(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
Gap junctions
(GJs) provide cell-to-cell
communication
of essential
metabolites and ions. GJs allow tissues to average responses, clear waste
products, and minimize the effects of xenobiotics by dilution and allowing
steady-state catabolism. Many chemicals can adversely affect the mem
brane GJ assembly causing reversible alterations in GJ intercellular com
munication. During toxicity essential metabolites, ions, and regulators are
not shared homeostatically throughout a tissue community. Alterations in
metabolic circuits are thought to interrupt organ integration. Persistent
GJ perturbation can cause chronic effects (e.g., cancer), and many tumor
promoters inhibit I..] intercellular communication. Liver precancerous
foci intracommunicate (but at a reduced level) and intercommunicate
improperly (or not at all) across the foci boundary to normal cells. In time,
foci can become less regulated and more isolated within the tissue. GJs
remain reduced quantitatively in the tumor progression stage and may be
qualitatively altered in metastasis since connections are made between the
primary tumor cells and foreign host cells at the secondary metastatic site.
Cell sorting and binding mechanisms by the cell adhesion molecules and
integrins may also be altered at secondary sites. This may allow the
relocation of primary tumor cells and nurturance via GJs at the secondary
Introduction
Scope. GJIC1 is widely believed to play a key role in tissue de
velopment and maintenance in multicellular organisms by homeostatic control of cooperative, interacting cells within tissues and or
gans. In recent years, data have suggested that toxicants can modulate
normal GJ functions in growth control, development, and differenti
ation. Such modulation is thought to be involved in various disease
states, but the specific role remains to be delineated. Mounting evi
dence, however, indicates that modulation of GJIC may be involved at
certain stages in the etiology of chemical carcinogenesis (1).
To evaluate the relevance of GJ mechanisms to disease, the United
States Environmental Protection Agency, with supporting contribu
tions from the National Institute of Environmental Health Sciences
and NSI Technology Services, Inc., sponsored a conference on Mech
anisms of Gap Junction Function and Relevance to Disease; the con
ference 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 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
or policies of the United States Environmental Protection Agency or the National Institute
of 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 and
Development (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 pore
contributed 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€¢Conference
held in Washington, DC, February 27-28. 1990. Presentors included:
relevance of normal GJ function and modulation in disease was re
viewed, the discussion often centered on the relevance to cancer
because of GJ involvement in the tumor promotion stage (1, 2). The
following discussion on GJs is topical and not comprehensive. It is
based in part on the presentations and discussions held at the Envi
ronmental Protection Agency meeting and in part on the organizers'
views of current GJ research since that meeting and the future direc
tions for mechanistic research and applications to the relevance of GJ
function to disease.
Overview. In this review, we have organized the material to
present the structural characteristics of GJs, which include morpho
logical and molecular aspects, the roles of GJs in cellular function and
maintenance, and how GJs are involved in cancer. Finally, we discuss
GJIC controls and xenobiotic alterations of those controls.
Role of Gap Junctions in Intercellular Communication
Gap Junction Definition. GJs are specialized intercellular chan
nels between apposing plasma membranes that can be open or closed
(gated). When open, these GJ channels allow metabolite exchange
between cells. Each GJ channel is composed of two connexons; one
contributed from each communicating cell. Each connexon is com
posed of six Cxs that act in concert in order to form a pore in the
center of the assembly. Connexons float laterally in the plasma mem
brane 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 are
conjoined at that locus so as to form a channel between the two cells
(Fig. 1). Conjoined cells are metabolic systems partially open to each
other with GJs providing channels of cell-to-cell communication.
Spatial patterns of interconnected GJs in a tissue form patterns of
communication in a tissue. These channels convey tissue metabolites
control chemicals, critical ions, and xenobiotic chemicals, but only
hydrophilic molecules with molecular weights of < 1000 are shared by
passive diffusion through the GJ conduits. Whether all such cellular
molecules are shared equally among connected cells is still uncertain,
but passive diffusion is assumed with the presumption of ionic and
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
plaques 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 Med
icine, Indianapolis, IN), R. Loch-Caruso (School of Public Health. University of Michi
gan. 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, East
Lansing, 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 of
Health and Environmental Assessment. United States Environmental Protection Agency,
Washington. DC), and J. W. Holder (Genetic Toxicology Assessment Branch. Office of
Health and Environmental Assessment. Office of Research and Development. United
States Environmental Protection Agency.
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GAP JUNCTION
INTERCELLULAR
FUNCTION
LVI/EM
LIPID BILAYER
HEMICONNEXON
CALCIUM IONS
NUTRIENTS
METABOLITES
BICARBONATE
BUFFERINQ
COMPOUNDS
_J
'•
/
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 GJ
pores is assumed to take place in mass transfer. Transferring molecules must he water
soluble with molecular weights <100(). Cytoplasmic bridges and active ion pumps also
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
open or close by a rapid (ms) and reversible process called gating in which a portion of
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
permission).
duits between cells for bulk exchange of small molecular weight ions
and molecules such as H2O, Ca~ '. and cAMP. The number of plaques
with adjacent cells is thought to be proportional to the metabolic
cooperation among these cells. Thus, fewer plaques indicate less
communication, which in turn suggests that the cells may act more
independently of each other. However, modes of intercellular com
munication other than GJs, such as paracrine functions and other ion
channels, continue to functionally couple cells, even when plaques are
few. The relative importance among the different modes of intercel
lular communication is not known.
CAMs can facilitate local GJIC in epidermal cells (3). CAMs can
also act as cell morphogenetic regulators conferring specific cell-tocell adhesions and cell sorting mechanisms (4). CAMs likely assist
locus-specific cell-to-cell docking processes in epidermal cells, pos
sibly by drawing and holding the adjoining membranes together in a
Ca2+-dependent process. This permits close intermemhrane apposi
tion, thereby facilitating cell-to-cell interactions that include connexon-to-connexon (GJ) interactions (3). Progressive loss of GJs (and
thus GJIC) and loss of E-cadherin (a type of CAM) have been ob
served to occur in advancing metastatic disease, which suggests the
importance of functioning GJs and CAMs to proper cell control (5-7).
The universal occurrence of gap junctions in multicellular organ
isms (metazoa) indicates that gap junctions play a ubiquitous, essen
tial role in collective cell interactions (8). It is through these interac
tions that cellular physiological balances and controls are cumulated
into regional tissue function in the corpus of multicellular organisms.
Comparative structural and functional differences in GJs among var
ious members of metazoa are not well understood.
Gap Junction Function. One of the most fundamental theories of
modern 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 organisms
within themselves which are separate from, but related to. the organs
in which they reside. The whole organism is constituted from collec
tions of cells and organs that are ordered within the organism accord
ing to definite laws. These early works and the pathology studies of
AND CANCER
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 the
accepted cell theory: the cell is a unit, but a reproductive and bio
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
bilities locally and allows the integration of regional tissues into organ
function. The syncytium may be a higher and more relevant physio
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
regional tissue-specific functions (11). These regional adaptations,
with specific interactive communication occurring, allow for integra
tion and phcnotypic expression of the cell collective in organ function.
The corpus is the next higher level of organization: the vasculature
and nervous systems provide corporeal integration. Corporeal integra
tion physiologically unites and relates the various organs, tissues
within 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 xenobiotic
chemicals. This buffering is achieved by dispersing xenobiotic mol
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
be catabolized in more cells and in a steady-state fashion. Another
GJ-mediated function is: (/)) nourishing of sick or deprived cells by
healthy neighboring cells (reciprocity). This concept is basic to tissue
homeostasis. The inuring process offers plasticity to cells during in
jurious processes and responses. As long as GJIC is not compromised
and the toxicity limit is not exceeded, the cell is remediated with
assistance from healthy neighboring cells. Hence, a tissue can reversibly recover or remodel following a toxic insult. However, it is thought
that if toxic limits are exceeded then cells slow or stop communicating
(GJIC i ). Disorganization can then occur which can be followed by
necrosis and cellular turnover. Another regional function mediated by
GJs is: (c) rapid exchange of critical ionic electrical signals (<'.#.,
Ca2^ ions) and their regulators. For instance, when a cellular mem
brane is sufficiently disturbed, waves of Ca2 ' ions can move from the
disturbance point in a field of cells through cells to neighboring cells
in order to signal the disturbance ( 12). This wave action is hypothe
sized to happen so that regional tissue reaction can occur in a coor
dinated manner. GJs also participate in regional function by: (r/)
allowing the distributions of critical metabolites (<'.,(;..cAMP) among
tissue cells. Cells benefit from GJ-mediated sharing of essential me
tabolites because all of the cells in a syncytium do not possess the
same metabolic capacity (13). Cells of lesser capacity benefit from
those with ample capacity. If some cell fields are under stress and
some are not. GJ sharing is beneficial to the tissue as a w hole. Lastly,
another regional GJ function is: (?) the elimination of unwanted
byproducts. Elimination is facilitated by GJs in a gradient fashion
from tissue interior cells to the vascular system for purposes of ex
cretion.
Gap Junction Proteins and Membrane Structure. Isolation of
GJs is facilitated by their relative resistance to alkali and detergent
solubilization. Development of antibodies against different purified
Cx proteins has made possible topologica! mapping of GJs. The most
detailed biochemical characterization is from Cx purified from liver.
Lipid analyses of GJ isolates have indicated an enrichment of choles
terol, depletion of sphingomyelin. and significant levels of phosphatidylinositol and arachidonic acid ( 14). The role, if any. of these lipids
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GAP JUNCTION
Kl'NCTION
is noi clear. It appears that GJ proteins from liver, as well as from
heart, can he fatty acylated but not glycosylatecl. Palmitic or myristic
additions may assist posttranslational transport of the connexins
through the Golgi cisternae (15).
GJs isolated from heart consist of a single protein with an apparent
molecular 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 ( = lO—LWi
) ( 15). Isolated GJ from lens
fiber cells consist of MP26 and MP70 ( 15). MP26 is unrelated to heart
and liver proteins. While detailed information has not been presented,
it appears that MP70 is related to liver and heart GJ Cxs. Some
relatively large size differences do exist in the variable cytoplasmic
regions among connexin proteins, such as Cx.32 and Cx43. These
different sequences form different Cx types (15, 16). Each type is
thought to basically act the same in GJIC no matter in which tissue the
type occurs but fine differences of GJ control and function may exist
(17). More new sequences for additional Cx have been found by
amplification using ihe polymerase chain reaction and low stringency
hybridi/.ation; new Cx proteins in rat liver have been predicted: Cx40,
Cx37. C.\33, and Cx3l.l (18). These new sequences exhibit the typ
ical sequence pattern of the GJ protein family, i.e.. highly conserved
transmembrane and less conserved extracellular domains, but variable
cytoplasmic domains (tails).
Cx mRNA seem to be made from only one exon ( 19). This is unlike
most cellular proteins found to date that are made from mRNAs.
which are assembled from heterogeneous RNAs containing dispersed
intron sequences among exon sequences. The rat Cx32 and human
Cx43 heterogeneous RNAs each have uninterrupted Cx reading
frames (no introns). and each exon has a 5'-nontranslated region (20,
found that expression of pp60%src in Xenopus oocytes blocks GJIC of
Cx43. but not Cx32. This GJ inhibition is caused by tyrosine residue
265 phosphorylation in Cx43, whereas Cx32 is not similarly phos
phorylated. Interestingly, the pp60%"slx'-promoted phosphorylation of
Cx43 does not inhibit the Cx assembly into GJs, only their function in
communication once in the membrane (32). Site-directed mutants
with shortened tail regions (where Cx phosphorylation occurs) show
no altered voilage sensilivity but do show altered unitary conductance
differences when compared to unmutated Cx (33). However, the tailmodified sites do not seem to interfere with insertion of mutated Cx
into membrane-associated connexons (33). This conclusion was con
firmed by Musil and Goodenough who showed that unlabeled GJs
occur intracellularly. whereas '2PO4 labeling occurred only in the
membrane. Phosphorylation of GJs occurs after membrane insertion
of Cx which leads to Cx deposition by insolubility and ability to
associate into plaques (34). This and the above studies suggest thai
phosphorylation 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 from
X-ray diffraction studies (36). The emerging picture is one in which
each 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 be
identical, organized in a circular rosette array and aligned en face
across an intercellular space. This space is the gap of GJ seen in
electron microscopy. The schematic structure of GJs in cells is shown
in Fig. 1. The Cx proteins are thought to be the only proteins in GJ.
Each Cx protein traverses the bilayer four times, which mnemonically
configures the letter "M" in cross-section. The Cx sequences that
traverse the membrane are highly conserved among species ( 16). This
conservation pattern is consistent with most channel proteins. The Cx
cytosol sequences (visualized as two bottom legs of an "M." corre
21 ). The human chromosomal locations of Cx genes are not clustered
on a particular chromosome, such as the globin genes, but rather occur
on different chromosomes ( 18. 22). A number of Cx DNA sequences
occur in humans: Cx26. Cx32. Cx43, and Cx46 on chromosomes 13,
X. 6. and 13. respectively (22). Similar coding sequences exist in the
mouse and rat. Another mouse connexin protein. Cx37. has been
reported (23). The Cx genes isolated thus far as complementary DNA
hybridize as distinct mRNA species; therefore connexin proteins make
up a multigene family (24). Such a structural diversity of related Cx
genes suggests different connexon functions, e.g., during differentia
tion (24). Genetic drift and reorganization should also be considered
since Cxs are thought to constitute a set of evolutionarily old proteins.
Cxs have been found to occur and function in mesozoa. which are
transition marine parasites biologically organized between unicellular
(protozoa) and multicellular (metazoa) organisms (8. 16). There seems
to be no exception to the hypothesis that all metazoa utilize GJ
channels. These conserved Cxs apparently provide an essential set of
functional cellular proteins in higher organisms.
Besides the cytoplasmic tail Cx sequence differences, chemical
modification of the Cx protein also can take place. For example, Cx
phosphorylation can be stimulated by cAMP. which acts as a cofactor
for kinase A phosphorylation. and such phosphorylations can produce
increases in GJIC (25. 26). Tumor-promoting agents (e.g., TPA) down
regulate PKC while also down-regulating GJIC (27, 28). Presumably
the Cx is structurally altered when the Cx is phosphorylated. which is
likely 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 in
short times (min); this process is considered part of the fast GJ control
responses (29). Since Cx are proteins that have t,,, of only a few h,
coordination of phosphate attachment and removal is essential to Cx
function (30). Proteins other than Cx can be phosphorylated and yet
affect GJIC; e.g.. in rat liver phosphorylation of src tyrosine residue
527 has been related to GJIC inhibition (31). Swenson et al. (32) have
AND CANCER
sponding to the NH2 and COOH termini, and an intervening middle
loop) vary. These variable regions suggest that different types of
connexons may be due to Cx heterododecylhedral mixes within con
nexons in GJ, but these mixes have yet to be demonstrated. These
mixes, if present, could present fine structural differences in assem
bled GJ channels.
Any agent that alters the connexin protein assembly, other connexon-associated componenis. or local membrane morphology can po
tentially affect GJ function. Proximal desmosomes seem to strengthen
local cell connections. It now appears in the cell systems tested thus
far that neither GJs nor the adhesion molecule E-cadherin function
well in low Ca2 ' . It has been suggested thai E-cadherin and GJs need
adequate levels of Ca2 ' to function optimally (3, 4). What Ca2 ' ion
levels are adequate likely depends on the cell type, stage of develop
ment, or disease state. CAMs like uvomorulin, which is identical to
E-cadherin and homologous to liver-CAM, may facilitate GJ docking
and subsequent stabilization of cell-to-cell interactions (3, 6). The
integrins also may play a role in specific cell loci matching up and
allowing GJ formation between specific compartments of the two
conjoined cells (37). Other extracellular elements such as glycosaminoglycans and proleoglycans have been observed to proffer slabilizing
influences and affect increased GJs (1. 4. 6. 38). The explicit rela
tionship 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 of
connexons could be a passive process; i.e.. the cell-to-cell pore struc
ture is thermodynamically unstable at 37°Cand low Ca2 '. This con
nexon separalion could also be an active process, due to actin atlachment to ihe plasma membrane microvilli proximal to the connexons
(40). Notably, cytochalasin B. vincristine. colchicine. vinblastine, or
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GAP JUNCTION
FUNCTION
low temperatures (0°C),all of which disrupt cytoskeletal structures,
each can increase GJ plaque areas (40). The emerging concept from
this work suggests that the actin cytoskeletal system regulates GJIC by
first polarizing the fibrillar apparatus toward the outer cell face and
modulating concentration and polarization of microvilli at the cell
surface (Ref. 40) and references therein). By this mechanism, the
number and positioning of GJ plaques are coordinated by cell orga
nization (cellular polarity), which contributes first to the anisotropic
organization within the cell and secondly to the collocation of the cell
within the tissue (2, 40).
Some Gap Junction Examples in Cellular Communication.
Electrical coupling occurs among communicating cells due to ion flow
measured in conductance units of picosiemens (pS). Cellular coupling
establishes a ground state potential, along with Na+, K +, Ca2+, and
H+/~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 for
propagation of depolarization in myocardial and smooth muscle tis
sues and is thought to provide a means for coordinated contraction
(43, 44). For example, at parturition, a rapid, incipient increase in GJ
Cx protein in myometrial smooth muscle enhances syncytial activity
and coordinated contraction necessary for fetal expulsion in parturi
tion (2, 45). The brain has many GJs, but little is understood about
how 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 of
electronically coupled neuronal pools (16). Neural activity in glial
cells causes focal K+ transient loads that are dissipated in a recovery
process mediated by GJ (16). Retinal horizontal cell conductance in
the eye can be modulated via GJs that transmit dopamine, which in
turn affects retinal cAMP-dependent protein kinase (49).
Another example of the integration of tissue activity and metabolic
function is the coordination of cells in a hormone-responsive tissue
( 16, 50). Coupling via GJs plays a role in secretory systems, and it has
been demonstrated that secondary messengers (of primary hormone
stimulation) such as cAMP, cGMP, Ca2 +. and IP, pass through GJ
channels (51 ). It is likely that many other critical molecules are shared
via GJs. Insulin production in pancreatic islet cells correlates with
prior secondary messenger activity (cAMP). bursts of pancreatic Cx43
mRNA activity and Golgi and endosomal vesicular exportation of the
Cx43 into islet cell membranes (52). Such a sequence of events
suggests that GJs are made and assembled in order to assist insulin
synthesis and release from pancreatic cells. The mechanism by which
this is achieved remains obscure. The examples given above suggest
that the initial sharing among cells of effector molecules via GJs can
lead to integrated tissue responses (15).
Another interesting case where cells share effector molecules via
GJs is in the ovaries. Oocytes are maintained in meiotic arrest by
granulosa cells to which they are coupled by GJ via filopodial pro
cesses projecting through the zona pellucida (53). These GJs mediate
the intercellular flow of the ovulatory meiosis inhibitor between the
membrana granulosa and cumulus oophorus cells with stasis of oocyte
meiotic cell division as a result (54). An ovulatory stimulus, provided
by luteinizing hormone, leads to disruption of GJIC in which inhib
iting granulosa cells can no longer transport ovulatory meiosis to the
arrested oocyte cells (55). Accordingly, the oocytes are no longer
arrested and ovulation ensues.
GJs play an essential role in embryonal cells during development
and differentiation (56. 57). Certain teratogens seem to act by altering
GJIC (57). A potential role for GJs exists in providing a mechanism
for transmitting timed-growth and positional signals involved in de
AND CANCER
42). Coordinated GJ appearance prior to critical events in differenti
ation is consistent with a role for GJs in signaling and maintenance or
disruption of morphogen transmission ( 15). Vitamin A analogues and
glucocorticoids, both known to be involved with cellular differentia
tion, are linked to the increase in the number of GJs (60). Selective
disruption of GJIC either by introducing Cx antibodies or by injecting
Cx antisense mRNA has led to developmental defects in both inver
tebrates and vertebrates, thereby implicating GJ in these processes
(16, 61). Correlations are now being made between genes that are
central to development, e.g., pattern formation, and those that stimu
late cancer (oncogenes) (62-64). Changes in development involving
GJs have been correlated with loss of cell growth control, which can
lead to cancer (56. 65. 66). The relationship between cancer and GJIC
is discussed below.
Modulation of Gap Junction Function
Temporal Modulation of Cap Junctions
Changes in GJs are essential for allowing, or attenuating, the in
tercommunication in a field of cells. Three kinetic courses of GJ
control are beginning to emerge: (a) fast control by gating responses
(=ms); (b) intermediate control by vesicular insertion or withdrawal
of 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. Compara
tive differences among species of these times are not known.
Fast Control. The transport of electrolytes or small low molecular
weight substances from cell to cell depends on the number of GJs
connecting these cells that are open at any given time, the specific
conductance of the type of shared connexons. and the tissue environ
ment. Different structural models have been proposed to account for
the gating (quick closure) of GJs. In one model it is suggested that
bulky, hydrophobic phenylalanines are introduced into the aqueous
channel 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 this
process by M3 a-helix rotation in the opposite direction reintroduces
small hydrophilic amino acids into the channel, which clears the way
and reopens the channel to aqueous transport (pS ÃŽ
) (67). It is also
possible that if the subunit rotation were progressively twisted in a
cooperative manner along the pore axis, a gating from open to closed
states would occur without resolvable intermediates, much like the
aperture of a camera lens. The kinetics of gating, which in part
involves M3 rotation, has been estimated to be =ms (68). The energy
difference 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. Since
the average energy of kinetic motion ^=0.6 kcal/mol at 37°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 tempera
ture and can accommodate rapid ion signal (voltage) changes.
Some agents effect almost immediate GJ channel closure ( 16). For
example, immediate effects can result from rapid changes in intracellular pH. ion transients, protein kinases, and even changes in GJ
membrane lipid composition (14, 70). Examples are acidic pH and
PKC and protein kinase A activators, inhibitors of cAMP. Ca2 ' spike-
inducing agents, and high lipophilic agents (such as unsaturated fatty
acids). 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 Cxantibody-reactive material exists in vesicles proximate to the plasma
velopment (58, 59). Several observations based on ultrastructural
membrane. It has been observed that Cx can be inserted into the
analyses indicate that GJs do appear or disappear at various times (16,
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plasma membrane even in the presence of protein synthesis inhibitors
(71 ). This suggests that a peri membrane pool of preexisting connexins
exists and may buffer the Cx supply (16. 40, 72, 73). It is not known
if the intermediate pool is under physiological control. The presence
of the intermediate pool underscores the essentiality of Cx to cellular
function.
It is known that Cx are not phosphorylated in the pool. Evidence
exists to suggest that Cx43 is phosphorylated after being inserted into
the plasma membrane (34). This phosphorylation seems to accompany
Triton X-100 insolubility of Cx43 (34). Phosphorylation does not
determine cellular stability of Cx43 but has been correlated with Cx43
accumulation into plaques (34). Such a mechanism suggests phos
phorylation control of mass transport.
Long-Term Control. Evidence for longer term control of GJIC is
still emerging (16). In liver cultures, the apparent half-life for Cx32
and 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 levels
of GJs by Cx mRNA synthesis rates and stability. In primary hepatocyte culture, GJs disappear 10 h after plating but reappear within 72
h; this disappearance and appearance also occur after partial hepatectomy (16). These recoveries are preceded by Cx mRNA and are
accelerated by cAMP (75).
Cyclic nucleotides affect GJ in most tissues and cell lines that have
been studied to date, and these agents are the likely candidates for the
major set point of physiological GJ regulation. cAMP analogues can
cause either increases in GJs, e.g., liver cells, or, in some cases,
decreases in GJs (76-78). Inhibitor experiments suggest that these GJ
changes are attributable to effects mediated through cAMP-dependent
protein kinase A, which can phosphorylate Cx (as well as regulatory
proteins) and thereby regulate GJs. For example, liver Cx32 can be
phosphorylated at a serine residue on its cytoplasmic tail, with kinetics
similar 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 GJIC
between sympathetic neurons, which is blocked by inhibitors of
mRNA and protein synthesis (16). In hepatocytes, cAMP extends the
lifetime of GJ between dissociated cell groups, which is attributed
largely toan increase in stability of Cx32 mRNA (16). Transcriptional
regulation is expected as well, based on the cAMP response element
sequence in the genomic clone of Cx32. As discussed previously,
components of the intercellular and extracellular matrices influence
GJs and seem to provide stability to these structures and enhance
GJIC. For example, in hepatocytes, there is an interaction of matrix
components and glucagon: treatment of cultured cells with glucagon
together with proteoglycans leads to a marked increase in Cx32 tran
scription as well as increased stability of Cx mRNA (38). Downregulation of GJIC has been reported in uterine smooth muscle cells
treated with cAMP derivatives (2).
Tumor promoters such as TPA stimulate the signal transduction
mediated by PKC by mimicking the normal substrate of PKC, diacylglycerol (80). PKC exists in multiforms (in as many as six genes) that
change in relative gene expression upon TPA binding to PKC or
transformation (81, 82). Like many tumor promoters, TPA inhibits
GJIC (27, 28) and may do so by altering one of the PKC isoforms with
respect to GJ phosphorylation (83-85). In several cell lines, active
phorbol esters inhibit GJIC in approximately 10-20 min, a time course
similar to that of PKC activation. Chronic treatment of the tumor
promoter phénobarbitalcan lead to long-term cellular changes, e.g., a
decreased occurrence of GJs in altered hepatic foci (86). Such GJ
decreases have been observed to be the result of a lessened synthesis
of Cx32 in liver and are possibly linked to the preneoplastic nature of
the AHF (86).
AND CANCER
Toxicant Modulation of Gap Junctions
Involvement of GJs in the toxic response of cells to chemicals has
been an overlooked area and there are few systematic studies on this
topic (2, 87). GJ perturbations may be involved in the toxicity caused
by many chemicals, but the precise mechanisms are not yet known
and can only be inferred in certain cases.
Generally it is known that when a cell is damaged, the membrane
can become leaky to ions such as H+, Ca2+, and K+. If the damaged
cell remains coupled to contiguous undamaged cells, essential metab
olites are lost at the damaged cell site. GJs are thought to attenuate
during these periods of damage for self-correction by the cell (88).
The mechanism of attenuation is not known but is expected to be
primitive and an intrinsic aspect of the Cx molecular engine and
membrane assembly. The mechanism of closure may also respond to
paracrine control. pH. and critical ion balances. Closing agents would
induce 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 the
cells are repaired and survive, then they can later recouple using GJ
channels to become once again functional participants in the syncytium in which they reside. Such regional attenuation of GJIC with
remedial recruitment could also be under tissue physiological control
about which we can only speculate at this time. Toxicity-induced
closure is reminiscent of GJIC reduction (not ablation) during mitosis
when cells withdraw to the internal program of cell duplication and
division (88, 89). However, these mitotic cells remain electrically
coupled to some extent to surrounding ¡nterphasecells (90). In agree
ment with this is the observation that regenerating tissue shows first a
loss 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 /¡-heptanol or /¡-octanol)may reversibly close GJs by physically reacting with the GJ plasma mem
brane assembly and abolishing cell-to-cell coupling (44, 94, 95). Ca2 +
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
been thought that inhalation of anesthetics may act reversibly by
inhibiting GJIC (97). Many anesthetics are small lipid molecules with
high fugacity and are accessible to many tissue compartments. These
properties could cause reversible membrane perturbations in various
tissues including neural GJs thereby causing reversible sensory inter
ruption. Moreover, chronic ethanol consumption (a C2 alcohol com
pared to C7 and Cx alcohols above) may persistently perturb GJs by
excessive membrane solvency and perturbant effects along with ex
cessive acetaldehyde generated upon ethanol metabolism. The persis
tent influence of perturbants on GJs can be thought to lead to persis
tent dyscommunication in the affected field of cells, which can then
lead to either (a) facultative accommodation by the cells or (¿)disease
progression. In the second pathway, chronic disease follows when
deleterious acute effects become irreversible, which can occur from
repetitive acute or subchronic insults (2).
In Vitro Assays for Gap Junction Toxicity
Data on GJIC following toxic injury have been obtained from
various types of assays for GJIC: (a) cooperative sharing of a metab
olite; (b) fluorescent dye transfer; and (c) electrical coupling. Toxicological data (=200 agents) has been obtained in vitro in V79 met
abolic cooperation assays (98). One problem encountered in this in
vitro work is that there is a relatively low potential for V79 cells to
metabolically catabolize chemicals, a transformation that may be nec
essary for some induced toxicity. Moreover chemicals that are tumor
promoters are usually organ specific and would necessarily require the
appropriate cell culture to test GJIC effects. Another shortcoming is
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the lack of ECM effects manifest in solid tissues. Nonetheless, in vitro
V79 data have provided some insights into the direct pharmacological
actions of various types of chemical agents on GJIC and provide
useful comparisons of structure-activity relationships. Whether or not
these reactions on GJs occur in vivo has to be assessed on a chemicalby-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 co
operation, eight of these (89%) were correctly identified for their
carcinogenic potential based on their ability to inhibit GJIC (98). Not
all known animal carcinogens tested in V79 cells inhibited metabolic
cooperation. Given the lack of metabolic activation, however, it is
intriguing that up to 75% of the chemicals tested with known carci
nogenic activity produced some inhibition in the V79 metabolic co
operation assay (98).
The activity of alcohols and lipophilic agents in the V79 assay
appears to be directly related to their octanol/water partition coeffi
cients (98). Fatty acids demonstrate activity as well. Strong mutagens
such as diethylnitrosamine and ethylnitrosourea also inhibit metabolic
cooperation. Certain toxicant classes, such as pesticides including
aldrin, mirex, kepone. DDT. DDE, heptachlor, methoxychlor, and
lindane, inhibit metabolic cooperation in the in vitro V79 assay (98).
Thus, a wide variety of toxic, pharmacologically active agents have
activity in modulating GJ function in an in vitro assay. These effects
may 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 GJ
channel.
For agents associated with noncancerous disease end points, the
following correlations were found; 80% of all teratogens tested in
hibited metabolic cooperation, whereas 73% of all neurotoxins tested
were detected (98). While these V79 in vitro data cannot be considered
unbiased, due to the partial selection of agents for study based on their
known activity as pharmacologically active toxicants in one system or
another, the selectivity does imply the usefulness of GJIC inhibition
assays to detect chemicals associated with toxic responses.
Gap Junctions and Cancer
Experimental animal carcinogenesis studies as well as epidemiological studies of human carcinogenesis strongly indicate that the
process of carcinogenesis is multistaged and sequential (99, 100).
Some stages in the sequence are not always the same which means
that a number of different overall mechanisms can cause cancer (101,
102). This has made the explicit identification of the necessary and
sufficient components a complex matter of biochemical and logical
analysis. While mutations often play a role in the multistage nature of
carcinogenesis, nongenotoxic mechanisms also play key roles in car
cinogenesis (103-105). Examples of nongenotoxic mechanistic types
are changes in gene expression, cell proliferation, cell organization,
cell interrelationships (including GJIC), cell death (including apopto-
AND CANCER
first reports implicating GJIC inhibition by tumor promoters were
published in the same year (27, 28). Since then, objective correlation
has been made in a number of laboratories between the promotion and
progression stages of carcinogenesis and decreased GJIC (1.2, 106111). It is thought that chronic inhibition of GJIC in carcinogeninitiated 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 been
substantiated both I'Mvitro ( 1, 113) and/'»vivo (\\2, 114). The role of
GJIC disruption and chemical carcinogenesis has been reviewed pre
viously (115).
There is an inverse correlation of cell growth and GJIC in trans
formed 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 transforma
tion 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 tumor
promoters reversibly down-regulate GJIC /';; vitro and in vivo (1, 27,
28). GJ inhibition by
few tumor promoters
do not down-regulate
studied (1, 118-120).
tumor-promoting chemicals is not universal. A
studied thus far. e.g., okadaic acid and TCDD,
GJIC in the systems in which they have been
These observations suggest that some tumor
promotion mechanisms proceed without altering GJIC, which agrees
with the growing evidence that there are qualitatively different mech
anisms for tumor promotion (121). Different mechanisms could also
relate to different adaptive phenotypic defense mechanisms with
which cells respond to carcinogen exposure (102, 122).
While it is universally agreed that chemical mutagens react with
DNA and can cause lesions that lead to hereditary diseases as well as
contribute to somatic diseases (including cancer), all toxicants do not
act directly on DNA and all carcinogens do not necessarily react
directly with DNA (87. 105, 109). Toxicants working on "other than
DNA" are often referred to as epigenetic. A number of epigenetic
events can conjunctionally cause persistent toxicity. Among these
events, chemical modulation of GJIC may play an important role in
the areas of reproductive toxicology and teratogenesis, as well as
carcinogenesis (2, 87, 115, 123). By perturbing GJIC, chemical ex
posures can lead to a loss in tissue integration via distortion, or even
ablation, 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 of
cell growth are shared among normal communicating cells via GJs
(56, 66). Moreover, as the hypothesis goes, these shared repressors
keep cells in an orderly growth-arrest state, except those cells gener
ated to replace normal cell loss. Growth ensues when conditions cause
a diminution of sharing of these negative repressors (56. 124).
Ca2 + ion oscillation takes place within cells, which is not well
understood, but seems to be frequency-modulated signaling. Ca2 + ion
oscillation is an intracellular process which does not disturb neigh
boring cells. Ca2' ion oscillations has been related to: (a) enhance
ment of cellular signal recognition; (b) reloading of Ca~ + ion storage
sis), and cell removal. There are a plethora of potential interventions
that a xenobiotic chemical can bring about to alter the cellular phesites; and (c) directional ion transport (125. 126). In contradistinction,
a Ca2+ ion wave is a form of transmitted Ca2 ' communication orig
notypic expression of an exposed field of cells. Although affecting
inating from a cellular membrane trauma. The Ca2 f wave propagates
GJIC is only a subset of all potential interventions, adversely affecting
through
not around, cells (12). The hypothesis has been that the Ca2 +
GJIC may be a more common toxic event than heretofore thought
ion wave is moved from cell to cell by a "leader" substance which
(2, 87).
progressively causes Ca2+ ion pulse in each cell (126). This substance
Because cancer has been described as a stem cell disease, a disease
has proven to be IP, (12, 126). The IP, moves through cells, causes a
of differentiation, and a disease of cell regulation or growth control
and because GJIC has been linked in normal cells to control of Ca2+ pulse, and then diffuses onto neighboring cells via GJs. Hence
the wave of Ca2 + ions moves from the point of disturbance and is
development, differentiation, and growth, it might be supposed that
directed to neighboring cells which must be alerted. The cybernetics
there may be a relationship between cancer and GJIC. Loewenstein
of the Ca2+ wave signal is unknown. Chemical toxicant disruption
(56) predicted this relationship in a prescient 1979 review, while the
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may scramble the signal before it is translated. When wave signals are
interrupted, distal tissue cells would not be alerted and thus would not
interact cooperatively. Damage can ensue due to the lack of homeostatically controlled repair.
When hormones, secondary messengers, and essential metabolites
are not shared to their normal extent due to impaired GJIC. then
excess cell divisions can occur. It persistent, this condition can lead to
dysplasia. Trauma, for instance, can lead to cell loss and regenerative
hyperplasia. This loss of growth control is temporary because hyperplasia can regress in a reversible manner when the inducing stimuli are
removed. GJIC is thought to be important to remodeling but the
mechanism of this process is not clear (2. 127). If toxic stimulation is
persistent and 1+ cells are present, then 1+ cells can be clonally
expanded as can other cells in the field. Not all 1+ cells are equally
committed to cancer susceptibility. However, if a critical clonal size of
a deleterious 1+ cell population is ever reached, the growth-suppressive effects of surrounding normal cells on these particular 1+ cells
can be overwhelmed (1). 1+ cells can become disconnected from the
surrounding normal cells to form islands of dysregulation. Regulatory
controls are thus circumvented, at least partially, and the unrestricted
1+ cells continue to divide and eventually form genetically and phenotypical ly heterogenous preneoplastic foci (102, 122. 128-130).
Foci are preneoplastic and are only one set of cellular events oc
curring in the tumor promotion stage of cancer evolution (127, 128,
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
organs is not as clear. Many preneoplastic foci show GJIC among foci
cells (homologous communication) but fail to communicate properly
with normal cells surrounding these foci (heterologous communica
tion) (I, 110). This segregation of communication creates different
subcommunities which, in effect, can interrupt the integration of the
tissue. These foci are genetically variable, or at least evolve hetero
geneity, and they exhibit different phenotypic patterns of enzyme
deficiencies at the foci stage and beyond. The outcome of early and
continuing generation of heterogeneity is successive cell selection to
progressively higher levels of adaptation to the altered (transformed)
microenvironment ( 102, 111) that appears to include decreased GJIC
of foci to normal cells. Various posttranslation mechanisms lead to
increased cellular phenotypic variability. The phenotypic variability
occurs at a rate far exceeding the genetic mutation rates (111, 133).
The evolving phenotypic variability is an important component in
cellular responses to xenobiotics. One such cellular variability in
volves decreased GJIC among various cells as metastatic disease
progresses (5).
Many transformed cells have fewer GJs than their corresponding
normal tissue cells (83, 134). Since transformation takes place early in
neoplasia, GJIC reduction may be an early, fundamental lesion in the
origin of cancer. Species and strain differences to the liver tumorpromoting effects of various chemicals seem to correlate with their
ability to down-regulate GJIC in vivo in liver (135). As malignancy
progresses, a decrease in GJIC levels in mouse epidermal and liver
cells is observed (136. 137). Tumor metastasis seems to correlate with
decreases 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 than
those cells with weak potential. The decreased number of GJs found
in more cancerous cells suggests that GJs tend to be lost in cancer
development and accompany disturbances in tissue growth control
just as Loewenstein (56) predicted in 1979. The long-standing concept
in cancer technology, that early cancer cells somehow become differ
ent than their neighbors and are accordingly "outlaw" cells, could now
AND CANCER
cellular communication or even removal of focal cell communities
from normal local tissue communication patterns.
Although GJ expression may be altered by certain oncogenes and
tumor promoters in many highly metastatic cells. Kanno (65) reported
that tumor cells can remain coupled by GJs. at least to some extent.
Thus, a complete lack of coupling is not necessarily an obligatory
requirement tor conditions leading to loss of growth control. Rather,
a critical level of GJIC may have to exist in order to avoid cancer, but
such a level is not yet known. Furthermore, it may be that total GJIC
ablation causes premature toxic cell death and does not allow time for
cancer to evolve. Relative cell kinetics is important. For example, if
the rate of loss of 1+ cells in a tissue (through premature death) is
balanced by the rate of newly generated 1+ cells (through persistent
initiator activity), then the 1+ cell pool would be constant; but if the
loss of 1+ cells exceeds the 1+ cell supply, eventually the field will be
devoid of a significant amount of 1+ cells to cause cancer. In the case
where a persistent carcinogen produces more 1+ cells than are lost,
the chances of cancer increase as 1+ cells accrue. The risk of cancer
increases in the latter case. During the course of cancer evolution, the
processes of tumor promotion, conversion, and progression act on
these accruing 1+ cells and the disease develops. However, it is
necessary to minimally nurture these advancing 1+ cells via some
GJIC in order to from a tumor.
Cancer is likely the stochastic result of the evolution of 1+ cells and
the surrounding focal cells which is a collective of altered (trans
formed) cells. Foci are usually identified histologically by an enzyme
alteration (usually a deletion); it should be remembered that these
focal cells also differ in other phenotypic traits. Foci are heteroge
neous (genetic and phenotypic) populations. Thus, if certain combi
nations occur within foci (transformants + I+ cells) and certain en
vironmental conditions occur surrounding these foci (all which
interact at the micro level), 1+ cells can ultimately emerge to be
malignant. Carcinogenicity in fact begins when these combinatorial
biological interactions become irreversible; i.e., the adaptations can
not readapt. Since focal cells may be selective in communication with
themselves and at a reduced GJIC rate compared with surrounding
normal cells (137). the expectation is that focal cells have a different
qualitative GJ apparatus. This prediction should be investigated. Re
striction of communication to surrounding tissue cells dissociates
these focal cells from the normal flow of tissue growth regulators. A
recent suggestion is that reduced GJIC in carcinogenesis may be
primarily due to reduced homologous communication and less due to
reduced heterologous communication ( 137). Thus, whether it is func
tionally most significant that focal cells do not either properly ¡ntracommunicate or intercommunicate with surrounding normal cells, or
both, remains a mystery. More work on the patterns of chemically
induced GJIC inhibition should prove fruitful.
Overall tumor growth is influenced by organ size constraints (sim
ilar to normal organs), albeit the plateau size has pathologically been
reset upward (hence tumor which simply means swelling). This con
tinued size constraint is explained by the "integrated organ hypothe
sis" (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 liver
size and lobular conformation. Accordingly, organs "know" their size,
but continued normal use of the organ GJs may be necessary to
maintain that knowledge. Gap junctional disturbances may lead to loss
in the cybernetics of size restraint: hyperplasia results which is the
combination of increased cell number and cellular volume. If contin
ued, a condition sets up where tumorigenesis begins. Carcinogenesis
can evolve out of persistent tumorigenesis. which can then allow
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
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GAP JUNCTION
FUNCTION
phenotype where early mutations lead to enhanced and progressive
susceptibility to more fixed mutations ( 140). This type of phenotype
can "conven" transformants into growing neoplasms that in turn can
Cx43 is the principal connexin protein of the heart (where it was first
observed), which is an electrically active tissue that has extensive
GJIC. Why cells //; vitn> allow dereprcssion of one member in the Cx
imiltigene family while allowing suppression of another member is
not known, but environmental conditions have been implicated in the
liver phcnotypic switching between (Cx32, Cx26| <=>|Cx43| (146,
147). TPA reduces GJIC in 1AR 20 liver epithelial cells (as TPA does
in many other tissues and cell lines), but Cx43 mRNA does not
decrease ( 146). Thus if the Cx43 mediates IAR 20 GJIC. the reduction
seems to be caused by posttranslational Cx alterations (146). The
relative importance of Cx mRNA transcription and translation versus
Cx protein posttranslational transport through the ER and Golgi to the
perimembrane pool and then membrane processing into assembled
connexons is not yet known.
CAMs [and other ECM molecules (7)| can play a role in controlling
GJIC (3) and have been linked with invusiveness in the progression
phase of cancer (5, 7). Progressive loss of GJs and loss of E-cadherin
has 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-regulate GJIC in a tumor cell line having low levels of GJIC (26, 38).
Interestingly, one of the deleted genes on chromosome 18q in human
colorcctul cancer (this deletion found in ==70% of cases) has a se
quence similar to certain neural adhesion sequences ( 150). This indi
cates that deletion of CAMs by mutation may lead to a lack of proper
cell-to-cell contacts, including GJs (1.2, 4).
be converted into malignant cells during the progression stage of
cancer. Hence, progressive alterations and aberrant growth occurring
in carcinogenesis could affect the GJ systems among other changes. If
so. this could include altering both the quality and the quantity of
tumor cell GJs. We have objective bases for quantitative changes in
GJ1C. but thus far. qualitative changes in GJs during carcinogenesis
have been elusive. It is important to establish the precise relationship
of GJIC to the etiology of cancer (2).
Some further relationships between cancer and GJIC have been
presented, nix T24 protein accumulates in rat liver cells after trans
formation by /-</.vand is inversely related to GJIC (141). This obser
vation suggests that an oncogene may operate, at least in part, by
inhibiting GJ function. Mehta et cil. (142) showed in mouse trans
formed l()T'/2 cells (which had low GJIC) that a transfected gene for
Cx43 protein restored GJIC communication. Following GJIC resto
ration, 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 de
ficiency, i.e.. adding back Cx43 genes to the transformants. In another
investigation. Naus et til. (143) showed that transformed C6 glioma
cells can cause brain tumors. C6 glioma cells grow fast in culture but
are greatly slowed when transfected with complementary DNA for
Cx43. This investigation, like the studies in l()T'/2 cells described
above, indicate that lack of expression of Cx43 can be associated with
tumor growth, but C.\43 expression can suppress tumor growth.
Metastatic colonization at a distal organ site is initiated by the
attachment of blood- or lymph-borne tumor cells to organ-specific
adhesion molecules which are expressed on the surface of microvascular endothelial cells. The establishment of communication by the
invading cell to the host endothelial cells is essential for tumor growth
and 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 endothelial
cells at the secondary site ( 144). Low metastatic or nonmetastatic cells
did not similarly establish GJIC with the endothelial cells ( 144). These
authors conclude that GJIC was necessary for metastatic establish
ment and may play a role in tumor cell extravasation at secondary sites
( 144). Although some form of GJIC is required for the further nurturance of the tumor cells, it may be that (here is something different
about these GJs in establishing GJIC at secondary sites.
It is clear that tumor promoters such as TPA inhibit GJs, whereas
retinoids (which inhibit TPA-induced tumors) enhance GJs (60, 83).
Are these actions juxtaposed through their direct interaction with GJ
pore assemblies or are the effects indirect'.' Or is it both? Evidence for
direct effects on connexons is not available, but evidence for an
indirect metabolic effect comes from studies on altered hepatic foci
Cx32 mRNA levels. Cx32 is attenuated in rat liver as a result of tumor
promoter treatment (86). Less Cx protein results from fewer Cx mRNAs, 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 short
turnover 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 there
are various Cx proteins: to replace other Cx types and alter connexon
function when, and for as long as, the need exists for a different GJ
mode. More work on this point is needed.
Investigations on some liver in vitro epithelial cell lines show that
liver Cx32 (made in vivo) is not synthesi/ed but rather Cx43 ( 146).
AND C'ANC'KR
Summary
Gap Junctions, Toxicity, and Cancer. In this review on gap junc
tion function and its relevance to cancer we have considered the
following: («)gap junctions play an essential role in the integrated
regulation of growth, differentiation, and function of multicellular
organisms: (/?) perturbation of gap junction function, whether direct or
indirect, can result in GJIC changes that may provide the beginning of
toxicity because the synergism. and in some cases the synchronism, of
a field of cells is adversely affected. Persistence of GJIC abrogation
ultimately can cause irreversible damages to the integrity of a tissue.
This irreversibility can bring on the latter stages of various disease
states, but the etiology of any disease process is yet to be elucidated:
(c) tumor promoters may inhibit cell-to-cell communication by («)
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 altering
GJIC, (c) down-regulation of Cx synthesis and connexon assembly, or
(d) change in phosphorylation connexins. All of these could contrib
ute to tumor promotion by promoting local cell isolation. This would
be a stochastically critical microenvironmental defect if the field of
cells includes particular initiated (1+ ) cells; (d) while unequivocal
evidence for a causal relationship between modulation of gap junction
function and cancer etiology is not yet available, there is increasing
evidence that modulation of gap junction function is often associated
with the mechanism of action of many carcinogens and tumor pro
moters (2). Tumor progression also may be propagated by faulty
GJIC.
Conclusion and Future Directions. Considered in into, short- and
long-term regulatory mechanisms allow gap junction channels to ex
hibit enormous plasticity in response to intracellular and extracellular
signals. The range of permeant signaling molecules allows gap junc
tions to serve as major conduits for (a) ions setting the basic ground
state of a cell and signals and (h) the exchange of hydrophilic second
messenger molecules, metabolites, and substances that might serve as
either triggers or brakes (suppressors) for various cellular processes.
Tools are now available for pursuing the nature of the regulation of
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channel conductance in the context of short-term gating and long-term
expression. At the level of the gap junction channel itself, alterations
in structure of the connexins and the use of exogenous expression
systems should allow identification of the molecular domains respon
sible for sensitivities to various gating stimuli (151-154). The neces
sary probes are also now available and should lead to elucidation of
other levels of regulation (including precursor synthesis, membrane
trafficking and GJ insertion/degradation). These approaches should
also illuminate the relationships among various types of membrane
channels. Cells use both gap junctions and cytoplasmic bridges as
conduits for exchange of ions, metabolites, and signaling molecules.
The investigation of the gap junction channel may serve as a key to
identifying one class of intercellular channels and distinguishing them
from other cellular transport channels. Further understanding of the
molecular and cellular basis of GJIC will allow insights into the roles
that tumor promoters play in perturbing the tissue signaling networks
during the course of toxicity and disease progression.
Problems exist in considering assays of GJIC inhibition for pur
poses of evaluating chemical hazard. Evidence to date suggests that no
particular in vitro assay, based on a given species or cell type, can
serve as a universal predictor of a potential modulator of GJIC. Ad
ditionally, in vitro conditions may not simulate in vivo delivery of
tested chemicals to solid tissues insofar as the native extracellular
matrix scaffolding is missing. Furthermore, interactions of chemicals
and chemical mixtures, in terms of modulating GJIC, can either an
tagonize or synergize, as well as be additive. Because multiple GJ
proteins exist, often in different cells in the same organ, and several
mechanisms regulate GJIC. predicting the effect of a chemical based
on results with one cell type in vitro on another cell type or on the
same cell type in vivo is not yet possible. Establishing such correla
tions (including extrapolations to humans) is important for evaluating
teratogenic, reproductive, and carcinogenic risks of environmental
chemicals that disrupt GJ.
Acknowledgments
The authors wish to thank the meeting contributors and William Farland,
United States Environmental Protection Agency, and the National Institute of
Environmental Health Sciences for assistance in sponsoring the meeting. We
especially thank James Trosko for his early encouragement, Elliot Hertzberg
for his cooperation and for reading the manuscript, and David Spray and Mark
Neveu for helpful discussions.
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Gap Junction Function and Cancer
James W. Holder, Eugene Elmore and J. Carl Barrett
Cancer Res 1993;53:3475-3485.
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