Gap Junction Function and Cancer...munication; GJ. gap junction: connexon, one-half of the assembled dodecylhedral pore contributed by a cell: channel, low-resistance pore allowing

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

Perspectives in Cancer Research

Gap Junction Function and CancerJames 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 Â¡J.W. H.Â¡:National Institute for the Advancement of in Vitro Sciences, Irvine, California 92715 Â¡E.Â£./; and Laboratory of Molecular Carcinogenesis, National Institute of Environmental

Health Sciences. Research Triangle Park. North Carolina 27709 Â¡J.C. B.Â¡

Abstract

Gap junctions (GJs) provide cell-to-cell communication of essential

metabolites and ions. GJs allow tissues to average responses, clear wasteproducts, and minimize the effects of xenobiotics by dilution and allowingsteady-state catabolism. Many chemicals can adversely affect the membrane GJ assembly causing reversible alterations in GJ intercellular communication. During toxicity essential metabolites, ions, and regulators arenot shared homeostatically throughout a tissue community. Alterations inmetabolic circuits are thought to interrupt organ integration. PersistentGJ perturbation can cause chronic effects (e.g., cancer), and many tumorpromoters inhibit I..] intercellular communication. Liver precancerousfoci intracommunicate (but at a reduced level) and intercommunicateimproperly (or not at all) across the foci boundary to normal cells. In time,foci can become less regulated and more isolated within the tissue. GJsremain reduced quantitatively in the tumor progression stage and may bequalitatively altered in metastasis since connections are made between theprimary tumor cells and foreign host cells at the secondary metastatic site.Cell sorting and binding mechanisms by the cell adhesion molecules andintegrins may also be altered at secondary sites. This may allow therelocation of primary tumor cells and nurturance via GJs at the secondary

IntroductionScope. GJIC1 is widely believed to play a key role in tissue de

velopment and maintenance in multicellular organisms by homeo-

static control of cooperative, interacting cells within tissues and organs. In recent years, data have suggested that toxicants can modulatenormal GJ functions in growth control, development, and differentiation. Such modulation is thought to be involved in various diseasestates, but the specific role remains to be delineated. Mounting evidence, however, indicates that modulation of GJIC may be involved atcertain stages in the etiology of chemical carcinogenesis (1).

To evaluate the relevance of GJ mechanisms to disease, the UnitedStates Environmental Protection Agency, with supporting contributions from the National Institute of Environmental Health Sciencesand NSI Technology Services, Inc., sponsored a conference on Mechanisms of Gap Junction Function and Relevance to Disease; the conference included 12 speakers and discussants."1 While the general

Received 12/18/92: accepted 5/26/93.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 with18 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

or policies of the United States Environmental Protection Agency or the National Instituteof Environmental Health Sciences.

: To whom requests for reprints should be addressed, at Genetic Toxicology Assess

ment Branch. Office of Health and Environmental Assessment. Office of Research andDevelopment (RD-689). United States Environmental Protection Agency, Washington.

DC 20460.'The abbreviations and definitions used are: GJIC. gap junctional intercellular com

munication; GJ. gap junction: connexon, one-half of the assembled dodecylhedral porecontributed by a cell: channel, low-resistance pore allowing bulk water flow made up of

two connexons; plaques, multiple gap junctions aggregated in a plasma membrane locus;Cx, a connexin protein (<'.,!,'.,noted as Cx32 the predominant liver gap junction protein);ECM. extracellular matrix; TPA. l2-tetradecanoylphorbol-13-acetate; 1+ cells, initiated

cells; cAMP, cyclic AMP; CAM. cell adhesion molecule; PKC. protein kinase C; IP.,.inositol triphosphate.

â€¢¿�â€¢Conferenceheld in Washington, DC, February 27-28. 1990. Presentors included:

relevance of normal GJ function and modulation in disease was reviewed, the discussion often centered on the relevance to cancerbecause of GJ involvement in the tumor promotion stage (1, 2). Thefollowing discussion on GJs is topical and not comprehensive. It isbased in part on the presentations and discussions held at the Environmental Protection Agency meeting and in part on the organizers'

views of current GJ research since that meeting and the future directions for mechanistic research and applications to the relevance of GJfunction to disease.

Overview. In this review, we have organized the material topresent the structural characteristics of GJs, which include morphological and molecular aspects, the roles of GJs in cellular function andmaintenance, and how GJs are involved in cancer. Finally, we discussGJIC controls and xenobiotic alterations of those controls.

Role of Gap Junctions in Intercellular Communication

Gap Junction Definition. GJs are specialized intercellular channels between apposing plasma membranes that can be open or closed(gated). When open, these GJ channels allow metabolite exchangebetween cells. Each GJ channel is composed of two connexons; onecontributed from each communicating cell. Each connexon is composed of six Cxs that act in concert in order to form a pore in thecenter of the assembly. Connexons float laterally in the plasma membrane until a match is made to a proximal connexon in an adjacent cell(Fig. 1). When a match is made, the membranes of the cells areconjoined at that locus so as to form a channel between the two cells(Fig. 1). Conjoined cells are metabolic systems partially open to eachother with GJs providing channels of cell-to-cell communication.

Spatial patterns of interconnected GJs in a tissue form patterns ofcommunication in a tissue. These channels convey tissue metabolitescontrol chemicals, critical ions, and xenobiotic chemicals, but onlyhydrophilic molecules with molecular weights of < 1000 are shared bypassive diffusion through the GJ conduits. Whether all such cellularmolecules are shared equally among connected cells is still uncertain,but passive diffusion is assumed with the presumption of ionic andmolecular transients. Large molecules or lipid molecules, which require carrier macromolecules for transport, would not be transferredthrough GJs. Many GJs can aggregate with each other to form GJplaques at a membrane locus. Plaques are localized, high-traffic con-

E. Beyer (Division of Hematology. Washington University School of Medicine. St. Louis.MO). E. Elmore (National Institute for the Advancement of //( Vitro Sciences, Irvine, CA),E. Hertzberg (Department of Neuroscience. Albert Einstein College of Medicine. Bronx,NY). J. Klaunig (Department of Pharmacology and Toxicology. Indiana School of Medicine, Indianapolis, IN), R. Loch-Caruso (School of Public Health. University of Michigan. Ann Arbor, Ml), M. Neveu (Dana Farber Cancer Institute. Boston. MA), D. Spray(Department of Neuroscience, Albert Einstein College of Medicine. Bronx. NY). J. Trosko(Department of Pediatrics and Human Development. Michigan State University, EastLansing, MI), H. Yamasaki (International Agency for Research on Cancer. Lyon. France).Discussants included: J. C. Berrett (moderator. Laboratory of Molecular Carcinogenesis.National Institute of Environmental Health Sciences. Research Triangle Park. NO. R.Binder (Proctor & Gamble Co.. Cincinnati. OH) W. Farland (meeting sponsor. Office ofHealth and Environmental Assessment. United States Environmental Protection Agency,Washington. DC), and J. W. Holder (Genetic Toxicology Assessment Branch. Office ofHealth and Environmental Assessment. Office of Research and Development. UnitedStates Environmental Protection Agency.

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GAP JUNCTION FUNCTION AND CANCER

INTERCELLULAR LVI/EMLIPID BILAYER

HEMICONNEXON

CALCIUM IONS

NUTRIENTS

METABOLITES

BICARBONATEBUFFERINQ _JCOMPOUNDS 'â€¢

/CYTOPLASMIC PRESENTATION OF C

CEll-TO-CElL CONNECTION

Fig. I. Diagrammatic model ot'GJs in the plasma membrane of two apposed cells. The

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 GJpores is assumed to take place in mass transfer. Transferring molecules must he watersoluble with molecular weights <100(). Cytoplasmic bridges and active ion pumps alsotranspon material across plasma membranes in addition to gap junctions. The interactivekinetics among these transport mechanisms are not well understood. The GJ channel canopen or close by a rapid (ms) and reversible process called gating in which a portion ofthe connexon protein blocks the bulk water flou in the closure mode, whereas this proteinsequence quickly moves out of the GJ channel upon opening (adapted from Ref. 115 withpermission).

duits between cells for bulk exchange of small molecular weight ionsand molecules such as H2O, Ca~ '. and cAMP. The number of plaques

with adjacent cells is thought to be proportional to the metaboliccooperation among these cells. Thus, fewer plaques indicate lesscommunication, which in turn suggests that the cells may act moreindependently of each other. However, modes of intercellular communication other than GJs, such as paracrine functions and other ionchannels, continue to functionally couple cells, even when plaques arefew. The relative importance among the different modes of intercellular communication is not known.

CAMs can facilitate local GJIC in epidermal cells (3). CAMs canalso act as cell morphogenetic regulators conferring specific cell-to-

cell adhesions and cell sorting mechanisms (4). CAMs likely assistlocus-specific cell-to-cell docking processes in epidermal cells, pos

sibly by drawing and holding the adjoining membranes together in aCa2+-dependent process. This permits close intermemhrane apposi

tion, thereby facilitating cell-to-cell interactions that include connex-on-to-connexon (GJ) interactions (3). Progressive loss of GJs (andthus GJIC) and loss of E-cadherin (a type of CAM) have been ob

served to occur in advancing metastatic disease, which suggests theimportance of functioning GJs and CAMs to proper cell control (5-7).

The universal occurrence of gap junctions in multicellular organisms (metazoa) indicates that gap junctions play a ubiquitous, essential role in collective cell interactions (8). It is through these interactions that cellular physiological balances and controls are cumulatedinto regional tissue function in the corpus of multicellular organisms.Comparative structural and functional differences in GJs among various members of metazoa are not well understood.

Gap Junction Function. One of the most fundamental theories ofmodern biology is the "cell theory" first proposed in the mid-18()()s by

Mathias Schieiden (plants) and by Theodor Schwann (animals) (9).They proposed that plants and animals possess cells that are organismswithin themselves which are separate from, but related to. the organsin which they reside. The whole organism is constituted from collections of cells and organs that are ordered within the organism according to definite laws. These early works and the pathology studies of

Rudolf Virchow ( 10) formed the basis of the hypothesis that the cell,with its nucleus, is the functional biological unit.

Our current understanding of GJs suggests an extension of theaccepted cell theory: the cell is a unit, but a reproductive and biochemical unit within the local tissue. The tissue is integrated in part bymeans of the GJ connections and forms a syncytium. a connectedarray of cells. The syncytium averages cellular variances and capabilities locally and allows the integration of regional tissues into organfunction. The syncytium may be a higher and more relevant physiological unit within each tissue of an organ than individual cells. At theorgan level, the GJ interconnected cell fields are often heterogeneous.For example, natural physiological barriers, such as fascia, bone, andvarious extracellular matrix elaborations, allow the creation of localenvironments in GJ-interconnected cell communities, which manifestregional tissue-specific functions (11). These regional adaptations,

with specific interactive communication occurring, allow for integration and phcnotypic expression of the cell collective in organ function.The corpus is the next higher level of organization: the vasculatureand nervous systems provide corporeal integration. Corporeal integration physiologically unites and relates the various organs, tissueswithin these organs, and regional syncytia within tissues.

GJs seem to provide a number of regional functions within tissues.One such function is: (a) buffering the harmful effects of xenobioticchemicals. This buffering is achieved by dispersing xenobiotic molecules from the exposure entrance point outward into the tissue via anumber of interconnected cells. Dispersal in effect dilutes the localconcentration of the potentially offending xenobiotic. which can nowbe catabolized in more cells and in a steady-state fashion. AnotherGJ-mediated function is: (/)) nourishing of sick or deprived cells by

healthy neighboring cells (reciprocity). This concept is basic to tissuehomeostasis. The inuring process offers plasticity to cells during injurious processes and responses. As long as GJIC is not compromisedand the toxicity limit is not exceeded, the cell is remediated withassistance from healthy neighboring cells. Hence, a tissue can revers-

ibly recover or remodel following a toxic insult. However, it is thoughtthat if toxic limits are exceeded then cells slow or stop communicating(GJIC i ). Disorganization can then occur which can be followed bynecrosis and cellular turnover. Another regional function mediated byGJs is: (c) rapid exchange of critical ionic electrical signals (<'.#.,Ca2^ ions) and their regulators. For instance, when a cellular membrane is sufficiently disturbed, waves of Ca2 ' ions can move from the

disturbance point in a field of cells through cells to neighboring cellsin order to signal the disturbance ( 12). This wave action is hypothesized to happen so that regional tissue reaction can occur in a coordinated manner. GJs also participate in regional function by: (r/)allowing the distributions of critical metabolites (<'.,(;..cAMP) amongtissue cells. Cells benefit from GJ-mediated sharing of essential me

tabolites because all of the cells in a syncytium do not possess thesame metabolic capacity (13). Cells of lesser capacity benefit fromthose with ample capacity. If some cell fields are under stress andsome are not. GJ sharing is beneficial to the tissue as a whole. Lastly,another regional GJ function is: (?) the elimination of unwantedbyproducts. Elimination is facilitated by GJs in a gradient fashionfrom tissue interior cells to the vascular system for purposes of excretion.

Gap Junction Proteins and Membrane Structure. Isolation ofGJs is facilitated by their relative resistance to alkali and detergentsolubilization. Development of antibodies against different purifiedCx proteins has made possible topologica! mapping of GJs. The mostdetailed biochemical characterization is from Cx purified from liver.Lipid analyses of GJ isolates have indicated an enrichment of cholesterol, depletion of sphingomyelin. and significant levels of phosphati-

dylinositol and arachidonic acid ( 14). The role, if any. of these lipids

3476



GAP JUNCTION Kl'NCTION AND CANCER

is noi clear. It appears that GJ proteins from liver, as well as fromheart, can he fatty acylated but not glycosylatecl. Palmitic or myristicadditions may assist posttranslational transport of the connexinsthrough the Golgi cisternae (15).

GJs isolated from heart consist of a single protein with an apparentmolecular weight of about 43,(XX)-45,0(X) (Cx43). Other organs such

as liver have connexin mixtures: the major liver connexin is Cx32.with a lesser amount of Cx26 (= lOâ€”LWi) ( 15). Isolated GJ from lens

fiber cells consist of MP26 and MP70 ( 15). MP26 is unrelated to heartand liver proteins. While detailed information has not been presented,it appears that MP70 is related to liver and heart GJ Cxs. Somerelatively large size differences do exist in the variable cytoplasmicregions among connexin proteins, such as Cx.32 and Cx43. Thesedifferent sequences form different Cx types (15, 16). Each type isthought to basically act the same in GJIC no matter in which tissue thetype occurs but fine differences of GJ control and function may exist(17). More new sequences for additional Cx have been found byamplification using ihe polymerase chain reaction and low stringencyhybridi/.ation; new Cx proteins in rat liver have been predicted: Cx40,Cx37. C.\33, and Cx3l.l (18). These new sequences exhibit the typical sequence pattern of the GJ protein family, i.e.. highly conservedtransmembrane and less conserved extracellular domains, but variablecytoplasmic domains (tails).

Cx mRNA seem to be made from only one exon ( 19). This is unlikemost cellular proteins found to date that are made from mRNAs.which are assembled from heterogeneous RNAs containing dispersedintron sequences among exon sequences. The rat Cx32 and humanCx43 heterogeneous RNAs each have uninterrupted Cx readingframes (no introns). and each exon has a 5'-nontranslated region (20,

21 ). The human chromosomal locations of Cx genes are not clusteredon a particular chromosome, such as the globin genes, but rather occuron different chromosomes ( 18. 22). A number of Cx DNA sequencesoccur in humans: Cx26. Cx32. Cx43, and Cx46 on chromosomes 13,X. 6. and 13. respectively (22). Similar coding sequences exist in themouse and rat. Another mouse connexin protein. Cx37. has beenreported (23). The Cx genes isolated thus far as complementary DNAhybridize as distinct mRNA species; therefore connexin proteins makeup a multigene family (24). Such a structural diversity of related Cxgenes suggests different connexon functions, e.g., during differentiation (24). Genetic drift and reorganization should also be consideredsince Cxs are thought to constitute a set of evolutionarily old proteins.Cxs have been found to occur and function in mesozoa. which aretransition marine parasites biologically organized between unicellular(protozoa) and multicellular (metazoa) organisms (8. 16). There seemsto be no exception to the hypothesis that all metazoa utilize GJchannels. These conserved Cxs apparently provide an essential set offunctional cellular proteins in higher organisms.

Besides the cytoplasmic tail Cx sequence differences, chemicalmodification of the Cx protein also can take place. For example, Cxphosphorylation can be stimulated by cAMP. which acts as a cofactorfor kinase A phosphorylation. and such phosphorylations can produceincreases in GJIC (25. 26). Tumor-promoting agents (e.g., TPA) downregulate PKC while also down-regulating GJIC (27, 28). Presumably

the Cx is structurally altered when the Cx is phosphorylated. which islikely related to the down-regulation of GJIC and would be expected

to contribute to the pleotrophic tumor promotion mechanism ( 1, 16).Specific phosphatases remove attached phosphate groups from Cx inshort times (min); this process is considered part of the fast GJ controlresponses (29). Since Cx are proteins that have t,,, of only a few h,coordination of phosphate attachment and removal is essential to Cxfunction (30). Proteins other than Cx can be phosphorylated and yetaffect GJIC; e.g.. in rat liver phosphorylation of src tyrosine residue527 has been related to GJIC inhibition (31). Swenson et al. (32) have

found that expression of pp60%src in Xenopus oocytes blocks GJIC of

Cx43. but not Cx32. This GJ inhibition is caused by tyrosine residue265 phosphorylation in Cx43, whereas Cx32 is not similarly phosphorylated. Interestingly, the pp60%"slx'-promoted phosphorylation of

Cx43 does not inhibit the Cx assembly into GJs, only their function incommunication once in the membrane (32). Site-directed mutants

with shortened tail regions (where Cx phosphorylation occurs) showno altered voilage sensilivity but do show altered unitary conductancedifferences when compared to unmutated Cx (33). However, the tail-

modified sites do not seem to interfere with insertion of mutated Cxinto membrane-associated connexons (33). This conclusion was con

firmed by Musil and Goodenough who showed that unlabeled GJsoccur intracellularly. whereas '2PO4 labeling occurred only in the

membrane. Phosphorylation of GJs occurs after membrane insertionof Cx which leads to Cx deposition by insolubility and ability toassociate into plaques (34). This and the above studies suggest thaiphosphorylation affects GJIC but not insertion into the membrane.

Lone GJs and GJ plaques are readily visualized by freeze-fracture

electron microscopy on closely apposed membranes of adjacent cells(35). The morphological fine structure of GJs has been deduced fromX-ray diffraction studies (36). The emerging picture is one in whicheach connexon (i.e., one-half of the channel contributed by each cell)

is made of six protein subunits called Cxs, which may or may not beidentical, organized in a circular rosette array and aligned en faceacross an intercellular space. This space is the gap of GJ seen inelectron microscopy. The schematic structure of GJs in cells is shownin Fig. 1. The Cx proteins are thought to be the only proteins in GJ.Each Cx protein traverses the bilayer four times, which mnemonicallyconfigures the letter "M" in cross-section. The Cx sequences that

traverse the membrane are highly conserved among species ( 16). Thisconservation pattern is consistent with most channel proteins. The Cxcytosol sequences (visualized as two bottom legs of an "M." corre

sponding to the NH2 and COOH termini, and an intervening middleloop) vary. These variable regions suggest that different types ofconnexons may be due to Cx heterododecylhedral mixes within connexons in GJ, but these mixes have yet to be demonstrated. Thesemixes, if present, could present fine structural differences in assembled GJ channels.

Any agent that alters the connexin protein assembly, other connex-on-associated componenis. or local membrane morphology can po

tentially affect GJ function. Proximal desmosomes seem to strengthenlocal cell connections. It now appears in the cell systems tested thusfar that neither GJs nor the adhesion molecule E-cadherin functionwell in low Ca2 ' . It has been suggested thai E-cadherin and GJs needadequate levels of Ca2 ' to function optimally (3, 4). What Ca2 ' ion

levels are adequate likely depends on the cell type, stage of development, or disease state. CAMs like uvomorulin, which is identical toE-cadherin and homologous to liver-CAM, may facilitate GJ dockingand subsequent stabilization of cell-to-cell interactions (3, 6). The

integrins also may play a role in specific cell loci matching up andallowing GJ formation between specific compartments of the twoconjoined cells (37). Other extracellular elements such as glycosami-

noglycans and proleoglycans have been observed to proffer slabilizinginfluences and affect increased GJs (1. 4. 6. 38). The explicit relationship of GJs and the ECM remains to be delineated. Inside the cell,the actin cyloskelelal network may be associated wilh GJs (39. 40).Preliminary work suggests that when cells are placed in low Ca2 *,

connexons are pulled apart by retraction (41, 42). The separation ofconnexons could be a passive process; i.e.. the cell-to-cell pore structure is thermodynamically unstable at 37Â°Cand low Ca2 '. This con

nexon separalion could also be an active process, due to actin atlach-

ment to ihe plasma membrane microvilli proximal to the connexons(40). Notably, cytochalasin B. vincristine. colchicine. vinblastine, or

3477




low temperatures (0Â°C),all of which disrupt cytoskeletal structures,

each can increase GJ plaque areas (40). The emerging concept fromthis work suggests that the actin cytoskeletal system regulates GJIC byfirst polarizing the fibrillar apparatus toward the outer cell face andmodulating concentration and polarization of microvilli at the cellsurface (Ref. 40) and references therein). By this mechanism, thenumber and positioning of GJ plaques are coordinated by cell organization (cellular polarity), which contributes first to the anisotropicorganization within the cell and secondly to the collocation of the cellwithin the tissue (2, 40).

Some Gap Junction Examples in Cellular Communication.Electrical coupling occurs among communicating cells due to ion flowmeasured in conductance units of picosiemens (pS). Cellular couplingestablishes a ground state potential, along with Na+, K +, Ca2+, andH+/~HCO, pumps, among cells in the community, all of which pro

vides the background on which pulse signals such as Ca2 + ions are

generated and interpreted. Ionic coupling among cells is critical forpropagation of depolarization in myocardial and smooth muscle tissues and is thought to provide a means for coordinated contraction(43, 44). For example, at parturition, a rapid, incipient increase in GJCx protein in myometrial smooth muscle enhances syncytial activityand coordinated contraction necessary for fetal expulsion in parturition (2, 45). The brain has many GJs, but little is understood abouthow brain GJs participate in neural cell function and maintenance(46-48). Electronic coupling takes place in the nervous system (in

addition to synaptic transmission) and can synchronize outputs ofelectronically coupled neuronal pools (16). Neural activity in glialcells causes focal K+ transient loads that are dissipated in a recovery

process mediated by GJ (16). Retinal horizontal cell conductance inthe eye can be modulated via GJs that transmit dopamine, which inturn affects retinal cAMP-dependent protein kinase (49).

Another example of the integration of tissue activity and metabolicfunction is the coordination of cells in a hormone-responsive tissue

( 16, 50). Coupling via GJs plays a role in secretory systems, and it hasbeen demonstrated that secondary messengers (of primary hormonestimulation) such as cAMP, cGMP, Ca2 +. and IP, pass through GJ

channels (51 ). It is likely that many other critical molecules are sharedvia GJs. Insulin production in pancreatic islet cells correlates withprior secondary messenger activity (cAMP). bursts of pancreatic Cx43mRNA activity and Golgi and endosomal vesicular exportation of theCx43 into islet cell membranes (52). Such a sequence of eventssuggests that GJs are made and assembled in order to assist insulinsynthesis and release from pancreatic cells. The mechanism by whichthis is achieved remains obscure. The examples given above suggestthat the initial sharing among cells of effector molecules via GJs canlead to integrated tissue responses (15).

Another interesting case where cells share effector molecules viaGJs is in the ovaries. Oocytes are maintained in meiotic arrest bygranulosa cells to which they are coupled by GJ via filopodial processes projecting through the zona pellucida (53). These GJs mediatethe intercellular flow of the ovulatory meiosis inhibitor between themembrana granulosa and cumulus oophorus cells with stasis of oocytemeiotic cell division as a result (54). An ovulatory stimulus, providedby luteinizing hormone, leads to disruption of GJIC in which inhibiting granulosa cells can no longer transport ovulatory meiosis to thearrested oocyte cells (55). Accordingly, the oocytes are no longerarrested and ovulation ensues.

GJs play an essential role in embryonal cells during developmentand differentiation (56. 57). Certain teratogens seem to act by alteringGJIC (57). A potential role for GJs exists in providing a mechanismfor transmitting timed-growth and positional signals involved in de

velopment (58, 59). Several observations based on ultrastructuralanalyses indicate that GJs do appear or disappear at various times (16,

42). Coordinated GJ appearance prior to critical events in differentiation is consistent with a role for GJs in signaling and maintenance ordisruption of morphogen transmission ( 15). Vitamin A analogues andglucocorticoids, both known to be involved with cellular differentiation, are linked to the increase in the number of GJs (60). Selectivedisruption of GJIC either by introducing Cx antibodies or by injectingCx antisense mRNA has led to developmental defects in both invertebrates and vertebrates, thereby implicating GJ in these processes(16, 61). Correlations are now being made between genes that arecentral to development, e.g., pattern formation, and those that stimulate cancer (oncogenes) (62-64). Changes in development involving

GJs have been correlated with loss of cell growth control, which canlead to cancer (56. 65. 66). The relationship between cancer and GJICis discussed below.

Modulation of Gap Junction Function

Temporal Modulation of Cap Junctions

Changes in GJs are essential for allowing, or attenuating, the intercommunication in a field of cells. Three kinetic courses of GJcontrol are beginning to emerge: (a) fast control by gating responses(=ms); (b) intermediate control by vesicular insertion or withdrawalof connexins in or out of the plasma membrane (= min); and (r)long-term control, where transcription of Cx mRNAs is turned on or

off or is attenuated and Cx protein pools are adjusted accordingly (h).Times are empirical and often reflect rodent liver studies. Comparative differences among species of these times are not known.

Fast Control. The transport of electrolytes or small low molecularweight substances from cell to cell depends on the number of GJsconnecting these cells that are open at any given time, the specificconductance of the type of shared connexons. and the tissue environment. Different structural models have been proposed to account forthe gating (quick closure) of GJs. In one model it is suggested thatbulky, hydrophobic phenylalanines are introduced into the aqueouschannel by an a-helix rotation of the third transverse region (M3) of

the Cx protein. The channel is thus occluded for aqueous transport,thereby causing a restricted or closed channel (pS J, ). Reversal of thisprocess by M3 a-helix rotation in the opposite direction reintroduces

small hydrophilic amino acids into the channel, which clears the wayand reopens the channel to aqueous transport (pS ÃŽ) (67). It is alsopossible that if the subunit rotation were progressively twisted in acooperative manner along the pore axis, a gating from open to closedstates would occur without resolvable intermediates, much like theaperture of a camera lens. The kinetics of gating, which in partinvolves M3 rotation, has been estimated to be =ms (68). The energydifference between the open and closed states is =1-2 kcal/mol (68,

69), which is on the order of the strength of the hydrogen bond. Sincethe average energy of kinetic motion ^=0.6 kcal/mol at 37Â°C,then

gating energies may require only 0.4-1.4 kcal/mol. Such low energy

requirements suggest that GJs can be gated readily at body temperature and can accommodate rapid ion signal (voltage) changes.

Some agents effect almost immediate GJ channel closure ( 16). Forexample, immediate effects can result from rapid changes in intracell-

ular pH. ion transients, protein kinases, and even changes in GJmembrane lipid composition (14, 70). Examples are acidic pH andPKC and protein kinase A activators, inhibitors of cAMP. Ca2 ' spike-

inducing agents, and high lipophilic agents (such as unsaturated fattyacids). The study of actual GJ structure-activity relationships of rap

idly acting agents has just begun.Intermediate Control. Evidence for the intermediate control

mechanism is scant and rests mainly on the observations that Cx-antibody-reactive material exists in vesicles proximate to the plasma

membrane. It has been observed that Cx can be inserted into the

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CAP JUNCTION FUNCTION AND CANCER

plasma membrane even in the presence of protein synthesis inhibitors(71 ). This suggests that a peri membrane pool of preexisting connexinsexists and may buffer the Cx supply (16. 40, 72, 73). It is not knownif the intermediate pool is under physiological control. The presenceof the intermediate pool underscores the essentiality of Cx to cellularfunction.

It is known that Cx are not phosphorylated in the pool. Evidenceexists to suggest that Cx43 is phosphorylated after being inserted intothe plasma membrane (34). This phosphorylation seems to accompanyTriton X-100 insolubility of Cx43 (34). Phosphorylation does not

determine cellular stability of Cx43 but has been correlated with Cx43accumulation into plaques (34). Such a mechanism suggests phosphorylation control of mass transport.

Long-Term Control. Evidence for longer term control of GJIC isstill emerging (16). In liver cultures, the apparent half-life for Cx32and Cx26 is estimated at 2 h with about the same Cx half-life found

in liver in vivo (74). This turnover is rapid enough to regulate levelsof GJs by Cx mRNA synthesis rates and stability. In primary hepa-

tocyte culture, GJs disappear 10 h after plating but reappear within 72h; this disappearance and appearance also occur after partial hepatec-

tomy (16). These recoveries are preceded by Cx mRNA and areaccelerated by cAMP (75).

Cyclic nucleotides affect GJ in most tissues and cell lines that havebeen studied to date, and these agents are the likely candidates for themajor set point of physiological GJ regulation. cAMP analogues cancause either increases in GJs, e.g., liver cells, or, in some cases,decreases in GJs (76-78). Inhibitor experiments suggest that these GJchanges are attributable to effects mediated through cAMP-dependent

protein kinase A, which can phosphorylate Cx (as well as regulatoryproteins) and thereby regulate GJs. For example, liver Cx32 can bephosphorylated at a serine residue on its cytoplasmic tail, with kineticssimilar to that associated with increased conductance ( f pS) (79).

cAMPcan be a mediator of up-regulation of GJs in a variety of cell

types. Within 6 h. cAMP exposure leads to an increase in GJICbetween sympathetic neurons, which is blocked by inhibitors ofmRNA and protein synthesis (16). In hepatocytes, cAMP extends thelifetime of GJ between dissociated cell groups, which is attributedlargely toan increase in stability of Cx32 mRNA (16). Transcriptionalregulation is expected as well, based on the cAMP response elementsequence in the genomic clone of Cx32. As discussed previously,components of the intercellular and extracellular matrices influenceGJs and seem to provide stability to these structures and enhanceGJIC. For example, in hepatocytes, there is an interaction of matrixcomponents and glucagon: treatment of cultured cells with glucagontogether with proteoglycans leads to a marked increase in Cx32 transcription as well as increased stability of Cx mRNA (38). Down-

regulation of GJIC has been reported in uterine smooth muscle cellstreated with cAMP derivatives (2).

Tumor promoters such as TPA stimulate the signal transductionmediated by PKC by mimicking the normal substrate of PKC, diacyl-

glycerol (80). PKC exists in multiforms (in as many as six genes) thatchange in relative gene expression upon TPA binding to PKC ortransformation (81, 82). Like many tumor promoters, TPA inhibitsGJIC (27, 28) and may do so by altering one of the PKC isoforms withrespect to GJ phosphorylation (83-85). In several cell lines, activephorbol esters inhibit GJIC in approximately 10-20 min, a time course

similar to that of PKC activation. Chronic treatment of the tumorpromoter phÃ©nobarbitalcan lead to long-term cellular changes, e.g., a

decreased occurrence of GJs in altered hepatic foci (86). Such GJdecreases have been observed to be the result of a lessened synthesisof Cx32 in liver and are possibly linked to the preneoplastic nature ofthe AHF (86).

Toxicant Modulation of Gap Junctions

Involvement of GJs in the toxic response of cells to chemicals hasbeen an overlooked area and there are few systematic studies on thistopic (2, 87). GJ perturbations may be involved in the toxicity causedby many chemicals, but the precise mechanisms are not yet knownand can only be inferred in certain cases.

Generally it is known that when a cell is damaged, the membranecan become leaky to ions such as H+, Ca2+, and K+. If the damaged

cell remains coupled to contiguous undamaged cells, essential metabolites are lost at the damaged cell site. GJs are thought to attenuateduring these periods of damage for self-correction by the cell (88).

The mechanism of attenuation is not known but is expected to beprimitive and an intrinsic aspect of the Cx molecular engine andmembrane assembly. The mechanism of closure may also respond toparacrine control. pH. and critical ion balances. Closing agents wouldinduce structural changes in Cxs by rotating M3 into the channel (cf."Fast Control"). Localized closures can prevent spreading of toxic

injury in the tissue and allow energy to be switched to repair. If thecells are repaired and survive, then they can later recouple using GJchannels to become once again functional participants in the syncy-

tium in which they reside. Such regional attenuation of GJIC withremedial recruitment could also be under tissue physiological controlabout which we can only speculate at this time. Toxicity-induced

closure is reminiscent of GJIC reduction (not ablation) during mitosiswhen cells withdraw to the internal program of cell duplication anddivision (88, 89). However, these mitotic cells remain electricallycoupled to some extent to surrounding Â¡nterphasecells (90). In agreement with this is the observation that regenerating tissue shows first aloss in GJs but later the recovery and recoupling with contiguous cells(91-93). The community of cells is thereby established once again.

Toxicants (e.g., the perturbants /Â¡-heptanolor /Â¡-octanol)may re-

versibly close GJs by physically reacting with the GJ plasma membrane assembly and abolishing cell-to-cell coupling (44, 94, 95). Ca2 +

and pH must be optimum for GJs to function well (15), althoughhumoral factors can alter the sensitivity to Ca2+ and pH (96). It has

been thought that inhalation of anesthetics may act reversibly byinhibiting GJIC (97). Many anesthetics are small lipid molecules withhigh fugacity and are accessible to many tissue compartments. Theseproperties could cause reversible membrane perturbations in varioustissues including neural GJs thereby causing reversible sensory interruption. Moreover, chronic ethanol consumption (a C2 alcohol compared to C7 and Cx alcohols above) may persistently perturb GJs byexcessive membrane solvency and perturbant effects along with excessive acetaldehyde generated upon ethanol metabolism. The persistent influence of perturbants on GJs can be thought to lead to persistent dyscommunication in the affected field of cells, which can thenlead to either (a) facultative accommodation by the cells or (Â¿)diseaseprogression. In the second pathway, chronic disease follows whendeleterious acute effects become irreversible, which can occur fromrepetitive acute or subchronic insults (2).

In Vitro Assays for Gap Junction Toxicity

Data on GJIC following toxic injury have been obtained fromvarious types of assays for GJIC: (a) cooperative sharing of a metabolite; (b) fluorescent dye transfer; and (c) electrical coupling. Toxi-cological data (=200 agents) has been obtained in vitro in V79 met

abolic cooperation assays (98). One problem encountered in this invitro work is that there is a relatively low potential for V79 cells tometabolically catabolize chemicals, a transformation that may be necessary for some induced toxicity. Moreover chemicals that are tumorpromoters are usually organ specific and would necessarily require theappropriate cell culture to test GJIC effects. Another shortcoming is

3479




the lack of ECM effects manifest in solid tissues. Nonetheless, in vitroV79 data have provided some insights into the direct pharmacologicalactions of various types of chemical agents on GJIC and provideuseful comparisons of structure-activity relationships. Whether or notthese reactions on GJs occur in vivo has to be assessed on a chemical-by-chemical basis considering the pharmacokinetics and metabolism

of xenobiotic exposures in various species.Several examples of the usefulness of the V79 assay exist. When

nine phenylenediamine congeners were evaluated for metabolic cooperation, eight of these (89%) were correctly identified for theircarcinogenic potential based on their ability to inhibit GJIC (98). Notall known animal carcinogens tested in V79 cells inhibited metaboliccooperation. Given the lack of metabolic activation, however, it isintriguing that up to 75% of the chemicals tested with known carcinogenic activity produced some inhibition in the V79 metabolic cooperation assay (98).

The activity of alcohols and lipophilic agents in the V79 assayappears to be directly related to their octanol/water partition coefficients (98). Fatty acids demonstrate activity as well. Strong mutagenssuch as diethylnitrosamine and ethylnitrosourea also inhibit metaboliccooperation. Certain toxicant classes, such as pesticides includingaldrin, mirex, kepone. DDT. DDE, heptachlor, methoxychlor, andlindane, inhibit metabolic cooperation in the in vitro V79 assay (98).Thus, a wide variety of toxic, pharmacologically active agents haveactivity in modulating GJ function in an in vitro assay. These effectsmay correlate to in vivo exposures since dose delivery is likely: (a)direct external delivery to GJ is assured from the ECM; and (b)internal delivery is likely from transponed molecules through the GJchannel.

For agents associated with noncancerous disease end points, thefollowing correlations were found; 80% of all teratogens tested inhibited metabolic cooperation, whereas 73% of all neurotoxins testedwere detected (98). While these V79 in vitro data cannot be consideredunbiased, due to the partial selection of agents for study based on theirknown activity as pharmacologically active toxicants in one system oranother, the selectivity does imply the usefulness of GJIC inhibitionassays to detect chemicals associated with toxic responses.

Gap Junctions and Cancer

Experimental animal carcinogenesis studies as well as epidemio-

logical studies of human carcinogenesis strongly indicate that theprocess of carcinogenesis is multistaged and sequential (99, 100).Some stages in the sequence are not always the same which meansthat a number of different overall mechanisms can cause cancer (101,102). This has made the explicit identification of the necessary andsufficient components a complex matter of biochemical and logicalanalysis. While mutations often play a role in the multistage nature ofcarcinogenesis, nongenotoxic mechanisms also play key roles in carcinogenesis (103-105). Examples of nongenotoxic mechanistic types

are changes in gene expression, cell proliferation, cell organization,cell interrelationships (including GJIC), cell death (including apopto-

sis), and cell removal. There are a plethora of potential interventionsthat a xenobiotic chemical can bring about to alter the cellular phe-

notypic expression of an exposed field of cells. Although affectingGJIC is only a subset of all potential interventions, adversely affectingGJIC may be a more common toxic event than heretofore thought(2, 87).

Because cancer has been described as a stem cell disease, a diseaseof differentiation, and a disease of cell regulation or growth controland because GJIC has been linked in normal cells to control ofdevelopment, differentiation, and growth, it might be supposed thatthere may be a relationship between cancer and GJIC. Loewenstein(56) predicted this relationship in a prescient 1979 review, while the

first reports implicating GJIC inhibition by tumor promoters werepublished in the same year (27, 28). Since then, objective correlationhas been made in a number of laboratories between the promotion andprogression stages of carcinogenesis and decreased GJIC (1.2, 106-111). It is thought that chronic inhibition of GJIC in carcinogen-

initiated tissues, which contain 1+ cells as a result of being initiated,can bring about the tumor promotion stage of chemical carcinogenesis(1. 27, 28. 112). Tumor promoter interference with GJIC has beensubstantiated both I'Mvitro ( 1, 113) and/'Â»vivo (\\2, 114). The role of

GJIC disruption and chemical carcinogenesis has been reviewed previously (115).

There is an inverse correlation of cell growth and GJIC in transformed lines, i.e., induction of GJIC leads to growth inhibition,whereas blockage of GJIC leads to uninhibited cell growth (116).Antitumor agents such as retinoic acid or retinol reduce transformation and up-regulate GJIC (60). Several activated oncogenes (e.g., ras,\-mos, neu. src. little and large T, and EIA, but not v-myc or v-fos)

reduce GJIC in some cells (66. 117). Although most known tumorpromoters reversibly down-regulate GJIC /';; vitro and in vivo (1, 27,

28). GJ inhibition by tumor-promoting chemicals is not universal. A

few tumor promoters studied thus far. e.g., okadaic acid and TCDD,do not down-regulate GJIC in the systems in which they have beenstudied (1, 118-120). These observations suggest that some tumor

promotion mechanisms proceed without altering GJIC, which agreeswith the growing evidence that there are qualitatively different mechanisms for tumor promotion (121). Different mechanisms could alsorelate to different adaptive phenotypic defense mechanisms withwhich cells respond to carcinogen exposure (102, 122).

While it is universally agreed that chemical mutagens react withDNA and can cause lesions that lead to hereditary diseases as well ascontribute to somatic diseases (including cancer), all toxicants do notact directly on DNA and all carcinogens do not necessarily reactdirectly with DNA (87. 105, 109). Toxicants working on "other thanDNA" are often referred to as epigenetic. A number of epigenetic

events can conjunctionally cause persistent toxicity. Among theseevents, chemical modulation of GJIC may play an important role inthe areas of reproductive toxicology and teratogenesis, as well ascarcinogenesis (2, 87, 115, 123). By perturbing GJIC, chemical exposures can lead to a loss in tissue integration via distortion, or evenablation, of essential biological signaling networks (2, 11, 108, 109).The hypothesis of GJ-mediated control of cell growth was introduced

by Loewenstein, who advanced the concept that negative repressors ofcell growth are shared among normal communicating cells via GJs(56, 66). Moreover, as the hypothesis goes, these shared repressorskeep cells in an orderly growth-arrest state, except those cells gener

ated to replace normal cell loss. Growth ensues when conditions causea diminution of sharing of these negative repressors (56. 124).

Ca2 + ion oscillation takes place within cells, which is not wellunderstood, but seems to be frequency-modulated signaling. Ca2 + ion

oscillation is an intracellular process which does not disturb neighboring cells. Ca2' ion oscillations has been related to: (a) enhancement of cellular signal recognition; (b) reloading of Ca~ + ion storage

sites; and (c) directional ion transport (125. 126). In contradistinction,a Ca2+ ion wave is a form of transmitted Ca2 ' communication originating from a cellular membrane trauma. The Ca2 f wave propagatesthrough not around, cells (12). The hypothesis has been that the Ca2 +ion wave is moved from cell to cell by a "leader" substance which

progressively causes Ca2+ ion pulse in each cell (126). This substance

has proven to be IP, (12, 126). The IP, moves through cells, causes aCa2+ pulse, and then diffuses onto neighboring cells via GJs. Hencethe wave of Ca2 + ions moves from the point of disturbance and is

directed to neighboring cells which must be alerted. The cyberneticsof the Ca2+ wave signal is unknown. Chemical toxicant disruption

3480



CIAF' JUNCTION FUNCTION AND CANCER

may scramble the signal before it is translated. When wave signals areinterrupted, distal tissue cells would not be alerted and thus would notinteract cooperatively. Damage can ensue due to the lack of homeo-

statically controlled repair.When hormones, secondary messengers, and essential metabolites

are not shared to their normal extent due to impaired GJIC. thenexcess cell divisions can occur. It persistent, this condition can lead todysplasia. Trauma, for instance, can lead to cell loss and regenerativehyperplasia. This loss of growth control is temporary because hyper-

plasia can regress in a reversible manner when the inducing stimuli areremoved. GJIC is thought to be important to remodeling but themechanism of this process is not clear (2. 127). If toxic stimulation ispersistent and 1+ cells are present, then 1+ cells can be clonallyexpanded as can other cells in the field. Not all 1+ cells are equally

committed to cancer susceptibility. However, if a critical clonal size ofa deleterious 1+ cell population is ever reached, the growth-suppres-

sive effects of surrounding normal cells on these particular 1+ cellscan be overwhelmed (1). 1+ cells can become disconnected from thesurrounding normal cells to form islands of dysregulation. Regulatorycontrols are thus circumvented, at least partially, and the unrestricted1+ cells continue to divide and eventually form genetically and phe-notypical ly heterogenous preneoplastic foci (102, 122. 128-130).

Foci are preneoplastic and are only one set of cellular events occurring in the tumor promotion stage of cancer evolution (127, 128,131, 132). This is true at least in liver where the observation of alteredfoci was first made; the presence of foci in other cancer-susceptible

organs is not as clear. Many preneoplastic foci show GJIC among focicells (homologous communication) but fail to communicate properlywith normal cells surrounding these foci (heterologous communication) (I, 110). This segregation of communication creates differentsubcommunities which, in effect, can interrupt the integration of thetissue. These foci are genetically variable, or at least evolve heterogeneity, and they exhibit different phenotypic patterns of enzymedeficiencies at the foci stage and beyond. The outcome of early andcontinuing generation of heterogeneity is successive cell selection toprogressively higher levels of adaptation to the altered (transformed)microenvironment ( 102, 111) that appears to include decreased GJICof foci to normal cells. Various posttranslation mechanisms lead toincreased cellular phenotypic variability. The phenotypic variabilityoccurs at a rate far exceeding the genetic mutation rates (111, 133).The evolving phenotypic variability is an important component incellular responses to xenobiotics. One such cellular variability involves decreased GJIC among various cells as metastatic diseaseprogresses (5).

Many transformed cells have fewer GJs than their correspondingnormal tissue cells (83, 134). Since transformation takes place early inneoplasia, GJIC reduction may be an early, fundamental lesion in theorigin of cancer. Species and strain differences to the liver tumor-

promoting effects of various chemicals seem to correlate with theirability to down-regulate GJIC in vivo in liver (135). As malignancy

progresses, a decrease in GJIC levels in mouse epidermal and livercells is observed (136. 137). Tumor metastasis seems to correlate withdecreases in homologous as well as heterologous GJIC (1, 136, 138).Nicolson (111) found that, when metastatic potentials were compared,those cancer cells with strong metastatic potential had fewer GJs thanthose cells with weak potential. The decreased number of GJs foundin more cancerous cells suggests that GJs tend to be lost in cancerdevelopment and accompany disturbances in tissue growth controljust as Loewenstein (56) predicted in 1979. The long-standing concept

in cancer technology, that early cancer cells somehow become different than their neighbors and are accordingly "outlaw" cells, could now

find its explanation in the GJ mechanisms allowing for differential

cellular communication or even removal of focal cell communitiesfrom normal local tissue communication patterns.

Although GJ expression may be altered by certain oncogenes andtumor promoters in many highly metastatic cells. Kanno (65) reportedthat tumor cells can remain coupled by GJs. at least to some extent.Thus, a complete lack of coupling is not necessarily an obligatoryrequirement tor conditions leading to loss of growth control. Rather,a critical level of GJIC may have to exist in order to avoid cancer, butsuch a level is not yet known. Furthermore, it may be that total GJICablation causes premature toxic cell death and does not allow time forcancer to evolve. Relative cell kinetics is important. For example, ifthe rate of loss of 1+ cells in a tissue (through premature death) isbalanced by the rate of newly generated 1+ cells (through persistentinitiator activity), then the 1+ cell pool would be constant; but if theloss of 1+ cells exceeds the 1+ cell supply, eventually the field will bedevoid of a significant amount of 1+ cells to cause cancer. In the casewhere a persistent carcinogen produces more 1+ cells than are lost,the chances of cancer increase as 1+ cells accrue. The risk of cancerincreases in the latter case. During the course of cancer evolution, theprocesses of tumor promotion, conversion, and progression act onthese accruing 1+ cells and the disease develops. However, it isnecessary to minimally nurture these advancing 1+ cells via someGJIC in order to from a tumor.

Cancer is likely the stochastic result of the evolution of 1+ cells andthe surrounding focal cells which is a collective of altered (transformed) cells. Foci are usually identified histologically by an enzymealteration (usually a deletion); it should be remembered that thesefocal cells also differ in other phenotypic traits. Foci are heterogeneous (genetic and phenotypic) populations. Thus, if certain combinations occur within foci (transformants + I+ cells) and certain en

vironmental conditions occur surrounding these foci (all whichinteract at the micro level), 1+ cells can ultimately emerge to bemalignant. Carcinogenicity in fact begins when these combinatorialbiological interactions become irreversible; i.e., the adaptations cannot readapt. Since focal cells may be selective in communication withthemselves and at a reduced GJIC rate compared with surroundingnormal cells (137). the expectation is that focal cells have a differentqualitative GJ apparatus. This prediction should be investigated. Restriction of communication to surrounding tissue cells dissociatesthese focal cells from the normal flow of tissue growth regulators. Arecent suggestion is that reduced GJIC in carcinogenesis may beprimarily due to reduced homologous communication and less due toreduced heterologous communication ( 137). Thus, whether it is functionally most significant that focal cells do not either properly Â¡ntra-

communicate or intercommunicate with surrounding normal cells, orboth, remains a mystery. More work on the patterns of chemicallyinduced GJIC inhibition should prove fruitful.

Overall tumor growth is influenced by organ size constraints (similar to normal organs), albeit the plateau size has pathologically beenreset upward (hence tumor which simply means swelling). This continued size constraint is explained by the "integrated organ hypothesis" (139). We suggest this may be based on the systems integration

provided by the GJ-coupled network within organs. A case in point is

rat partial hepatectomy which stimulates regrowth to the proper liversize and lobular conformation. Accordingly, organs "know" their size,

but continued normal use of the organ GJs may be necessary tomaintain that knowledge. Gap junctional disturbances may lead to lossin the cybernetics of size restraint: hyperplasia results which is thecombination of increased cell number and cellular volume. If continued, a condition sets up where tumorigenesis begins. Carcinogenesiscan evolve out of persistent tumorigenesis. which can then allowfurther mutations or selection of deleterious phenotypes. Overall tumor growth can also be influenced by the generation of a mutator

3481



GAP JUNCTION FUNCTION AND C'ANC'KR

phenotype where early mutations lead to enhanced and progressivesusceptibility to more fixed mutations ( 140). This type of phenotypecan "conven" transformants into growing neoplasms that in turn can

be converted into malignant cells during the progression stage ofcancer. Hence, progressive alterations and aberrant growth occurringin carcinogenesis could affect the GJ systems among other changes. Ifso. this could include altering both the quality and the quantity oftumor cell GJs. We have objective bases for quantitative changes inGJ1C. but thus far. qualitative changes in GJs during carcinogenesishave been elusive. It is important to establish the precise relationshipof GJIC to the etiology of cancer (2).

Some further relationships between cancer and GJIC have beenpresented, nix T24 protein accumulates in rat liver cells after transformation by /-</.vand is inversely related to GJIC (141). This obser

vation suggests that an oncogene may operate, at least in part, byinhibiting GJ function. Mehta et cil. (142) showed in mouse transformed l()T'/2 cells (which had low GJIC) that a transfected gene for

Cx43 protein restored GJIC communication. Following GJIC restoration, growth of the mouse transformed IOT'/2 cells was slowed,

saturation density was reduced, and focus formation was suppressed(142). These results in lOT'/i cells indicate how impaired GJIC can

cause transformation, which can be reversed by overcoming the deficiency, i.e.. adding back Cx43 genes to the transformants. In anotherinvestigation. Naus et til. (143) showed that transformed C6 gliomacells can cause brain tumors. C6 glioma cells grow fast in culture butare greatly slowed when transfected with complementary DNA forCx43. This investigation, like the studies in l()T'/2 cells described

above, indicate that lack of expression of Cx43 can be associated withtumor growth, but C.\43 expression can suppress tumor growth.

Metastatic colonization at a distal organ site is initiated by theattachment of blood- or lymph-borne tumor cells to organ-specificadhesion molecules which are expressed on the surface of microvas-

cular endothelial cells. The establishment of communication by theinvading cell to the host endothelial cells is essential for tumor growthand maintenance at the secondary site. El-Sabban and Pauli showed

that highly malignant rat mammary carcinoma or melanoma cells,preloaded with fluorescent dye. readily transfer the dye to endothelialcells at the secondary site ( 144). Low metastatic or nonmetastatic cellsdid not similarly establish GJIC with the endothelial cells ( 144). Theseauthors conclude that GJIC was necessary for metastatic establishment and may play a role in tumor cell extravasation at secondary sites( 144). Although some form of GJIC is required for the further nur-

turance of the tumor cells, it may be that (here is something differentabout these GJs in establishing GJIC at secondary sites.

It is clear that tumor promoters such as TPA inhibit GJs, whereasretinoids (which inhibit TPA-induced tumors) enhance GJs (60, 83).

Are these actions juxtaposed through their direct interaction with GJpore assemblies or are the effects indirect'.' Or is it both? Evidence for

direct effects on connexons is not available, but evidence for anindirect metabolic effect comes from studies on altered hepatic fociCx32 mRNA levels. Cx32 is attenuated in rat liver as a result of tumorpromoter treatment (86). Less Cx protein results from fewer Cx mR-

NAs, which in turn produce fewer GJ insertions into membranes.Since the turnover time for connexins is relatively short (=2 h), the

net number of GJs in the plasma membrane decreases (145). The shortturnover time for Cx proteins suggests that long-term control (as

opposed to rapid gating and vesicular insertion/withdrawal processes)is exerted by Cx removal and resynthesis. This may explain why thereare various Cx proteins: to replace other Cx types and alter connexonfunction when, and for as long as, the need exists for a different GJmode. More work on this point is needed.

Investigations on some liver in vitro epithelial cell lines show thatliver Cx32 (made in vivo) is not synthesi/ed but rather Cx43 ( 146).

Cx43 is the principal connexin protein of the heart (where it was firstobserved), which is an electrically active tissue that has extensiveGJIC. Why cells //; vitn> allow dereprcssion of one member in the Cximiltigene family while allowing suppression of another member isnot known, but environmental conditions have been implicated in theliver phcnotypic switching between (Cx32, Cx26| <=>|Cx43| (146,147). TPA reduces GJIC in 1AR 20 liver epithelial cells (as TPA doesin many other tissues and cell lines), but Cx43 mRNA does notdecrease ( 146). Thus if the Cx43 mediates IAR 20 GJIC. the reductionseems to be caused by posttranslational Cx alterations (146). Therelative importance of Cx mRNA transcription and translation versusCx protein posttranslational transport through the ER and Golgi to theperimembrane pool and then membrane processing into assembledconnexons is not yet known.

CAMs [and other ECM molecules (7)| can play a role in controllingGJIC (3) and have been linked with invusiveness in the progressionphase of cancer (5, 7). Progressive loss of GJs and loss of E-cadherinhas been observed to occur in advancing metastatic disease (5-7).

Furthermore. CAM genes can act as tumor suppressor genes (148.149). Cadherins. proteoglycans, and glycosaminoglycans can up-reg-

ulate GJIC in a tumor cell line having low levels of GJIC (26, 38).Interestingly, one of the deleted genes on chromosome 18q in humancolorcctul cancer (this deletion found in ==70% of cases) has a se

quence similar to certain neural adhesion sequences ( 150). This indicates that deletion of CAMs by mutation may lead to a lack of propercell-to-cell contacts, including GJs (1.2, 4).

Summary

Gap Junctions, Toxicity, and Cancer. In this review on gap junction function and its relevance to cancer we have considered thefollowing: (Â«)gap junctions play an essential role in the integratedregulation of growth, differentiation, and function of multicellularorganisms: (/?) perturbation of gap junction function, whether direct orindirect, can result in GJIC changes that may provide the beginning oftoxicity because the synergism. and in some cases the synchronism, ofa field of cells is adversely affected. Persistence of GJIC abrogationultimately can cause irreversible damages to the integrity of a tissue.This irreversibility can bring on the latter stages of various diseasestates, but the etiology of any disease process is yet to be elucidated:(c) tumor promoters may inhibit cell-to-cell communication by (Â«)

plasma membrane toxic effects (ion displacement. H:O imbibition,critical lipid relocation, etc.). which results in the loss of cell-to-cell

contact, (b) specific binding of the GJ assembly, thereby alteringGJIC, (c) down-regulation of Cx synthesis and connexon assembly, or

(d) change in phosphorylation connexins. All of these could contribute to tumor promotion by promoting local cell isolation. This wouldbe a stochastically critical microenvironmental defect if the field ofcells includes particular initiated (1+ ) cells; (d) while unequivocalevidence for a causal relationship between modulation of gap junctionfunction and cancer etiology is not yet available, there is increasingevidence that modulation of gap junction function is often associatedwith the mechanism of action of many carcinogens and tumor promoters (2). Tumor progression also may be propagated by faultyGJIC.

Conclusion and Future Directions. Considered in into, short- andlong-term regulatory mechanisms allow gap junction channels to ex

hibit enormous plasticity in response to intracellular and extracellularsignals. The range of permeant signaling molecules allows gap junctions to serve as major conduits for (a) ions setting the basic groundstate of a cell and signals and (h) the exchange of hydrophilic secondmessenger molecules, metabolites, and substances that might serve aseither triggers or brakes (suppressors) for various cellular processes.Tools are now available for pursuing the nature of the regulation of

3482



CAP JUNCTION FUNCTION AND CANCER

channel conductance in the context of short-term gating and long-term

expression. At the level of the gap junction channel itself, alterationsin structure of the connexins and the use of exogenous expressionsystems should allow identification of the molecular domains responsible for sensitivities to various gating stimuli (151-154). The neces

sary probes are also now available and should lead to elucidation ofother levels of regulation (including precursor synthesis, membranetrafficking and GJ insertion/degradation). These approaches shouldalso illuminate the relationships among various types of membranechannels. Cells use both gap junctions and cytoplasmic bridges asconduits for exchange of ions, metabolites, and signaling molecules.The investigation of the gap junction channel may serve as a key toidentifying one class of intercellular channels and distinguishing themfrom other cellular transport channels. Further understanding of themolecular and cellular basis of GJIC will allow insights into the rolesthat tumor promoters play in perturbing the tissue signaling networksduring the course of toxicity and disease progression.

Problems exist in considering assays of GJIC inhibition for purposes of evaluating chemical hazard. Evidence to date suggests that noparticular in vitro assay, based on a given species or cell type, canserve as a universal predictor of a potential modulator of GJIC. Additionally, in vitro conditions may not simulate in vivo delivery oftested chemicals to solid tissues insofar as the native extracellularmatrix scaffolding is missing. Furthermore, interactions of chemicalsand chemical mixtures, in terms of modulating GJIC, can either antagonize or synergize, as well as be additive. Because multiple GJproteins exist, often in different cells in the same organ, and severalmechanisms regulate GJIC. predicting the effect of a chemical basedon results with one cell type in vitro on another cell type or on thesame cell type in vivo is not yet possible. Establishing such correlations (including extrapolations to humans) is important for evaluatingteratogenic, reproductive, and carcinogenic risks of environmentalchemicals that disrupt GJ.

Acknowledgments

The authors wish to thank the meeting contributors and William Farland,United States Environmental Protection Agency, and the National Institute ofEnvironmental Health Sciences for assistance in sponsoring the meeting. Weespecially thank James Trosko for his early encouragement, Elliot Hertzbergfor his cooperation and for reading the manuscript, and David Spray and MarkNeveu for helpful discussions.

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1993;53:3475-3485. Cancer Res James W. Holder, Eugene Elmore and J. Carl Barrett Gap Junction Function and Cancer

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Gap Junction Function and Cancer...munication; GJ. gap junction: connexon, one-half of the assembled dodecylhedral pore contributed by a cell: channel, low-resistance pore allowing