role of disrupted gap junctional intercellular communication in detection and characterization of...

E L S E V I E R Mutation Research 365 (1996) 91-105 Reviews in Genetic Toxicology

Role of disrupted gap junctional intercellular communication in detection and characterization of carcinogens

H i r o s h i Y a m a s a k i *

Unit of Multistage Carcinogenesis, International Agency for Research on Cancer, 150, cours Albert Thomas, 69372 Lyon cedex 08, France

Abstract

Results from short-term tests for carcinogens and our advanced knowledge on cellular and molecular mechanisms of carcinogenesis strongly suggest that carcinogens do not induce genetic changes necessarily by directly interacting with DNA. Therefore, it is not surprising to see that many carcinogens are not detectable by available genetic toxicology tests. Thus, it has become necessary to study nongenotoxic mechanisms of carcinogenesis and to provide methods to predict those carcinogens which escape from conventional mutation tests. One possible nongenotoxic mechanism of carcinogenesis which is supported by abundant experimental evidence is inhibition of gap junctional intercellular communication. Many, but not all, tumor-promoting agents have been shown to inhibit the communication of cultured cells as well as in vivo. Molecular mechanisms of gap junctional intercellular communication control revealed that connexin (gap junction) genes form a family of tumor suppressor genes. Control mechanisms of expression as well as function of connexins are vulnerable to various carcinogenic insults, notably to nongenetoxic carcinogens. Thus, studies on the role of connexins in cell growth and carcinogenesis may prove to be useful for establishing a mechanism-based test to detect certain types of nongenotoxic carcinogens.

1. Introduct ion

Any reliable short-term test to detect carcinogens must be based on solid scientific information. During the past two decades, numerous short-term test systems have been developed, almost all are based on the assumption that carcinogenesis requires mutational events. Recent molecular studies of human cancers have supported the idea that multiple genetic changes must be cumulated before a normal cell develops into a malignant tumor (Fearon and Vogel- stein, 1990), so, therefore, short-term tests based on mutational events may well be justified. On the other

* Corresponding author. Tel.: (33) 72 73 84 85; Fax: (33) 72 73 84 42.

hand, it has been revealed that standard short-term test results identify no more than 65% of rodent carcinogens (Ashby and Tennant, 1991).

Does such a low predictivity suggest that the assumption that mutational events are critical in carcinogenesis is wrong? The answer is clearly no. What is wrong, however, is to assume that genetic changes required for carcinogenesis are always directly induced by exogenous carcinogens and thus detectable by short-term tests. The hypothesis that cancers arise from mutagenic events is based on the observations that malignant phenotypes are heredi- tary in terms of their cellular lineage and that molecular analysis of cancer cells indicates the presence of specific changes in oncogenes and tumor-suppressor genes. Because many environmental carcinogens

0165-1110/96/$15.00 Copyright © 1996 Published by Elsevier Science B.V. PII SO 165- 11 10(96)0001 8-8

92 H. Yamasaki / Mutation Research 365 (1996) 91-105

(e.g., tobacco smoke, X-rays, aflatoxin, sunlight etc.) have been implicated in human cancer causation and because the genetic basis of cancer is solid, it has been assumed that environmental carcinogens must have induced these genetic changes. That assumption is probably still correct, but it cannot be easily translated into simple mutation tests. For example,

exposure to agents that induce cell proliferation, block intercellular communication, or produce oxy- gen radicals may eventually lead to genetic damage, but these indirect mutational processes are not usually detected by existing short-term tests.

It is therefore important to find new short-term tests that will detect carcinogenic agents that do not

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PROPOSED TOPOLOGY OF CONNEXIN

COOH

GAP JUNCTION

emO ane

Involved in connexon / eonnexon interaction ( E1 and E2 ) Voltage gating ( E1 ) Recognition of connexon specificity ( E2 )

Conserved among different connexin molecules

Hydrophobic surface

Polar residues proposed to be involved in forming the wall of the channel

Fig. h Schematic view of gap junctions, connexons, and connexins (modified from Yamasaki, 1990).

H. Yamasaki / Mutation Research 365 (1996) 91-105 93

directly induce DNA damage but participate indirectly in the process of accumulating genetic changes. These carcinogens are usually called nongenotoxic carcinogens, since their primary target of interactions with cells is not the DNA. One possible nongenotoxic mechanism of carcinogenesis which is supported by abundant experimental evidence is inhibition of gap junctional intercellular communication (GJIC) (Loewenstein, 1979; Trosko and Chang, 1988; Yamasaki, 1990; Yamasaki et al., 1995b). In this article, current knowledge on the role of GJIC in carcinogenesis and its possible application to carcinogen detection is discussed.

2. Gap junctional intercellular communication

Among various forms of intercellular communication systems in multicellular organisms, GJIC is the only one by which cells exchange signals directly from the inside of one cell to that of neighboring cells (Pitts and Finbow, 1986; Bennett et al., 1991). GJIC is considered to play a crucial role in maintain- ing homeostasis by keeping growth control signals at equilibrium among GJIC-connected cells. GJIC-diffusible molecules include those involved in signal transduction; for example, calcium, cAMP and inositol triphosphate (Lawrence et al., 1978; Saez et al., 1989). Because GJIC is considered to play an essential role in homeostasis maintenance, it is reasonable to assume that its disruption will result in a variety of diseases. In fact, besides its significant role in carcinogenesis, aberrant GJIC is implicated in the etiology of heart malformations and malfunctions of the neurosystem in humans (Berghoffen et al., 1993; Britz-Cunningham et al., 1995).

GJIC is mediated by gap junction channels, which are composed of connexin molecules. Six connexin molecules form a connexon in one cell, and a gap junction channel is formed when a connexon in one cell links with one in a neighboring cell (Fig. 1). So far, 12 cDNAs coding for different connexin species have been cloned (Beyer, 1993). Connexins have four transmembrane domains and their C- and N- terminals in the cytoplasm. The amino acid sequences of these terminal portions and of the cytoplasmic loop vary significantly between different connexins. Protein kinase motifs are present at the C-terminal end of some of the connexins, phosphory-

lation is considered to play an important regulatory role in GJIC (Moreno et al., 1992). Connexin gene expression has a certain tissue specificity, and one cell type usually expresses several connexin gene species. It is known that gap junctions composed of one connexin species differ in their permeability from those made of another connexin species, and it is likely that the combination of different connexins may subtly differentiate the function of GJIC in specific tissues (Elfgang et al., 1995). Cis and trans

regulatory elements that control connexin gene transcription are not yet fully understood, although a few studies have indicated the presence of various elements in the upstream region of the connexin genes (Bai et al., 1995; Yu et al., 1994; Chen et al., 1995). It also has been suggested that promoter sequences of the Cx32 gene play a crucial role in its tissue- specific expression (Neuhaus et al., 1995).

GJIC can be modulated by various mechanisms. In addition to the usual regulatory mechanisms that apply for most proteins, such as transcription, mRNA stabilization and translational control, and posttranslational phosphorylation, the function of connexin proteins can also be modulated at other control levels. For example, connexins are assembled into connexons in a trans-golgi apparatus, and their translocation to the cytoplasmic membrane may also be regulated (Musil and Goodenough, 1995). Moreover, even when gap junctions are formed between two cells, it is known that gap junctional channels can be closed or opened under certain circumstances (Zampighi and Unwin, 1979). More indirectly, GJIC can be influenced by cell adhesion molecules (Jongen et al., 1991; Meyer et al., 1992) as well as by extracellular matrix (Spray et al., 1987) and growth factors (Madhuker et al., 1989; Van Zoelen and Tertoolen, 1991); mutations of connexin genes or other genes involved in the control of connexin function also may affect the regulation of GJIC (Britz-Cun- ningham et al., 1995). As described below, these various regulatory points appear to be vulnerable to carcinogenic insults.

3. Basis for using GJIC disruption as an endpoint for short-term tests for carcinogens

The first evidence for the involvement of aberrant GJIC in carcinogenesis was the observation that

94 1-1. Yamasaki / Mutation Research 365 (1996) 91-105

almost all tumors have aberrant GJIC (reviewed by Yamasaki, 1990). Cell lines established from tumors as well as in vitro transformed cells usually show lower GJIC ability. These include not only rodent but also human cell lines. With B A L B / c 3T3 cells, GJIC between transformed cells was unchanged, but between transformed cells and neighboring normal counterparts, it was clearly defective (Enomoto and Yamasaki, 1984). A similar lack of heterologous GJIC between transformed and normal cells was also observed with rat liver epithelial cell lines (Mesnil and Yamasaki, 1988). To extend these in vitro re- suits, we developed a simple method by which GJIC can be examined in slices of liver freshly removed from the rat (Krutovskikh and Yamasaki, 1995). Using this method, we have shown a reduced level of GJIC in rat liver tumors (Krutovskikh et al., 1991). We also applied such a method for measuring GJIC in human liver freshly removed surgically in a hospital. Again, we found that the GJIC level was much reduced in hepatocellular carcinoma cells in comparison with normal or surrounding lesions (Krutovskikh et al., 1994). It therefore appears that reduced GJIC is a common feature of many tumor cells. This is compatible with the idea that GJIC is essential for the maintenance of homeostasis and, because tumor formation inevitably requires the loss

of homeostasis, GJIC must be altered during carcinogenesis.

Further studies on changes of GJIC using multistage models of rat liver and mouse skin carcinogenesis have revealed that there is in general a progressive decrease in the level of GJIC during tumor progression. For example, in rat liver carcinogenesis, GJIC is already reduced in many preneoplastic foci and further reduction was evident in tumors (Krutovskikh et al., 1991). In some of these foci, we also noted a lack of heterologous communication between preneoplastic (GST-P-positive) and surrounding cells. In mouse skin carcinogenesis, cell lines established from papillomas usually communi- cate less than normal cells, but more than cells from carcinomas (Klann et al., 1989). When the levels of GJIC and tumorigenicity of a series of rat liver epithelial cell lines were examined, there was an inverse relationship (Mesnil et al., 1986), although no such relationship was observed in human esophageal cancer cell lines (Oyamada et al., 1994).

The above information indicates possible roles of GJIC blockage in carcinogenesis, but it is not clear whether the disruption of GJIC is a consequence or a cause of carcinogenesis. The most direct way to test whether GJIC is causally involved in carcinogenesis is to transfect connexin genes into GJIC-deficient

Table 1 Growth suppression by connexin genes

Gene Host cell line Growth suppression Ref.

In vitro In vivo

Cx32

Cx43

Cx43

Human hepatoma Rat g l ioma (C6) + Transformed 10T 1 / 2 cells +

+

Rat g l ioma (C6) +

HeLa cells + Rat WB-F344 cells a + Human rhabdomyosarcoma + Rat-1 cells b (antisense oligo transfection) +

+ Eghbali et al., 1991 + Bond et al., 1994

NT Mehta et al., 1991 + Rosen et al., 1993

+ Zhu et al., 1991 Naus et al., 1992

+ Mesnil et al., 1995 NT Esinduy et al., 1995 NT Lin et al., 1995 NT Goldberg et al., 1994

Cx40 HeLa cells - - Mesnil et al., 1995 Cx26 HeLa cells + + Mesnil et al., 1995

a Restoration of GJIC by Cx43 cDNA transfection was important to suppress growth of cocultured transformed cells. b Inhibition of GJIC by Cx43 antisense oligos reduced the ability of rat-I cells to suppress foci formation of cocultured transformed cells.

NT, not tested.


cancer cells; a number of such studies have been performed. As summarized in Table 1, such transfec- tants showed an increase in communication capacity and a decrease in tumorigenicity in vivo and /or in

growth in vitro. Rat glioma cells and chemically transformed mouse fibroblasts transfected with the connexin 43 gene (Cx43) showed reduced growth both in vivo and in vitro (Naus et al., 1992; Zhu et

Table 2 Examples of GJIC inhibition by carcinogenic stimuli and stimulation by chemopreventive agents

Tissues/cells studied Methods of GJIC assay a Selected refs.

INHIBITION Tumor-promoting agents

Phorbol esters Various cultured cells EC, MC, DT, BMA DDT Various cultured cells, rat liver MC, DT~ Many others Cultured cells MC, DT, BMA

Tumor-promoting stimuli Partial hepatectomy Rat liver DT, BMA Skin wounding Mouse skin EC DNA-reactive carcinogens Cultured cells MC, DT

ONCOGENES v-src NRK cells DT c-src NIH3T3 cells DT v-Ha-ras Rat liver cell line (WB) DT v-raf Cultured rat liver epithelial DT cells v-fps Rat-2 cells DT, BMA neu Rat liver cell line (WB) DT, BMA Polyoma-middle T B A L B / c 3T3 cells DT SV-40-T Human fibroblasts MC HPV 16-E5 Human keratinocyte cell line DT

(HaCaT)

GROWTH FACTORS/HORMONES EGF Ethinyl estradiol FGF b

PDGF Testosterone

Stimulation Retinoids cAMP elevating agents Glucocorticoids

Green tea extracts Flavones Fermented soy bean (Natto) TGF-~

Cholesterol Vitamin D

Swierenga and Yamasaki, 1992 c Budunova and Williams, 1994 c

Yamasaki et al., 1993 c Loewenstein and Penn, 1967 Budunova and Williams, 1994

Atkinson et al., 1981 Azarnia et al., 1988 De Feijter et al., 1990 Kalimi et al., 1992 Kurata and Lau, 1994 Jou et al., 1995 Katoh et al., 1993 Rosen et al., 1988 Oelze et al., 1995

v79 cells MC Trosko et al., 1982 v79 cells MC Trosko et al., 1982 Rat liver cell line (T51B) DT Kanematsu and Lau, 1993 C3H10T 1 /2 cells DT Pelletier and Boynton, 1994 Human transitional cell DT Kihara et al., 1990 Carcinoma cell lines

Various cells MC, DT, BMA Goldberg and Bertram, 1994 c Various cells MC, DT, BMA Mehta et al., 1992 B A L B / c 3T3 cells DT Yamasaki and Katoh, 1988 Primary hepatocyte culture DT Kwiatkowski et al., 1994 Rat liver cell line (WB) DT Sigler and Ruch, 1993 Rat liver cell lines (REL) DT Chaumontet et al., 1994 B A L B / c 3T3 cells DT Takahashi et al., 1995 NRK cells DT Van Zoelen and Tertoolen, 1991 C3H10T 1 /2 cells DT Ubeda et al., 1995 Novikoff hepatoma cells DT Meyer et al., 1990 C3HI0 1 / 2 cells DT Stahl et al., 1994

a DT, dye-transfer (see Table 3); EC, electrical coupling; MC, metabolic cooperation; BMA, biochemical and molecular assays (e.g., immunohistological staining, expression of connexin mRNA and proteins). b Synergistic with 12-0-tetradecanoylphorbol 13-acetate. c Representative reviews.

Tab

le 3

C

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n of

var

ious

ass

ay s

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ms

to m

easu

re G

JIC

Spec

ial

equi

pmen

t re

quir

emen

t T

echn

ical

sim

plic

ity

(+ +

+,

very

sim

ple,

-,

di

ffic

ult)

App

lica

tion

A

ppli

cati

on

Qua

ntif

icat

ion

Ava

ilab

le d

ata

to v

ario

us

to i

n vi

vo

of G

JIC

lev

el

base

for

car

cino

g.

cell

typ

es

( +

+,

good

, de

tect

ion

( + +

+

, (~

_+

,poo

r)

rich

,(~

,s

carc

e)

Oth

er c

hara

cter

isti

cs

Met

abol

ic c

oope

rati

on a

H

PR

T+

/HP

RT

C

onve

ntio

nal

cell

cul

ture

+

+ D

iffi

cult

N

ot p

ossi

ble

+ +

+ +

AS

S -

/AS

L-

+ +

Dif

ficu

lt

+ _+

A

K+

/AK

+

+ D

iffi

cult

+

- D

ye-t

rans

fer

Mic

roin

ject

ion

Mic

roin

ject

ion

+ E

asy

Suc

cess

ful

+ +

+ +

Scr

ape

load

ing

Con

vent

iona

l ce

ll c

ultu

re

+ +

+ E

asy

Not

don

e, b

ut p

ossi

ble

+ +

Pho

tobl

each

ing

Pho

tobl

each

ing

syst

em

+ E

asy

Not

don

e +

+ +

Prel

oadi

ng

Con

vent

iona

l ce

ll c

ultu

re

+ E

asy

Not

don

e +

+ -

Ele

ctri

cal

coup

ling

E

lect

roph

ysio

logi

cal

appa

ratu

s -

Eas

y S

ucce

ssfu

l +

+ _+

Nee

ds t

o ac

quir

e ap

prop

riat

e m

utan

t ce

ll l

ines

Stu

dies

on

dye-

tr

ansf

er k

inet

ics

are

poss

ible

Mea

sure

s io

n tr

ansf

er

I

~' F

or p

rinc

iple

s of

met

hods

, se

e Fi

g.

2.

Abb

revi

atio

ns o

f m

utan

ts a

re:

HP

RT

, hy

poxa

nthi

ne p

hosp

hori

bosy

ltra

nsfe

rase

; A

ss

, ar

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nosu

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synt

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.


al., 1991; Rose et al., 1993), but human liver tumor cells transfected with the Cx32 gene showed reduced tumorigenicity but no effect on growth in vitro (Eghbali et al., 1991). The transformed mouse cells also lost the ability to form foci on a normal cell monolayer after transfection of the Cx43 gene (Mehta et al., 1991). Our previous studies showed a more complicated picture when HeLa cells were transfected with Cx26, 40 or 43; only those transfected with the Cx26 gene became nontumorigenic and showed a reduced growth rate in vitro (Mesnil et al., 1995). Interestingly, the Cx26 gene is the major connexin gene expressed in the cervix, from which the HeLa cell line was originally isolated. These results suggest that connexin genes exert differential regulatory effects on cell growth, depending on the cell type in which they are expressed. This would be in agreement with the notion that connexin genes are in fact a family of tumor-suppressor genes, which is further supported by the emerging evidence that connexin genes are mutated in some tumors (Yamasaki et al., 1995a).

Another line of evidence that suggests a causal role of blockage of intercellular communication in carcinogenesis has come from studies in which agents or genes involved in carcinogenesis were shown to modulate GJIC. First, the mouse skin tumor-promoting agent 12-O-tetradecanoylphorbol 13-acetate (TPA) has been shown to inhibit GJIC in many types of cultured cells by various methods including metabolic cooperation, electrical coupling, and dye- transfer assays (Murray and Fitzgerald, 1979; Yotti et al., 1979; Enomoto et al., 1981 Enomoto et al., 1984). Many tumor-promoting agents were subse- quently reported to inhibit GJIC, and this led to the proposal that GJIC inhibition could be used as an endpoint for detecting tumor-promoting agents (Swierenga and Yamasaki, 1992). In addition to these tumor-promoting agents, other types of tumor- promoting stimuli such as partial hepatectomy and skin wounding also inhibit GJIC. Furthermore, it has been shown that various oncogenes also inhibit GJIC; such oncogenes include src, SV-40-T antigen, neu,

raf , f p s and ras oncogenes. On the other hand, certain growth factors such as PDGF, EGF, and TGF-[3 also modulate GJIC in cultured cells.

It has also been observed that chemopreventive agents enhance GJIC. Retinoids, glucocorticoids and

cAMP are known to antagonize the tumor-promoting ability of TPA in mouse skin, and they have been shown to upregulate GJIC. Other agents that upregulate GJIC include green tea extracts, flavones, vitamin D, and components of Japanese soybean fermented food (Natto).

Examples of inhibition and stimulation of GJIC by agents or factors and stimuli involved in carcinogenesis are summarized in Table 2. It is important to emphasize that only those studies that gave positive results are listed. There also are many studies that provided negative results. Budunova and Williams (1994) have reviewed this subject in detail. Taken together, there are sufficient lines of support for the use of GJIC disruption as a marker to identify poten- tial carcinogens.

4. Issues associated with assays of GJIC

In cultured cells, GJIC capacity can be measured by three main types of methods (1) metabolic cooperation, (2) dye-transfer, and (3) electrical coupling, as shown schematically in Fig. 2. Table 3 presents the different assays with their advantages and disad- vantages. Many laboratories are also using the micro- injection dye-transfer assay to quantitate GJIC. The biggest advantage of this procedure is that one can use any cell type that adheres to culture dishes. Because many tumor-promoting agents or so-called nongenotoxic carcinogens are likely to show a tissue-specific activity, it is important to study chemicals with some hypothesis regarding target tissues in which they may work. In this case, it is not practical to use a metabolic cooperation assay because one must prepare mutants of target cells before such an assay can be performed.

The dye-transfer assay also can be applied to measure GJIC in organ slices. This allows changes in GJIC to be followed in such slices after test chemicals are administered in vivo. Such a method has been developed in our laboratory and has already been successfully used in at least two other laboratories to test various tumor-promoting agents (Tateno et al., 1994; Krutovskikh et al., 1995). Alternatively, fresh tissue slices can be exposed to test chemicals in vitro before GJIC is measured. Primary cultures of human and rodent hepatocytes can be maintained

9g A. Metabolic co-operation

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H. Yamasaki / Mutation Research 365 (1996) 91-105

Table 4 Examples of mechanisms involved in GJIC modulation observed in tumors or induced by tumor-promoting agents

99

Examples in tumors or by carcinogenic factors Ref.

Mutation of connexin genes

Aberrant connexin gene expression

Reduced connexin protein expression

Aberrant phosphorylation of connexins

Aberrant localization (cytoplasmic) of connexins

Lack of cell adhesion molecules

Primary rat liver tumor Lewis lung carcinoma cell line

Primary human liver tumors (appearance of Cx43) Primary rat liver tumors (reduced Cx32) Human bladder cancer cells (reduced Cx26) Rat bladder tumors (increased Cx26 and Cx43) Reduction by the tumor-promoting agent, phenobarbital

Various human tumors Mouse skin Rat liver

By tumor-promoting agents (e.g., TPA, DDT, endosulfan, dieldrin, heptachlor epoxide)

By oncogenes

Primary human liver tumors Primary rat liver tumors By tumor-promoting agents in rat liver By tumor-promoting agents in cultured cells

Mouse skin tumor cells

Yamasaki, 1995 Mandelboim et al., 1994

Oyamada et al., 1990 Fitzgerald et al., 1989, Neveu et al., 1994 Grossman et al., 1994 Asamoto et al., 1994 Mesnil et al., 1988

Wilgenbus et al., 1992 Kamibayashi et al., 1995 Sakamoto et al., 1992

Berthoud et al., 1993, Mesnil et al., 1994, Rivedal et al., 1994, Kenne et al., 1994, Matesic et al., 1994 Loo et al., 1995, Kurata and Lau, 1994

Krutovskikh et al., 1994 Yamasaki, 1995 Krutovskikh et al., 1995 Asamoto et al., 1991, Rivedal et al., 1994, Matesic et al., 1994

Jongen et al., 1991

wi th c o n t i n u e d e x p r e s s i o n o f d i f f e ren t i a t ed func t i ons

for a long t ime on ly w h e n they are cocu l tu red wi th

b i l ia ry ep i the l ia l cells, m i m i c k i n g the in v ivo si tua-

t ion ( G u g u e n - G u i l l o u z o et al., 1983). W e h a v e re-

cen t ly i m p r o v e d the m e t h o d so that p r i m a r y hepa to -

cytes can be m a i n t a i n e d in cocu l tu re wi th e s t ab l i s hed

cel l l ines and h a v e s h o w n tha t G J I C is m a i n t a i n e d in

the hepa tocy tes . G J I C was i nh ib i t ed b y p h e n o b a r b i t a l

bu t no t by T P A , s h o w i n g t i s sue-spec i f i c i nh ib i t i on

by t u m o r - p r o m o t i n g agen ts ( Mes n i l et al., 1993).

A n o t h e r in vi t ro s y s t e m is p r ima ry cu l ture of ha i r

fol l ic les . P lucked h u m a n ha i r fo l l ic les can be p l aced

in cu l ture and the g r o w i n g cel ls are su i t ab le for GJ IC

s tudy ( S w i e r e n g a et al., 1991). T h e s e m e t h o d s shou ld

ref lect in v ivo G J I C states m o r e appropr ia t e ly than

c o n v e n t i o n a l l y used cel l l ines and p r ima ry cu l ture o f

s ingle types of cells.

W h i l e it appea r s tha t the dye - t r ans fe r assay is

su i tab le for m a n y labora tor ies , it is adv i sab le no t to

fix any assay s y s t e m or pro tocol at th is s tage for

Fig. 2. (A) Metabolic cooperation. In the coculture of HPRT + and HPRT cells, only HPRT + cells metabolize 6-thioguanine to 6-thioguanine monophosphates, which kill cells upon incorporation into nucleic acids. Although HPRT- cells do not convert 6-thioguanine and could therefore survive in the presence of 6-thioguanine when they are cultured alone, in contact with HPRT + cells, 6-thioguanine monophosphates can be transferred from HPRT ÷ cells through gap junctions. Thus, in the presence of 6-thioguanine monophosphates can be transferred from HPRT + cells through gap junctions. Thus, in the presence of 6-thioguanine, both HPRT + and HPRT- cells are killed. (B) Electrical coupling. A microelectrode is inserted into each coupled cell (C1 and C2). Electrical current is then run from C1, and whether the electron transfer occurs from C1 to C2 through GJIC is measured. (C) Dye transfer. Tracer dye molecules (usually fluorescent) that are not membrane-diffusible, but gap junction-diffusible, are microinjected into a cell. Lucifer Yellow CH molecules are mostly used for this purpose. The spread of dye into neighboring cells through gap junctions is determined.


carcinogen screening. Because each chemical differs in its properties and modes of action, it is wiser to vary the methods and protocols accordingly.

5. Mechanisms of GJIC inhibition by carcinogens

Advances in understanding the molecular biology of GJIC have enabled us to characterize the molecular events associated with the inhibition of GJIC by carcinogens. Although relatively few chemicals have been studied for their mechanisms of action on GJIC, it appears that posttranslational modulation is important. For example, TPA-treated cultured rat liver epithelial cells do not show any changes in their level of mRNA for the Cx43 gene (Asamoto et al., 1991), and Cx43 protein was present in similar amounts before and after TPA treatment. However, its phosphorylated forms were changed, and the connexin proteins were localized in the cytoplasm rather than in cytoplasmic membranes. Therefore, it seems that TPA caused phosphorylation of connexin proteins, probably through protein kinase C activation, leading to their translocation from the cytoplasmic membrane to cytoplasm (Mesnil et al., 1994). Be- cause connexin proteins can function only when they are in the cytoplasmic membranes, this internaliza- tion of connexins may be responsible for GJIC inhibition. Changes in the phosphorylation pattern of connexins are also associated with GJIC inhibition by other tumor-promoting agents and oncogenes (Ta- ble 4).

In vivo studies also suggest that posttranslational modulation of connexins and /or aberrant localization is a common mechanism for carcinogen-induced downregulation of GJIC. We have submitted groups of Fischer-344 rats were subjected to repeated treatment with phenobarbital (PB), polychlorinated biphenyls (PCB), dichloro-diphenyltrichloroethane (DDT), and clofibrate (CF) for 5 weeks. All four tumor-promoting agents decreased dye coupling in rat liver. This decrease was associated with a reduced number of gap junctions and aberrant localization of some of the Cx32 proteins in hepatocytes; Cx32 was often observed in the cytoplasm of hepatocytes instead of at gap junctions in the plasma membrane. Western blot analysis indicated only slight changes in the level of Cx32 proteins. A1-

though levels of Cx26 proteins at gap junctions were usually decreased by tumor promoters in rat liver, local induction of Cx26 protein expression in cen- trilobular groups of hepatocytes after PCB and DDT treatment was observed. Expression of Cx43 was induced in hepatocytes after PCB, DDT, and CF exposure, but this protein was also localized intracy- toplasmically, suggesting that it had no functional role (Krutovskikh et al., 1995).

It has previously been shown that treatment of rats with phenobarbital lowers the level of Cx32 mRNA (Mesnil et al., 1988). It has also been shown that in many rat liver tumors, levels of Cx32 mRNA are decreased (Beer et al., 1988). However, in human hepatocellular carcinomas, the level of Cx32 mRNA was not changed, although that of Cx43 mRNA was significantly reduced (Oyamada et al., 1990). In contrast, carotenoids can upregulate the transcription of Cx43 mRNA (Zhang et al., 1992). These results suggest that GJIC can also be modulated at the transcriptional level.

Although the inhibition of GJIC in toxicology has always been approached as a nongenotoxic process, it is possible that genotoxic mechanisms such as mutations of connexin genes lead to GJIC impair- ment. However, among 20 human liver tumors and 22 human gastric tumors, we found no Cx32 gene mutation (Mironov et al., 1994; Krutovskikh et al., 1994). On the other hand, when several rat liver tumors induced by a nitrosamine were analyzed, we found one sample with a mutation at the 220th codon of the Cx32 gene (Yamasaki et al., 1995a). Our preliminary results also suggest that some human malignant meningiomas harbor a mutation of the Cx43 gene.

Interestingly, recent studies have shown that two human diseases are caused by germ cell mutations of connexin genes. In certain types of visceroatrial het- erotaxia syndrome, a Cx43 gene mutation was found (Britz-Cunningham et al., 1995). In X-linked Char- cot-Marie Tooth (CMTX) disease, Cx32 gene mutations have been found (Berghoffen et al., 1993; Fairweather et al., 1994). Thus, it seems that mutational events in connexin genes can lead to impair- ment of GJIC. This would imply that mutagenic carcinogens may also induce connexin gene mutations and inhibition of GJIC. We and others have shown that some Cx32 mutants found in CMTX


patients are indeed defective in forming functional GJIC (Bruzzone et al., 1994; Omori et al., unpublished data). Moreover, these mutants showed a strong dominant-negative effect on wild-type Cx32 proteins when both of them were transfected into HeLa cells (unpublished data). It is likely that such a dominant-negative effect is due to the formation of chimeric connexons containing both mutant and wild-type connexins. Thus, mutations in one allele of connexin genes may result in complete loss of the connexin function, suggesting that a mutational event may be a powerful mechanism for GJIC inhibition.

GJIC can thus be modulated by various mechanisms induced by various carcinogenic stimuli. Some examples are summarized in Table 4.

6. Implications for cancer risk assessment

Mutation assays were introduced with great hopes that they could predict most human carcinogens. This expectation was not fulfilled, and it has therefore become important to attempt to develop assays in which the endpoints are not mutations. In develop- ing new nongenotoxic mechanism-based test systems for carcinogens, we should learn from the experience of genetic toxicologists. In retrospect, it appears that validation of a protocol by assaying large numbers of numerous chemicals may not have been a sound approach. It is essential to examine the concept behind a proposed test system. For example, it is important to have solid scientific evidence that disrupted GJIC is causally related to carcinogenesis, rather than simply testing many chemicals in one of the GJIC assays using cultured cells. It is also un- wise to suppose that all nongenotoxic carcinogens will be detected by a single test system; it is more likely that different carcinogens will affect different cellular constituents and in different manners.

Approaches to hazard identification and risk assessment in cancer are changing. It is being increas- ingly recognized that mechanistic information should be considered in addition to conventional epidemio- logical data or short-term and long-term test results from model systems (IARC, 1991). Thus, in the context of this article, it would be important to know whether chemicals inhibit GJIC by modulation of gene expression or by post translational modification. It is also possible that mutation of connexin

genes will lead to inhibition of GJIC. If agents inhibit GJIC by mutations, the effect may be irre- versible, whereas posttranslational modification could be reversible. On the other hand, a mutagenic effect can occur in a very few cells and thus is localized, whereas a nongenotoxic carcinogen may affect the communication of the whole exposed cell popula- tion. Such information is important for accurate risk estimation of a test agent.

The fact that a given endpoint of a test, such as inhibition of GJIC, can be attained by either mutagenic or nonmutagenic events implies a certain diffi- culty in classifying carcinogens into genotoxic and nongenotoxic categories on the basis of short-term results. As with the example of GJIC inhibition, most of the apparently nongenotoxic endpoints can also be induced by genotoxic mechanisms (Yamasaki et al., 1996). Therefore, classification of carcinogens into genotoxic or nongenotoxic, or into initiating or promoting agents, may not only be unhelpful but even an impediment to risk assessment. Once enough relevant mechanistic information about individual chemicals is accumulated, it will be more reasonable to use this information directly for risk estimation, without expending effort in classifying them. A possible negative consequence of carcinogen classification for risk assessment is that a classification can be based on only one of various activities of an agent; for example, ultraviolet light may be considered to be genotoxic (induction of gene mutations) or nongenotoxic (immunomodulation). If carcinogens are classified, the level of risk tends to be associated with such classification, so that the classification of a given agent is based on only one type of activity, an overall assessment of its risk cannot be made. Thus, a more complete risk assessment of carcinogens may be carried out in the absence of a classification.

Most of the data on inhibition of GJIC by chemicals have not been produced with a single protocol. This is probably not a defect of the approach, but in fact a more appropriate way to address the problem. If the protocol is fixed with one method and one cell type, it is likely that many carcinogens will not be detected. Instead, if assay systems with various cell types are used, selected on the basis of a working hypothesis, more useful information may be ob- tained. At a crossroads of changes in carcinogen screening, it is probably better to study each chemi-


cal agent with hypotheses rather than simply test

them with a fixed protocol.

Acknowledgements

I wish to thank many of my colleagues for contin- uous helpful discussion; my special thanks go to Drs. V. Krutovskikh, M. Mesnil, and C. Naus. I am grateful to Mme. Chantal D6chaux for her efficient secretarial aid and to Dr. John Cheney for editing the manuscript.

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role of disrupted gap junctional intercellular communication in detection and characterization of...

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