(CANCER RESEARCH 53. 3475-3485. August I. 1993]

Perspectives in Cancer Research

Gap Junction Function and Cancer James W. Holder,1-2 Eugene Elmore, and J. Carl Barrett i

Genetic Tmicologv Assessment Branch, Office of Health and Environmental Assessment. Office of Research and Development (RD-689). EPA. Washington. DC 20460 ¡J.W. H.¡: National Institute for the Advancement of in Vitro Sciences, Irvine, California 92715 ¡E.£./; and Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences. Research Triangle Park. North Carolina 27709 ¡J.C. B.¡

Abstract relevance of normal GJ function and modulation in disease was re viewed, the discussion often centered on the relevance to cancer Gap junctions (GJs) provide cell-to-cell communication of essential because of GJ involvement in the tumor promotion stage (1, 2). The metabolites and ions. GJs allow tissues to average responses, clear waste following discussion on GJs is topical and not comprehensive. It is products, and minimize the effects of xenobiotics by dilution and allowing based in part on the presentations and discussions held at the Envi steady-state catabolism. Many chemicals can adversely affect the mem ronmental Protection Agency meeting and in part on the organizers' brane GJ assembly causing reversible alterations in GJ intercellular com munication. During toxicity essential metabolites, ions, and regulators are views of current GJ research since that meeting and the future direc not shared homeostatically throughout a tissue community. Alterations in tions for mechanistic research and applications to the relevance of GJ metabolic circuits are thought to interrupt organ integration. Persistent function to disease. GJ perturbation can cause chronic effects (e.g., cancer), and many tumor Overview. In this review, we have organized the material to promoters inhibit I..] intercellular communication. Liver precancerous present the structural characteristics of GJs, which include morpho foci intracommunicate (but at a reduced level) and intercommunicate logical and molecular aspects, the roles of GJs in cellular function and improperly (or not at all) across the foci boundary to normal cells. In time, maintenance, and how GJs are involved in cancer. Finally, we discuss foci can become less regulated and more isolated within the tissue. GJs GJIC controls and xenobiotic alterations of those controls. remain reduced quantitatively in the tumor progression stage and may be qualitatively altered in metastasis since connections are made between the Role of Gap Junctions in Intercellular Communication primary tumor cells and foreign host cells at the secondary metastatic site. Cell sorting and binding mechanisms by the cell adhesion molecules and Gap Junction Definition. GJs are specialized intercellular chan integrins may also be altered at secondary sites. This may allow the nels between apposing plasma membranes that can be open or closed relocation of primary tumor cells and nurturance via GJs at the secondary (gated). When open, these GJ channels allow metabolite exchange between cells. Each GJ channel is composed of two connexons; one Introduction contributed from each communicating cell. Each connexon is com Scope. GJIC1 is widely believed to play a key role in tissue de posed of six Cxs that act in concert in order to form a pore in the velopment and maintenance in multicellular organisms by homeo- center of the assembly. Connexons float laterally in the plasma mem static control of cooperative, interacting cells within tissues and or brane until a match is made to a proximal connexon in an adjacent cell gans. In recent years, data have suggested that toxicants can modulate (Fig. 1). When a match is made, the membranes of the cells are normal GJ functions in growth control, development, and differenti conjoined at that locus so as to form a channel between the two cells ation. Such modulation is thought to be involved in various disease (Fig. 1). Conjoined cells are metabolic systems partially open to each other with GJs providing channels of cell-to-cell communication. states, but the specific role remains to be delineated. Mounting evi dence, however, indicates that modulation of GJIC may be involved at Spatial patterns of interconnected GJs in a tissue form patterns of certain stages in the etiology of chemical carcinogenesis (1). communication in a tissue. These channels convey tissue metabolites To evaluate the relevance of GJ mechanisms to disease, the United control chemicals, critical ions, and xenobiotic chemicals, but only States Environmental Protection Agency, with supporting contribu hydrophilic molecules with molecular weights of < 1000 are shared by tions from the National Institute of Environmental Health Sciences passive diffusion through the GJ conduits. Whether all such cellular and NSI Technology Services, Inc., sponsored a conference on Mech molecules are shared equally among connected cells is still uncertain, anisms of Gap Junction Function and Relevance to Disease; the con but passive diffusion is assumed with the presumption of ionic and ference included 12 speakers and discussants."1 While the general molecular transients. Large molecules or lipid molecules, which re quire carrier macromolecules for transport, would not be transferred through GJs. Many GJs can aggregate with each other to form GJ Received 12/18/92: accepted 5/26/93. plaques at a membrane locus. Plaques are localized, high-traffic con- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1The views expressed in this paper are those of the authors and do reflect the views E. Beyer (Division of Hematology. Washington University School of Medicine. St. Louis. or policies of the United States Environmental Protection Agency or the National Institute MO). E. Elmore (National Institute for the Advancement of //( Vitro Sciences, Irvine, CA), of Environmental Health Sciences. E. Hertzberg (Department of Neuroscience. Albert Einstein College of Medicine. Bronx, : To whom requests for reprints should be addressed, at Genetic Toxicology Assess NY). J. Klaunig (Department of Pharmacology and Toxicology. Indiana School of Med ment Branch. Office of Health and Environmental Assessment. Office of Research and icine, Indianapolis, IN), R. Loch-Caruso (School of Public Health. University of Michi Development (RD-689). United States Environmental Protection Agency, Washington. gan. Ann Arbor, Ml), M. Neveu (Dana Farber Cancer Institute. Boston. MA), D. Spray DC 20460. (Department of Neuroscience, Albert Einstein College of Medicine. Bronx. NY). J. Trosko 'The abbreviations and definitions used are: GJIC. gap junctional intercellular com (Department of Pediatrics and Human Development. Michigan State University, East munication; GJ. gap junction: connexon, one-half of the assembled dodecylhedral pore Lansing, MI), H. Yamasaki (International Agency for Research on Cancer. Lyon. France). contributed by a cell: channel, low-resistance pore allowing bulk water flow made up of Discussants included: J. C. Berrett (moderator. Laboratory of Molecular Carcinogenesis. two connexons; plaques, multiple gap junctions aggregated in a plasma membrane locus; National Institute of Environmental Health Sciences. Research Triangle Park. NO. R. Cx, a connexin protein (<'.,!,'.,noted as Cx32 the predominant liver gap junction protein); Binder (Proctor & Gamble Co.. Cincinnati. OH) W. Farland (meeting sponsor. Office of ECM. extracellular matrix; TPA. l2-tetradecanoylphorbol-13-acetate; 1+ cells, initiated Health and Environmental Assessment. United States Environmental Protection Agency, cells; cAMP, cyclic AMP; CAM. cell adhesion molecule; PKC. protein kinase C; IP.,. Washington. DC), and J. W. Holder (Genetic Toxicology Assessment Branch. Office of inositol triphosphate. Health and Environmental Assessment. Office of Research and Development. United •¿â€¢Conferenceheldin Washington, DC, February 27-28. 1990. Presentors included: States Environmental Protection Agency. 3475

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Rudolf Virchow ( 10) formed the basis of the hypothesis that the cell, INTERCELLULAR LVI/EM LIPID BILAYER with its nucleus, is the functional biological unit. Our current understanding of GJs suggests an extension of the accepted cell theory: the cell is a unit, but a reproductive and bio HEMICONNEXON chemical unit within the local tissue. The tissue is integrated in part by means of the GJ connections and forms a syncytium. a connected array of cells. The syncytium averages cellular variances and capa CALCIUM IONS bilities locally and allows the integration of regional tissues into organ NUTRIENTS function. The syncytium may be a higher and more relevant physio METABOLITES logical unit within each tissue of an organ than individual cells. At the organ level, the GJ interconnected cell fields are often heterogeneous. For example, natural physiological barriers, such as fascia, bone, and various extracellular matrix elaborations, allow the creation of local environments in GJ-interconnected cell communities, which manifest BICARBONATE BUFFERINQ _J CEll-TO-CElL CONNECTION regional tissue-specific functions (11). These regional adaptations, COMPOUNDS '• / with specific interactive communication occurring, allow for integra CYTOPLASMIC PRESENTATION OF C tion and phcnotypic expression of the cell collective in organ function. Fig. I. Diagrammatic model ot'GJs in the plasma membrane of two apposed cells. The The corpus is the next higher level of organization: the vasculature channel is made up of two connected connexons from each of two contiguous cells. Clustering of many GJs in a confined area is called plaques. Passive hulk flow through GJ and nervous systems provide corporeal integration. Corporeal integra pores is assumed to take place in mass transfer. Transferring molecules must he water tion physiologically unites and relates the various organs, tissues soluble with molecular weights <100(). Cytoplasmic bridges and active ion pumps also within these organs, and regional syncytia within tissues. transpon material across plasma membranes in addition to gap junctions. The interactive kinetics among these transport mechanisms are not well understood. The GJ channel can GJs seem to provide a number of regional functions within tissues. open or close by a rapid (ms) and reversible process called gating in which a portion of One such function is: (a) buffering the harmful effects of xenobiotic the connexon protein blocks the bulk water flou in the closure mode, whereas this protein sequence quickly moves out of the GJ channel upon opening (adapted from Ref. 115 with chemicals. This buffering is achieved by dispersing xenobiotic mol permission). ecules from the exposure entrance point outward into the tissue via a number of interconnected cells. Dispersal in effect dilutes the local concentration of the potentially offending xenobiotic. which can now duits between cells for bulk exchange of small molecular weight ions be catabolized in more cells and in a steady-state fashion. Another and molecules such as H2O, Ca~ '. and cAMP. The number of plaques GJ-mediated function is: (/)) nourishing of sick or deprived cells by with adjacent cells is thought to be proportional to the metabolic healthy neighboring cells (reciprocity). This concept is basic to tissue cooperation among these cells. Thus, fewer plaques indicate less homeostasis. The inuring process offers plasticity to cells during in communication, which in turn suggests that the cells may act more jurious processes and responses. As long as GJIC is not compromised independently of each other. However, modes of intercellular com and the toxicity limit is not exceeded, the cell is remediated with munication other than GJs, such as paracrine functions and other ion assistance from healthy neighboring cells. Hence, a tissue can revers- channels, continue to functionally couple cells, even when plaques are ibly recover or remodel following a toxic insult. However, it is thought few. The relative importance among the different modes of intercel that if toxic limits are exceeded then cells slow or stop communicating lular communication is not known. (GJIC i ). Disorganization can then occur which can be followed by CAMs can facilitate local GJIC in epidermal cells (3). CAMs can necrosis and cellular turnover. Another regional function mediated by also act as cell morphogenetic regulators conferring specific cell-to- GJs is: (c) rapid exchange of critical ionic electrical signals (<'.#., cell adhesions and cell sorting mechanisms (4). CAMs likely assist Ca2^ ions) and their regulators. For instance, when a cellular mem locus-specific cell-to-cell docking processes in epidermal cells, pos brane is sufficiently disturbed, waves of Ca2 ' ions can move from the sibly by drawing and holding the adjoining membranes together in a disturbance point in a field of cells through cells to neighboring cells Ca2+-dependent process. This permits close intermemhrane apposi in order to signal the disturbance ( 12). This wave action is hypothe tion, thereby facilitating cell-to-cell interactions that include connex- sized to happen so that regional tissue reaction can occur in a coor on-to-connexon (GJ) interactions (3). Progressive loss of GJs (and dinated manner. GJs also participate in regional function by: (r/) thus GJIC) and loss of E-cadherin (a type of CAM) have been ob allowing the distributions of critical metabolites (<'.,(;..cAMP) among served to occur in advancing metastatic disease, which suggests the tissue cells. Cells benefit from GJ-mediated sharing of essential me importance of functioning GJs and CAMs to proper cell control (5-7). tabolites because all of the cells in a syncytium do not possess the The universal occurrence of gap junctions in multicellular organ same metabolic capacity (13). Cells of lesser capacity benefit from isms (metazoa) indicates that gap junctions play a ubiquitous, essen those with ample capacity. If some cell fields are under stress and tial role in collective cell interactions (8). It is through these interac some are not. GJ sharing is beneficial to the tissue as a whole. Lastly, tions that cellular physiological balances and controls are cumulated another regional GJ function is: (?) the elimination of unwanted into regional tissue function in the corpus of multicellular organisms. byproducts. Elimination is facilitated by GJs in a gradient fashion Comparative structural and functional differences in GJs among var from tissue interior cells to the vascular system for purposes of ex ious members of metazoa are not well understood. cretion. Gap Junction Function. One of the most fundamental theories of Gap Junction Proteins and Membrane Structure. Isolation of modern biology is the "cell theory" first proposed in the mid-18()()s by GJs is facilitated by their relative resistance to alkali and detergent Mathias Schieiden (plants) and by Theodor Schwann (animals) (9). solubilization. Development of antibodies against different purified They proposed that plants and animals possess cells that are organisms Cx proteins has made possible topologica! mapping of GJs. The most within themselves which are separate from, but related to. the organs detailed biochemical characterization is from Cx purified from liver. in which they reside. The whole organism is constituted from collec Lipid analyses of GJ isolates have indicated an enrichment of choles tions of cells and organs that are ordered within the organism accord terol, depletion of sphingomyelin. and significant levels of phosphati- ing to definite laws. These early works and the pathology studies of dylinositol and arachidonic acid ( 14). The role, if any. of these lipids 3476

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1993 American Association for Cancer Research. GAP JUNCTION Kl'NCTION AND CANCER is noi clear. It appears that GJ proteins from liver, as well as from found that expression of pp60%src in Xenopus oocytes blocks GJIC of heart, can he fatty acylated but not glycosylatecl. Palmitic or myristic Cx43. but not Cx32. This GJ inhibition is caused by tyrosine residue additions may assist posttranslational transport of the connexins 265 phosphorylation in Cx43, whereas Cx32 is not similarly phos phorylated. Interestingly, the pp60%"slx'-promoted phosphorylation of through the Golgi cisternae (15). GJs isolated from heart consist of a single protein with an apparent Cx43 does not inhibit the Cx assembly into GJs, only their function in molecular weight of about 43,(XX)-45,0(X) (Cx43). Other organs such communication once in the membrane (32). Site-directed mutants as liver have connexin mixtures: the major liver connexin is Cx32. with shortened tail regions (where Cx phosphorylation occurs) show with a lesser amount of Cx26 (= lO—LWi)( 15). Isolated GJ from lens no altered voilage sensilivity but do show altered unitary conductance fiber cells consist of MP26 and MP70 ( 15). MP26 is unrelated to heart differences when compared to unmutated Cx (33). However, the tail- and liver proteins. While detailed information has not been presented, modified sites do not seem to interfere with insertion of mutated Cx it appears that MP70 is related to liver and heart GJ Cxs. Some into membrane-associated connexons (33). This conclusion was con relatively large size differences do exist in the variable cytoplasmic firmed by Musil and Goodenough who showed that unlabeled GJs regions among connexin proteins, such as Cx.32 and Cx43. These occur intracellularly. whereas '2PO4 labeling occurred only in the different sequences form different Cx types (15, 16). Each type is membrane. Phosphorylation of GJs occurs after membrane insertion thought to basically act the same in GJIC no matter in which tissue the of Cx which leads to Cx deposition by insolubility and ability to type occurs but fine differences of GJ control and function may exist associate into plaques (34). This and the above studies suggest thai (17). More new sequences for additional Cx have been found by phosphorylation affects GJIC but not insertion into the membrane. amplification using ihe polymerase chain reaction and low stringency Lone GJs and GJ plaques are readily visualized by freeze-fracture hybridi/.ation; new Cx proteins in rat liver have been predicted: Cx40, electron microscopy on closely apposed membranes of adjacent cells Cx37. C.\33, and Cx3l.l (18). These new sequences exhibit the typ (35). The morphological fine structure of GJs has been deduced from ical sequence pattern of the GJ protein family, i.e.. highly conserved X-ray diffraction studies (36). The emerging picture is one in which transmembrane and less conserved extracellular domains, but variable each connexon (i.e., one-half of the channel contributed by each cell) cytoplasmic domains (tails). is made of six protein subunits called Cxs, which may or may not be Cx mRNA seem to be made from only one exon ( 19). This is unlike identical, organized in a circular rosette array and aligned en face most cellular proteins found to date that are made from mRNAs. across an intercellular space. This space is the gap of GJ seen in which are assembled from heterogeneous RNAs containing dispersed electron microscopy. The schematic structure of GJs in cells is shown intron sequences among exon sequences. The rat Cx32 and human in Fig. 1. The Cx proteins are thought to be the only proteins in GJ. Cx43 heterogeneous RNAs each have uninterrupted Cx reading Each Cx protein traverses the bilayer four times, which mnemonically frames (no introns). and each exon has a 5'-nontranslated region (20, configures the letter "M" in cross-section. The Cx sequences that 21 ). The human chromosomal locations of Cx genes are not clustered traverse the membrane are highly conserved among species ( 16). This on a particular chromosome, such as the globin genes, but rather occur conservation pattern is consistent with most channel proteins. The Cx on different chromosomes ( 18. 22). A number of Cx DNA sequences cytosol sequences (visualized as two bottom legs of an "M." corre occur in humans: Cx26. Cx32. Cx43, and Cx46 on chromosomes 13, sponding to the NH2 and COOH termini, and an intervening middle X. 6. and 13. respectively (22). Similar coding sequences exist in the loop) vary. These variable regions suggest that different types of mouse and rat. Another mouse connexin protein. Cx37. has been connexons may be due to Cx heterododecylhedral mixes within con reported (23). The Cx genes isolated thus far as complementary DNA nexons in GJ, but these mixes have yet to be demonstrated. These hybridize as distinct mRNA species; therefore connexin proteins make mixes, if present, could present fine structural differences in assem up a multigene family (24). Such a structural diversity of related Cx bled GJ channels. genes suggests different connexon functions, e.g., during differentia Any agent that alters the connexin protein assembly, other connex- tion (24). Genetic drift and reorganization should also be considered on-associated componenis. or local membrane morphology can po since Cxs are thought to constitute a set of evolutionarily old proteins. tentially affect GJ function. Proximal desmosomes seem to strengthen Cxs have been found to occur and function in mesozoa. which are local cell connections. It now appears in the cell systems tested thus transition marine parasites biologically organized between unicellular far that neither GJs nor the adhesion molecule E-cadherin function (protozoa) and multicellular (metazoa) organisms (8. 16). There seems well in low Ca2 ' . It has been suggested thai E-cadherin and GJs need to be no exception to the hypothesis that all metazoa utilize GJ adequate levels of Ca2 ' to function optimally (3, 4). What Ca2 ' ion channels. These conserved Cxs apparently provide an essential set of levels are adequate likely depends on the cell type, stage of develop functional cellular proteins in higher organisms. ment, or disease state. CAMs like uvomorulin, which is identical to Besides the cytoplasmic tail Cx sequence differences, chemical E-cadherin and homologous to liver-CAM, may facilitate GJ docking modification of the Cx protein also can take place. For example, Cx and subsequent stabilization of cell-to-cell interactions (3, 6). The phosphorylation can be stimulated by cAMP. which acts as a cofactor integrins also may play a role in specific cell loci matching up and for kinase A phosphorylation. and such phosphorylations can produce allowing GJ formation between specific compartments of the two increases in GJIC (25. 26). Tumor-promoting agents (e.g., TPA) down conjoined cells (37). Other extracellular elements such as glycosami- regulate PKC while also down-regulating GJIC (27, 28). Presumably noglycans and proleoglycans have been observed to proffer slabilizing the Cx is structurally altered when the Cx is phosphorylated. which is influences and affect increased GJs (1. 4. 6. 38). The explicit rela likely related to the down-regulation of GJIC and would be expected tionship of GJs and the ECM remains to be delineated. Inside the cell, to contribute to the pleotrophic tumor promotion mechanism ( 1, 16). the actin cyloskelelal network may be associated wilh GJs (39. 40). Specific phosphatases remove attached phosphate groups from Cx in Preliminary work suggests that when cells are placed in low Ca2 *, short times (min); this process is considered part of the fast GJ control connexons are pulled apart by retraction (41, 42). The separation of responses (29). Since Cx are proteins that have t,,, of only a few h, connexons could be a passive process; i.e.. the cell-to-cell pore struc coordination of phosphate attachment and removal is essential to Cx ture is thermodynamically unstable at 37°Cand low Ca2 '. This con function (30). Proteins other than Cx can be phosphorylated and yet nexon separalion could also be an active process, due to actin atlach- affect GJIC; e.g.. in rat liver phosphorylation of src tyrosine residue ment to ihe plasma membrane microvilli proximal to the connexons 527 has been related to GJIC inhibition (31). Swenson et al. (32) have (40). Notably, cytochalasin B. vincristine. colchicine. vinblastine, or 3477

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1993 American Association for Cancer Research. GAP JUNCTION FUNCTION AND CANCER low temperatures (0°C),all of which disrupt cytoskeletal structures, 42). Coordinated GJ appearance prior to critical events in differenti each can increase GJ plaque areas (40). The emerging concept from ation is consistent with a role for GJs in signaling and maintenance or this work suggests that the actin cytoskeletal system regulates GJIC by disruption of morphogen transmission ( 15). Vitamin A analogues and first polarizing the fibrillar apparatus toward the outer cell face and glucocorticoids, both known to be involved with cellular differentia modulating concentration and polarization of microvilli at the cell tion, are linked to the increase in the number of GJs (60). Selective surface (Ref. 40) and references therein). By this mechanism, the disruption of GJIC either by introducing Cx antibodies or by injecting number and positioning of GJ plaques are coordinated by cell orga Cx antisense mRNA has led to developmental defects in both inver nization (cellular polarity), which contributes first to the anisotropic tebrates and vertebrates, thereby implicating GJ in these processes organization within the cell and secondly to the collocation of the cell (16, 61). Correlations are now being made between genes that are within the tissue (2, 40). central to development, e.g., pattern formation, and those that stimu Some Gap Junction Examples in Cellular Communication. late cancer (oncogenes) (62-64). Changes in development involving Electrical coupling occurs among communicating cells due to ion flow GJs have been correlated with loss of cell growth control, which can measured in conductance units of picosiemens (pS). Cellular coupling lead to cancer (56. 65. 66). The relationship between cancer and GJIC establishes a ground state potential, along with Na+, K +, Ca2+, and is discussed below. H+/~HCO, pumps, among cells in the community, all of which pro vides the background on which pulse signals such as Ca2 + ions are Modulation of Gap Junction Function generated and interpreted. Ionic coupling among cells is critical for Temporal Modulation of Cap Junctions propagation of depolarization in myocardial and smooth muscle tis sues and is thought to provide a means for coordinated contraction Changes in GJs are essential for allowing, or attenuating, the in (43, 44). For example, at parturition, a rapid, incipient increase in GJ tercommunication in a field of cells. Three kinetic courses of GJ Cx protein in myometrial smooth muscle enhances syncytial activity control are beginning to emerge: (a) fast control by gating responses and coordinated contraction necessary for fetal expulsion in parturi (=ms); (b) intermediate control by vesicular insertion or withdrawal tion (2, 45). The brain has many GJs, but little is understood about of connexins in or out of the plasma membrane (= min); and (r) how brain GJs participate in neural cell function and maintenance long-term control, where transcription of Cx mRNAs is turned on or (46-48). Electronic coupling takes place in the nervous system (in off or is attenuated and Cx protein pools are adjusted accordingly (h). addition to synaptic transmission) and can synchronize outputs of Times are empirical and often reflect rodent liver studies. Compara electronically coupled neuronal pools (16). Neural activity in glial tive differences among species of these times are not known. cells causes focal K+ transient loads that are dissipated in a recovery Fast Control. The transport of electrolytes or small low molecular process mediated by GJ (16). Retinal horizontal cell conductance in weight substances from cell to cell depends on the number of GJs the eye can be modulated via GJs that transmit dopamine, which in connecting these cells that are open at any given time, the specific turn affects retinal cAMP-dependent protein kinase (49). conductance of the type of shared connexons. and the tissue environ Another example of the integration of tissue activity and metabolic ment. Different structural models have been proposed to account for function is the coordination of cells in a hormone-responsive tissue the gating (quick closure) of GJs. In one model it is suggested that ( 16, 50). Coupling via GJs plays a role in secretory systems, and it has bulky, hydrophobic phenylalanines are introduced into the aqueous been demonstrated that secondary messengers (of primary hormone channel by an a-helix rotation of the third transverse region (M3) of stimulation) such as cAMP, cGMP, Ca2 +. and IP, pass through GJ the Cx protein. The channel is thus occluded for aqueous transport, channels (51 ). It is likely that many other critical molecules are shared thereby causing a restricted or closed channel (pS J, ). Reversal of this via GJs. Insulin production in pancreatic islet cells correlates with process by M3 a-helix rotation in the opposite direction reintroduces prior secondary messenger activity (cAMP). bursts of pancreatic Cx43 small hydrophilic amino acids into the channel, which clears the way mRNA activity and Golgi and endosomal vesicular exportation of the and reopens the channel to aqueous transport (pS ÃŽ)(67). It is also Cx43 into islet cell membranes (52). Such a sequence of events possible that if the subunit rotation were progressively twisted in a suggests that GJs are made and assembled in order to assist insulin cooperative manner along the pore axis, a gating from open to closed synthesis and release from pancreatic cells. The mechanism by which states would occur without resolvable intermediates, much like the this is achieved remains obscure. The examples given above suggest aperture of a camera lens. The kinetics of gating, which in part that the initial sharing among cells of effector molecules via GJs can involves M3 rotation, has been estimated to be =ms (68). The energy lead to integrated tissue responses (15). difference between the open and closed states is =1-2 kcal/mol (68, Another interesting case where cells share effector molecules via 69), which is on the order of the strength of the hydrogen bond. Since GJs is in the ovaries. Oocytes are maintained in meiotic arrest by the average energy of kinetic motion ^=0.6 kcal/mol at 37°C,then granulosa cells to which they are coupled by GJ via filopodial pro gating energies may require only 0.4-1.4 kcal/mol. Such low energy cesses projecting through the zona pellucida (53). These GJs mediate requirements suggest that GJs can be gated readily at body tempera the intercellular flow of the ovulatory meiosis inhibitor between the ture and can accommodate rapid ion signal (voltage) changes. membrana granulosa and cumulus oophorus cells with stasis of oocyte Some agents effect almost immediate GJ channel closure ( 16). For meiotic cell division as a result (54). An ovulatory stimulus, provided example, immediate effects can result from rapid changes in intracell- by luteinizing hormone, leads to disruption of GJIC in which inhib ular pH. ion transients, protein kinases, and even changes in GJ iting granulosa cells can no longer transport ovulatory meiosis to the membrane lipid composition (14, 70). Examples are acidic pH and arrested oocyte cells (55). Accordingly, the oocytes are no longer PKC and protein kinase A activators, inhibitors of cAMP. Ca2 ' spike- arrested and ovulation ensues. inducing agents, and high lipophilic agents (such as unsaturated fatty GJs play an essential role in embryonal cells during development acids). The study of actual GJ structure-activity relationships of rap and differentiation (56. 57). Certain teratogens seem to act by altering idly acting agents has just begun. GJIC (57). A potential role for GJs exists in providing a mechanism Intermediate Control. Evidence for the intermediate control for transmitting timed-growth and positional signals involved in de mechanism is scant and rests mainly on the observations that Cx- velopment (58, 59). Several observations based on ultrastructural antibody-reactive material exists in vesicles proximate to the plasma analyses indicate that GJs do appear or disappear at various times (16, membrane. It has been observed that Cx can be inserted into the 3478

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1993 American Association for Cancer Research. CAP JUNCTION FUNCTION AND CANCER plasma membrane even in the presence of protein synthesis inhibitors Toxicant Modulation of Gap Junctions (71 ). This suggests that a peri membrane pool of preexisting connexins Involvement of GJs in the toxic response of cells to chemicals has exists and may buffer the Cx supply (16. 40, 72, 73). It is not known been an overlooked area and there are few systematic studies on this if the intermediate pool is under physiological control. The presence topic (2, 87). GJ perturbations may be involved in the toxicity caused of the intermediate pool underscores the essentiality of Cx to cellular by many chemicals, but the precise mechanisms are not yet known function. and can only be inferred in certain cases. It is known that Cx are not phosphorylated in the pool. Evidence Generally it is known that when a cell is damaged, the membrane exists to suggest that Cx43 is phosphorylated after being inserted into can become leaky to ions such as H+, Ca2+, and K+. If the damaged the plasma membrane (34). This phosphorylation seems to accompany cell remains coupled to contiguous undamaged cells, essential metab Triton X-100 insolubility of Cx43 (34). Phosphorylation does not olites are lost at the damaged cell site. GJs are thought to attenuate determine cellular stability of Cx43 but has been correlated with Cx43 during these periods of damage for self-correction by the cell (88). accumulation into plaques (34). Such a mechanism suggests phos The mechanism of attenuation is not known but is expected to be phorylation control of mass transport. primitive and an intrinsic aspect of the Cx molecular engine and Long-Term Control. Evidence for longer term control of GJIC is membrane assembly. The mechanism of closure may also respond to still emerging (16). In liver cultures, the apparent half-life for Cx32 paracrine control. pH. and critical ion balances. Closing agents would and Cx26 is estimated at 2 h with about the same Cx half-life found induce structural changes in Cxs by rotating M3 into the channel (cf. in liver in vivo (74). This turnover is rapid enough to regulate levels "Fast Control"). Localized closures can prevent spreading of toxic of GJs by Cx mRNA synthesis rates and stability. In primary hepa- injury in the tissue and allow energy to be switched to repair. If the tocyte culture, GJs disappear 10 h after plating but reappear within 72 cells are repaired and survive, then they can later recouple using GJ h; this disappearance and appearance also occur after partial hepatec- channels to become once again functional participants in the syncy- tomy (16). These recoveries are preceded by Cx mRNA and are tium in which they reside. Such regional attenuation of GJIC with accelerated by cAMP (75). remedial recruitment could also be under tissue physiological control Cyclic nucleotides affect GJ in most tissues and cell lines that have about which we can only speculate at this time. Toxicity-induced been studied to date, and these agents are the likely candidates for the closure is reminiscent of GJIC reduction (not ablation) during mitosis major set point of physiological GJ regulation. cAMP analogues can when cells withdraw to the internal program of cell duplication and cause either increases in GJs, e.g., liver cells, or, in some cases, division (88, 89). However, these mitotic cells remain electrically decreases in GJs (76-78). Inhibitor experiments suggest that these GJ coupled to some extent to surrounding ¡nterphasecells (90). In agree changes are attributable to effects mediated through cAMP-dependent ment with this is the observation that regenerating tissue shows first a protein kinase A, which can phosphorylate Cx (as well as regulatory loss in GJs but later the recovery and recoupling with contiguous cells proteins) and thereby regulate GJs. For example, liver Cx32 can be (91-93). The community of cells is thereby established once again. phosphorylated at a serine residue on its cytoplasmic tail, with kinetics Toxicants (e.g., the perturbants /¡-heptanolor /¡-octanol)may re- similar to that associated with increased conductance ( f pS) (79). versibly close GJs by physically reacting with the GJ plasma mem cAMPcan be a mediator of up-regulation of GJs in a variety of cell brane assembly and abolishing cell-to-cell coupling (44, 94, 95). Ca2 + types. Within 6 h. cAMP exposure leads to an increase in GJIC and pH must be optimum for GJs to function well (15), although humoral factors can alter the sensitivity to Ca2+ and pH (96). It has between sympathetic neurons, which is blocked by inhibitors of mRNA and protein synthesis (16). In hepatocytes, cAMP extends the been thought that inhalation of anesthetics may act reversibly by lifetime of GJ between dissociated cell groups, which is attributed inhibiting GJIC (97). Many anesthetics are small lipid molecules with largely toan increase in stability of Cx32 mRNA (16). Transcriptional high fugacity and are accessible to many tissue compartments. These regulation is expected as well, based on the cAMP response element properties could cause reversible membrane perturbations in various sequence in the genomic clone of Cx32. As discussed previously, tissues including neural GJs thereby causing reversible sensory inter components of the intercellular and extracellular matrices influence ruption. Moreover, chronic ethanol consumption (a C2 alcohol com GJs and seem to provide stability to these structures and enhance pared to C7 and Cx alcohols above) may persistently perturb GJs by GJIC. For example, in hepatocytes, there is an interaction of matrix excessive membrane solvency and perturbant effects along with ex components and glucagon: treatment of cultured cells with glucagon cessive acetaldehyde generated upon ethanol metabolism. The persis together with proteoglycans leads to a marked increase in Cx32 tran tent influence of perturbants on GJs can be thought to lead to persis scription as well as increased stability of Cx mRNA (38). Down- tent dyscommunication in the affected field of cells, which can then lead to either (a) facultative accommodation by the cells or (¿)disease regulation of GJIC has been reported in uterine smooth muscle cells progression. In the second pathway, chronic disease follows when treated with cAMP derivatives (2). deleterious acute effects become irreversible, which can occur from Tumor promoters such as TPA stimulate the signal transduction mediated by PKC by mimicking the normal substrate of PKC, diacyl- repetitive acute or subchronic insults (2). glycerol (80). PKC exists in multiforms (in as many as six genes) that In Vitro Assays for Gap Junction Toxicity change in relative gene expression upon TPA binding to PKC or transformation (81, 82). Like many tumor promoters, TPA inhibits Data on GJIC following toxic injury have been obtained from GJIC (27, 28) and may do so by altering one of the PKC isoforms with various types of assays for GJIC: (a) cooperative sharing of a metab respect to GJ phosphorylation (83-85). In several cell lines, active olite; (b) fluorescent dye transfer; and (c) electrical coupling. Toxi- phorbol esters inhibit GJIC in approximately 10-20 min, a time course cological data (=200 agents) has been obtained in vitro in V79 met similar to that of PKC activation. Chronic treatment of the tumor abolic cooperation assays (98). One problem encountered in this in promoter phénobarbitalcan lead to long-term cellular changes, e.g., a vitro work is that there is a relatively low potential for V79 cells to decreased occurrence of GJs in altered hepatic foci (86). Such GJ metabolically catabolize chemicals, a transformation that may be nec decreases have been observed to be the result of a lessened synthesis essary for some induced toxicity. Moreover chemicals that are tumor of Cx32 in liver and are possibly linked to the preneoplastic nature of promoters are usually organ specific and would necessarily require the the AHF (86). appropriate cell culture to test GJIC effects. Another shortcoming is 3479

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1993 American Association for Cancer Research. GAP JUNCTION FUNCTION AND CANCER the lack of ECM effects manifest in solid tissues. Nonetheless, in vitro first reports implicating GJIC inhibition by tumor promoters were V79 data have provided some insights into the direct pharmacological published in the same year (27, 28). Since then, objective correlation actions of various types of chemical agents on GJIC and provide has been made in a number of laboratories between the promotion and useful comparisons of structure-activity relationships. Whether or not progression stages of carcinogenesis and decreased GJIC (1.2, 106- these reactions on GJs occur in vivo has to be assessed on a chemical- 111). It is thought that chronic inhibition of GJIC in carcinogen- by-chemical basis considering the pharmacokinetics and metabolism initiated tissues, which contain 1+ cells as a result of being initiated, of xenobiotic exposures in various species. can bring about the tumor promotion stage of chemical carcinogenesis Several examples of the usefulness of the V79 assay exist. When (1. 27, 28. 112). Tumor promoter interference with GJIC has been nine phenylenediamine congeners were evaluated for metabolic co substantiated both I'Mvitro ( 1, 113) and/'»vivo (\\2, 114). The role of operation, eight of these (89%) were correctly identified for their GJIC disruption and chemical carcinogenesis has been reviewed pre carcinogenic potential based on their ability to inhibit GJIC (98). Not viously (115). all known animal carcinogens tested in V79 cells inhibited metabolic There is an inverse correlation of cell growth and GJIC in trans cooperation. Given the lack of metabolic activation, however, it is formed lines, i.e., induction of GJIC leads to growth inhibition, intriguing that up to 75% of the chemicals tested with known carci whereas blockage of GJIC leads to uninhibited cell growth (116). nogenic activity produced some inhibition in the V79 metabolic co Antitumor agents such as retinoic acid or retinol reduce transforma operation assay (98). tion and up-regulate GJIC (60). Several activated oncogenes (e.g., ras, The activity of alcohols and lipophilic agents in the V79 assay \-mos, neu. src. little and large T, and EIA, but not v-myc or v-fos) appears to be directly related to their octanol/water partition coeffi reduce GJIC in some cells (66. 117). Although most known tumor cients (98). Fatty acids demonstrate activity as well. Strong mutagens promoters reversibly down-regulate GJIC /';; vitro and in vivo (1, 27, such as diethylnitrosamine and ethylnitrosourea also inhibit metabolic 28). GJ inhibition by tumor-promoting chemicals is not universal. A cooperation. Certain toxicant classes, such as pesticides including few tumor promoters studied thus far. e.g., okadaic acid and TCDD, aldrin, mirex, kepone. DDT. DDE, heptachlor, methoxychlor, and do not down-regulate GJIC in the systems in which they have been lindane, inhibit metabolic cooperation in the in vitro V79 assay (98). studied (1, 118-120). These observations suggest that some tumor Thus, a wide variety of toxic, pharmacologically active agents have promotion mechanisms proceed without altering GJIC, which agrees activity in modulating GJ function in an in vitro assay. These effects with the growing evidence that there are qualitatively different mech may correlate to in vivo exposures since dose delivery is likely: (a) anisms for tumor promotion (121). Different mechanisms could also direct external delivery to GJ is assured from the ECM; and (b) relate to different adaptive phenotypic defense mechanisms with internal delivery is likely from transponed molecules through the GJ which cells respond to carcinogen exposure (102, 122). channel. While it is universally agreed that chemical mutagens react with For agents associated with noncancerous disease end points, the DNA and can cause lesions that lead to hereditary diseases as well as following correlations were found; 80% of all teratogens tested in contribute to somatic diseases (including cancer), all toxicants do not hibited metabolic cooperation, whereas 73% of all neurotoxins tested act directly on DNA and all carcinogens do not necessarily react were detected (98). While these V79 in vitro data cannot be considered directly with DNA (87. 105, 109). Toxicants working on "other than unbiased, due to the partial selection of agents for study based on their DNA" are often referred to as epigenetic. A number of epigenetic known activity as pharmacologically active toxicants in one system or events can conjunctionally cause persistent toxicity. Among these another, the selectivity does imply the usefulness of GJIC inhibition events, chemical modulation of GJIC may play an important role in assays to detect chemicals associated with toxic responses. the areas of reproductive toxicology and teratogenesis, as well as carcinogenesis (2, 87, 115, 123). By perturbing GJIC, chemical ex Gap Junctions and Cancer posures can lead to a loss in tissue integration via distortion, or even Experimental animal carcinogenesis studies as well as epidemio- ablation, of essential biological signaling networks (2, 11, 108, 109). logical studies of human carcinogenesis strongly indicate that the The hypothesis of GJ-mediated control of cell growth was introduced process of carcinogenesis is multistaged and sequential (99, 100). by Loewenstein, who advanced the concept that negative repressors of Some stages in the sequence are not always the same which means cell growth are shared among normal communicating cells via GJs that a number of different overall mechanisms can cause cancer (101, (56, 66). Moreover, as the hypothesis goes, these shared repressors 102). This has made the explicit identification of the necessary and keep cells in an orderly growth-arrest state, except those cells gener sufficient components a complex matter of biochemical and logical ated to replace normal cell loss. Growth ensues when conditions cause analysis. While mutations often play a role in the multistage nature of a diminution of sharing of these negative repressors (56. 124). carcinogenesis, nongenotoxic mechanisms also play key roles in car Ca2 + ion oscillation takes place within cells, which is not well cinogenesis (103-105). Examples of nongenotoxic mechanistic types understood, but seems to be frequency-modulated signaling. Ca2 + ion are changes in gene expression, cell proliferation, cell organization, oscillation is an intracellular process which does not disturb neigh cell interrelationships (including GJIC), cell death (including apopto- boring cells. Ca2' ion oscillations has been related to: (a) enhance sis), and cell removal. There are a plethora of potential interventions ment of cellular signal recognition; (b) reloading of Ca~+ ion storage that a xenobiotic chemical can bring about to alter the cellular phe- sites; and (c) directional ion transport (125. 126). In contradistinction, notypic expression of an exposed field of cells. Although affecting a Ca2+ ion wave is a form of transmitted Ca2 ' communication orig GJIC is only a subset of all potential interventions, adversely affecting inating from a cellular membrane trauma. The Ca2 f wave propagates GJIC may be a more common toxic event than heretofore thought through not around, cells (12). The hypothesis has been that the Ca2 + ion wave is moved from cell to cell by a "leader" substance which (2, 87). Because cancer has been described as a stem cell disease, a disease progressively causes Ca2+ ion pulse in each cell (126). This substance of differentiation, and a disease of cell regulation or growth control has proven to be IP, (12, 126). The IP, moves through cells, causes a and because GJIC has been linked in normal cells to control of Ca2+ pulse, and then diffuses onto neighboring cells via GJs. Hence development, differentiation, and growth, it might be supposed that the wave of Ca2 + ions moves from the point of disturbance and is there may be a relationship between cancer and GJIC. Loewenstein directed to neighboring cells which must be alerted. The cybernetics (56) predicted this relationship in a prescient 1979 review, while the of the Ca2+ wave signal is unknown. Chemical toxicant disruption 3480

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1993 American Association for Cancer Research. CIAF' JUNCTION FUNCTION AND CANCER may scramble the signal before it is translated. When wave signals are cellular communication or even removal of focal cell communities interrupted, distal tissue cells would not be alerted and thus would not from normal local tissue communication patterns. interact cooperatively. Damage can ensue due to the lack of homeo- Although GJ expression may be altered by certain oncogenes and statically controlled repair. tumor promoters in many highly metastatic cells. Kanno (65) reported When hormones, secondary messengers, and essential metabolites that tumor cells can remain coupled by GJs. at least to some extent. are not shared to their normal extent due to impaired GJIC. then Thus, a complete lack of coupling is not necessarily an obligatory excess cell divisions can occur. It persistent, this condition can lead to requirement tor conditions leading to loss of growth control. Rather, dysplasia. Trauma, for instance, can lead to cell loss and regenerative a critical level of GJIC may have to exist in order to avoid cancer, but hyperplasia. This loss of growth control is temporary because hyper- such a level is not yet known. Furthermore, it may be that total GJIC plasia can regress in a reversible manner when the inducing stimuli are ablation causes premature toxic cell death and does not allow time for removed. GJIC is thought to be important to remodeling but the cancer to evolve. Relative cell kinetics is important. For example, if mechanism of this process is not clear (2. 127). If toxic stimulation is the rate of loss of 1+ cells in a tissue (through premature death) is persistent and 1+ cells are present, then 1+ cells can be clonally balanced by the rate of newly generated 1+ cells (through persistent expanded as can other cells in the field. Not all 1+ cells are equally initiator activity), then the 1+ cell pool would be constant; but if the committed to cancer susceptibility. However, if a critical clonal size of loss of 1+ cells exceeds the 1+ cell supply, eventually the field will be a deleterious 1+ cell population is ever reached, the growth-suppres- devoid of a significant amount of 1+ cells to cause cancer. In the case sive effects of surrounding normal cells on these particular 1+ cells where a persistent carcinogen produces more 1+ cells than are lost, can be overwhelmed (1). 1+ cells can become disconnected from the the chances of cancer increase as 1+ cells accrue. The risk of cancer surrounding normal cells to form islands of dysregulation. Regulatory increases in the latter case. During the course of cancer evolution, the controls are thus circumvented, at least partially, and the unrestricted processes of tumor promotion, conversion, and progression act on 1+ cells continue to divide and eventually form genetically and phe- these accruing 1+ cells and the disease develops. However, it is notypical ly heterogenous preneoplastic foci (102, 122. 128-130). necessary to minimally nurture these advancing 1+ cells via some Foci are preneoplastic and are only one set of cellular events oc GJIC in order to from a tumor. Cancer is likely the stochastic result of the evolution of 1+ cells and curring in the tumor promotion stage of cancer evolution (127, 128, the surrounding focal cells which is a collective of altered (trans 131, 132). This is true at least in liver where the observation of altered foci was first made; the presence of foci in other cancer-susceptible formed) cells. Foci are usually identified histologically by an enzyme alteration (usually a deletion); it should be remembered that these organs is not as clear. Many preneoplastic foci show GJIC among foci focal cells also differ in other phenotypic traits. Foci are heteroge cells (homologous communication) but fail to communicate properly neous (genetic and phenotypic) populations. Thus, if certain combi with normal cells surrounding these foci (heterologous communica nations occur within foci (transformants + I+ cells) and certain en tion) (I, 110). This segregation of communication creates different vironmental conditions occur surrounding these foci (all which subcommunities which, in effect, can interrupt the integration of the interact at the micro level), 1+ cells can ultimately emerge to be tissue. These foci are genetically variable, or at least evolve hetero malignant. Carcinogenicity in fact begins when these combinatorial geneity, and they exhibit different phenotypic patterns of enzyme biological interactions become irreversible; i.e., the adaptations can deficiencies at the foci stage and beyond. The outcome of early and not readapt. Since focal cells may be selective in communication with continuing generation of heterogeneity is successive cell selection to themselves and at a reduced GJIC rate compared with surrounding progressively higher levels of adaptation to the altered (transformed) normal cells (137). the expectation is that focal cells have a different microenvironment ( 102, 111) that appears to include decreased GJIC qualitative GJ apparatus. This prediction should be investigated. Re of foci to normal cells. Various posttranslation mechanisms lead to striction of communication to surrounding tissue cells dissociates increased cellular phenotypic variability. The phenotypic variability these focal cells from the normal flow of tissue growth regulators. A occurs at a rate far exceeding the genetic mutation rates (111, 133). recent suggestion is that reduced GJIC in carcinogenesis may be The evolving phenotypic variability is an important component in primarily due to reduced homologous communication and less due to cellular responses to xenobiotics. One such cellular variability in reduced heterologous communication ( 137). Thus, whether it is func volves decreased GJIC among various cells as metastatic disease tionally most significant that focal cells do not either properly ¡ntra- progresses (5). communicate or intercommunicate with surrounding normal cells, or Many transformed cells have fewer GJs than their corresponding both, remains a mystery. More work on the patterns of chemically normal tissue cells (83, 134). Since transformation takes place early in induced GJIC inhibition should prove fruitful. neoplasia, GJIC reduction may be an early, fundamental lesion in the Overall tumor growth is influenced by organ size constraints (sim origin of cancer. Species and strain differences to the liver tumor- ilar to normal organs), albeit the plateau size has pathologically been promoting effects of various chemicals seem to correlate with their reset upward (hence tumor which simply means swelling). This con ability to down-regulate GJIC in vivo in liver (135). As malignancy tinued size constraint is explained by the "integrated organ hypothe progresses, a decrease in GJIC levels in mouse epidermal and liver sis" (139). We suggest this may be based on the systems integration cells is observed (136. 137). Tumor metastasis seems to correlate with provided by the GJ-coupled network within organs. A case in point is decreases in homologous as well as heterologous GJIC (1, 136, 138). rat partial hepatectomy which stimulates regrowth to the proper liver Nicolson (111) found that, when metastatic potentials were compared, size and lobular conformation. Accordingly, organs "know" their size, those cancer cells with strong metastatic potential had fewer GJs than but continued normal use of the organ GJs may be necessary to those cells with weak potential. The decreased number of GJs found maintain that knowledge. Gap junctional disturbances may lead to loss in more cancerous cells suggests that GJs tend to be lost in cancer in the cybernetics of size restraint: hyperplasia results which is the development and accompany disturbances in tissue growth control combination of increased cell number and cellular volume. If contin just as Loewenstein (56) predicted in 1979. The long-standing concept ued, a condition sets up where tumorigenesis begins. Carcinogenesis in cancer technology, that early cancer cells somehow become differ can evolve out of persistent tumorigenesis. which can then allow ent than their neighbors and are accordingly "outlaw" cells, could now further mutations or selection of deleterious phenotypes. Overall tu find its explanation in the GJ mechanisms allowing for differential mor growth can also be influenced by the generation of a mutator 3481

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1993 American Association for Cancer Research. GAP JUNCTION FUNCTION AND C'ANC'KR phenotype where early mutations lead to enhanced and progressive Cx43 is the principal connexin protein of the heart (where it was first susceptibility to more fixed mutations ( 140). This type of phenotype observed), which is an electrically active tissue that has extensive can "conven" transformants into growing neoplasms that in turn can GJIC. Why cells //; vitn> allow dereprcssion of one member in the Cx be converted into malignant cells during the progression stage of imiltigene family while allowing suppression of another member is cancer. Hence, progressive alterations and aberrant growth occurring not known, but environmental conditions have been implicated in the in carcinogenesis could affect the GJ systems among other changes. If liver phcnotypic switching between (Cx32, Cx26| <=>|Cx43| (146, so. this could include altering both the quality and the quantity of 147). TPA reduces GJIC in 1AR 20 liver epithelial cells (as TPA does tumor cell GJs. We have objective bases for quantitative changes in in many other tissues and cell lines), but Cx43 mRNA does not GJ1C. but thus far. qualitative changes in GJs during carcinogenesis decrease ( 146). Thus if the Cx43 mediates IAR 20 GJIC. the reduction have been elusive. It is important to establish the precise relationship seems to be caused by posttranslational Cx alterations (146). The of GJIC to the etiology of cancer (2). relative importance of Cx mRNA transcription and translation versus Some further relationships between cancer and GJIC have been Cx protein posttranslational transport through the ER and Golgi to the presented, nix T24 protein accumulates in rat liver cells after trans perimembrane pool and then membrane processing into assembled formation by /-Cadherins. proteoglycans, and glycosaminoglycans can up-reg- (142). These results in lOT'/i cells indicate how impaired GJIC can ulate GJIC in a tumor cell line having low levels of GJIC (26, 38). cause transformation, which can be reversed by overcoming the de Interestingly, one of the deleted genes on chromosome 18q in human ficiency, i.e.. adding back Cx43 genes to the transformants. In another colorcctul cancer (this deletion found in ==70% of cases) has a se investigation. Naus et til. (143) showed that transformed C6 glioma quence similar to certain neural adhesion sequences ( 150). This indi cells can cause brain tumors. C6 glioma cells grow fast in culture but cates that deletion of CAMs by mutation may lead to a lack of proper are greatly slowed when transfected with complementary DNA for cell-to-cell contacts, including GJs (1.2, 4). Cx43. This investigation, like the studies in l()T'/2 cells described Summary above, indicate that lack of expression of Cx43 can be associated with tumor growth, but C.\43 expression can suppress tumor growth. Gap Junctions, Toxicity, and Cancer. In this review on gap junc Metastatic colonization at a distal organ site is initiated by the tion function and its relevance to cancer we have considered the attachment of blood- or lymph-borne tumor cells to organ-specific following: («)gap junctions play an essential role in the integrated adhesion molecules which are expressed on the surface of microvas- regulation of growth, differentiation, and function of multicellular cular endothelial cells. The establishment of communication by the organisms: (/?) perturbation of gap junction function, whether direct or invading cell to the host endothelial cells is essential for tumor growth indirect, can result in GJIC changes that may provide the beginning of and maintenance at the secondary site. El-Sabban and Pauli showed toxicity because the synergism. and in some cases the synchronism, of that highly malignant rat mammary carcinoma or melanoma cells, a field of cells is adversely affected. Persistence of GJIC abrogation preloaded with fluorescent dye. readily transfer the dye to endothelial ultimately can cause irreversible damages to the integrity of a tissue. cells at the secondary site ( 144). Low metastatic or nonmetastatic cells This irreversibility can bring on the latter stages of various disease did not similarly establish GJIC with the endothelial cells ( 144). These states, but the etiology of any disease process is yet to be elucidated: authors conclude that GJIC was necessary for metastatic establish (c) tumor promoters may inhibit cell-to-cell communication by («) ment and may play a role in tumor cell extravasation at secondary sites plasma membrane toxic effects (ion displacement. H:O imbibition, ( 144). Although some form of GJIC is required for the further nur- critical lipid relocation, etc.). which results in the loss of cell-to-cell turance of the tumor cells, it may be that (here is something different contact, (b) specific binding of the GJ assembly, thereby altering about these GJs in establishing GJIC at secondary sites. GJIC, (c) down-regulation of Cx synthesis and connexon assembly, or It is clear that tumor promoters such as TPA inhibit GJs, whereas (d) change in phosphorylation connexins. All of these could contrib retinoids (which inhibit TPA-induced tumors) enhance GJs (60, 83). ute to tumor promotion by promoting local cell isolation. This would Are these actions juxtaposed through their direct interaction with GJ be a stochastically critical microenvironmental defect if the field of pore assemblies or are the effects indirect'.' Or is it both? Evidence for cells includes particular initiated (1+ ) cells; (d) while unequivocal direct effects on connexons is not available, but evidence for an evidence for a causal relationship between modulation of gap junction indirect metabolic effect comes from studies on altered hepatic foci function and cancer etiology is not yet available, there is increasing Cx32 mRNA levels. Cx32 is attenuated in rat liver as a result of tumor evidence that modulation of gap junction function is often associated promoter treatment (86). Less Cx protein results from fewer Cx mR- with the mechanism of action of many carcinogens and tumor pro NAs, which in turn produce fewer GJ insertions into membranes. moters (2). Tumor progression also may be propagated by faulty Since the turnover time for connexins is relatively short (=2 h), the GJIC. net number of GJs in the plasma membrane decreases (145). The short Conclusion and Future Directions. Considered in into, short- and turnover time for Cx proteins suggests that long-term control (as long-term regulatory mechanisms allow gap junction channels to ex opposed to rapid gating and vesicular insertion/withdrawal processes) hibit enormous plasticity in response to intracellular and extracellular is exerted by Cx removal and resynthesis. This may explain why there signals. The range of permeant signaling molecules allows gap junc are various Cx proteins: to replace other Cx types and alter connexon tions to serve as major conduits for (a) ions setting the basic ground function when, and for as long as, the need exists for a different GJ state of a cell and signals and (h) the exchange of hydrophilic second mode. More work on this point is needed. messenger molecules, metabolites, and substances that might serve as Investigations on some liver in vitro epithelial cell lines show that either triggers or brakes (suppressors) for various cellular processes. liver Cx32 (made in vivo) is not synthesi/ed but rather Cx43 ( 146). Tools are now available for pursuing the nature of the regulation of 3482

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1993 American Association for Cancer Research. CAP JUNCTION FUNCTION AND CANCER channel conductance in the context of short-term gating and long-term 1883: and Arch. Pathol. Anal. Physiol.. 8: 23. 1855. 11. Kam, E., Melville, L., and Pitts, J. Patterns of junctional communication in skin. J. expression. At the level of the gap junction channel itself, alterations Invest. Dermatol., 87: 748-753, 1986. in structure of the connexins and the use of exogenous expression 12. Charles, A. C-, Naus. C.. Zhu. D.. Kidder. G.. Dirksen, E., and Sanderson, M. systems should allow identification of the molecular domains respon Intercellular calcium signaling via gap junctions in glioma cells. J. Cell Biol., 118: sible for sensitivities to various gating stimuli (151-154). The neces 195-201, 1992. 13. Peterson. J. The widespread nature of phenotypic variability in hepatomas and cell sary probes are also now available and should lead to elucidation of lines, in the form of a geometric series. J. Theor. Biol., 702: 41-53. 1983. other levels of regulation (including precursor synthesis, membrane 14. Malewicz, B., Kumar. V., Johnson. R.. and Baumann, W. Lipids in gap junction assembly and function. Lipids, 25: 419^27. 1990. trafficking and GJ insertion/degradation). These approaches should 15. Hertzberg, E., and Johnson. R. (eds.). Modem Cell Biology, Gap Junctions, Vol. 7. also illuminate the relationships among various types of membrane pp. 1-548. New York: Alan R. Liss. Inc.. 1988. channels. Cells use both gap junctions and cytoplasmic bridges as 16. Bennett, M., Barrio. L.. Bargiello, T. Spray, D., Hertzberg. E., and Sáez.J. Gap junctions: new tools, new answers, new questions. Neuron, 6: 305-320, 1991. conduits for exchange of ions, metabolites, and signaling molecules. 17. Dermietzel. R.. Hertzberg. E.. Kessler. J.. and Spray. D. Gap junctions between The investigation of the gap junction channel may serve as a key to cultured astrocytes: immunocytochemical, molecular, and electrophysiological anal identifying one class of intercellular channels and distinguishing them ysis. J. Neurosa., //: 1421-1432, 1991. 18. Haeflinger, J.. Bruzzone, R.. Jenkins, N.. Gilben. D.. Copeland, N.. and Paul, D. from other cellular transport channels. Further understanding of the Molecular cloning, expression, and chromosome mapping. J. Biol. Chem., 267: molecular and cellular basis of GJIC will allow insights into the roles 2057-2064, 1992. that tumor promoters play in perturbing the tissue signaling networks 19. Kumar. N.. and Gilula. N. Cloning and characterization of human and rat liver cDNAs coding for a gap junction protein. J. Cell Biol., 103: 767-776, 1986. during the course of toxicity and disease progression. 20. Miller, T, Dahl. G.. and Werner. R. Structure of a gap junction gene: connexon 32. Problems exist in considering assays of GJIC inhibition for pur Biosci. Rep.. 8: 455-464, 1989. poses of evaluating chemical hazard. Evidence to date suggests that no 21. Fishman, G.. Spray. D.. and Leinwand. A. Molecular characterization and functional expression of the human cardiac gap junction channel. J. Cell Biol., ///: 589-598, particular in vitro assay, based on a given species or cell type, can 1990. serve as a universal predictor of a potential modulator of GJIC. Ad 22. Willecke, K.. Jungbluth, S., Dahl. E., Hennermann, H., Heynkes, R., and Grzeschik, ditionally, in vitro conditions may not simulate in vivo delivery of K. Six genes of the human connexin gene family coding for gap junctional proteins are assigned to four different human chromosomes. Eur. J. Cell Biol., 53: 275-280, tested chemicals to solid tissues insofar as the native extracellular 1990. matrix scaffolding is missing. Furthermore, interactions of chemicals 23. Willecke, K., Heynkes, R., Dahl, E., Stutenkemper, R., Hennemann. H., Jungbluth, and chemical mixtures, in terms of modulating GJIC, can either an S., Suchyna, T, and Nicholson. B. Mouse connexin 37: cloning and functional expression of a gap junction gene highly expressed in lung. J. Cell Biol., 114: tagonize or synergize, as well as be additive. Because multiple GJ 1049-1057, 1991. proteins exist, often in different cells in the same organ, and several 24. Willecke, K.. Hennemann, H.. Dahl, E., Jungbluth, S., and Heynkes, R. The diversity mechanisms regulate GJIC. predicting the effect of a chemical based of connexin genes encoding gap junctional proteins. Eur. J. Cell Biol., 56: 1-7, 1991. 25. Saez, J., Spray, D., Nairn, E., Hertzberg, E.. Greengard, P., and Bennett. M. cAMP on results with one cell type in vitro on another cell type or on the increases junctional conductance and stimulates phosphorylation of the 27-kDa same cell type in vivo is not yet possible. 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James W. Holder, Eugene Elmore and J. Carl Barrett

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