcinogenesis can be assessed. Indeed, if radiation-induced apoptosis is a biological protective mechanism to remove genetically damaged cells from tissue, an explanation for the findings to-date on the possible genetic damage done to the survivors of the atomic bombs, which so far have not shown any measurable increase (Neel et al., 1990; Neel and Lewis, 1993), might be that the germinal stem cells of the males have been eliminated.

While this analysis has not examined the phenomenon of the "adaptive response" or "hormesis" (Wolff, 1992; Lehnert, 1995), the possibility exists that a low dose of ionizing radiation, by inducing an increment of oxidative stress above normal background levels, could induce signal transduction for the transcription of genes needed to protect the cells from additional stress-inducing agents. If this includes induction of antioxidants, reductants, and cell cycle-checkpoint inhibitors to facilitate time for DNA repair—then one could hypothesize that there might be a mechanism to explain some of the reported "adaptive responses." The role of gap-junctional intercellular communication (Trosko, 1991) would be included. In tissues where cells are coupled by gap junctions, cell division would not be occurring, thereby facilitating the process of repair before lesions in DNA are "fixed" by replication errors.

These ideas were derived from research supported in part by grants from the National Cancer Institute (CA21104), the US Air Force Office of Scientific Research (No. 94-NL-196), and the Michigan Great Lakes Protection Fund. The author wishes to acknowledge the excellent word processing skills of Mrs. Robbyn Davenport.