Cancer Letters, 7 1 ( 1993) 1 1 - 18

Elsevier Scientific Publishers Ireland Ltd

Gap junctional intercellular communication in mouse lung epithklial cell lines: promoters

effects of cell transformation and tumor

Rakhi Chaudhuria, Kristi Sigler”, Emmanuel Dupontb, James E. Trosko b, Alvin M. Malkinson’ and Randall J. Ruth”

“Department of Pathology, Medical College of Ohio, Toledo. OH 43699. ‘Department of‘ Pediatrics and Human Drreiopment. Michigan State Universily. Easr Lansing, MI 48824. and ‘Molecular Toxicology and Colorado Cancer Center. University of Colorado, Denver, CO 80262 (USA)

(Received 5 January 1993)

(Revision received 18 March 1993)

(Accepted 19 March 1993)

Summary

Gap junctional intercellular communication (GJIC) is reduced by neoplastic transformation and treatment with tumor promoters in many types of cells but few data exist for the lung. GJIC was therefore evaluated in non-transformed (ClO) and transformed (E9, 82-l 32, and PCC4) mouse lung epithelial cell lines and in Cl0 cells treated with tumor promoters. GJIC was assessed by flu- orescent dye microinjection (dye-coupling). Dye- coupling levels were highest in Cl0 cells (SS-90% communicating cells) followed by 82-132 cells (40-50%), E9 cells (15-20%/o), and PCC4 cells (3-10X). Indirect immunofluorescent staining with anti-gap junction protein (connexin) anti- bodies revealed that Cl0 cells expressed gap junctions comprised of connexin43, but not connexin32 or connexin26. The tumor promoters, butylated hydroxytoluene (BHT), 12-O-tetradeca- noylphorbol-13-acetate (TPA), and p,p’-dichloro- diphenyltrichloroethane (DDT), inhibited dye- coupling in Cl0 cells but phenobarbital (PB) did not. BHT promotes mouse lung tumor formation,

Correspondence to: Randall J. Ruth. Department of Pathology,

Medical College of Ohio. HEB 202, 3000 Arlington Avenue, Toledo. OH 43699. USA.

PB does not, while the effects of TPA and DDT on lung tumor development have not been reported. These data indicate that cell transformation and certain tumor promoters reduce GJIC in mouse lung epithelial cells and demonstrate correlations between the in vitro inhibition of GJIC and lung tumor promotion.

Keywords: gap junctions; lung; mouse; tumor promotion; carcinogenesis

Introduction

Gap junctions are located in the plasma mem- branes between adjacent cells and are thought to serve as direct passageways for the exchange of low molecular weight ( < 1000) ions and small molecules between cells. This exchange of ionic and chemical information has been termed gap junctional intercellular communication (GJIC) [21]. One proposed role for GJIC is growth regula- tion via the cell-to-cell exchange of growth regula- tory factors [21]. In support of this hypothesis, many studies have documented that neoplastic transformation and treatment with tumor pro- moters can result in the reduction of both gap junction number and permeability [l&21,41]. The

0304-3835/93/$06.00 0 1993 Elsevier Scientific Publishers Ireland Ltd

Printed and Published in Ireland

12

reduction of GJIC by promoters and transforma- tion may enable cells to escape growth regulation [l&21,25,46].

Few studies have examined whether gap junc- tions might play a role in lung neoplasia and tumor promotion [l]. Heussen and Alink [13]

reported that the skin tumor promoter, 12-Otetradecanoylphorbol-13-acetate (TPA), and lung carcinogenic airborne particulate matter reduced GJIC in primary cultures of rat alveolar type II cells. In addition, cigarette smoke conden- sates have been shown to down-regulate GJIC in V-79 Chinese hamster lung fibroblasts [lo], as well as in hamster tracheal epithelial cells [34]. Because tumor promotion has been extensively studied in mouse lung [22,39,45], we have quantified the lev- els of GJIC in several non-transformed and trans- formed mouse lung epithelial cell lines and have also tested the effects of putative tumor promoters on lung epithelial cell GJIC.

Materials and Methods

Materials Unless otherwise noted, all reagents were pur-

chased from Sigma Chemical Co. (St. Louis, MO).

Cell lines and culture conditions Immortalized, non-tumorigenic (C 10) and

transformed, tumorigenic (E9, 82-l 32, and PCC4) mouse lung epithelial cell lines were cultured as previously described [5,6,28,37,38]. The neoplastic cells are anchorage-independent and contain mutations in the K-ras protooncogene [28]. Cl0 cells had properties of Type II alveolar epithelial cells at early passage [37]. E9 cells are a spon- taneously transformed clone of E 10 cells, a cell line related to Cl0 [38]. 82-l 32 cells originated from a Type II cell-derived, solid carcinoma [28]. PCC4 cells were established from a Clara cell-derived, papillary tumor [28].

Assay of GJIC GJIC was assayed in the lung cells following mi-

croinjection of Lucifer Yellow CH fluorescent dye into cells and quantification of the number of directly adjacent cells to which dye had spread (dye-coupling) as we have reported [32]. The cells

were cultured in 35-mm dishes and evaluated for dye-coupling at 40-50X and 90- 100% confluence. At least three dishes were tested and lo- 15 cells were injected per dish at each confluency level. In the figures, ‘the dye-coupling percentage’ is reported. This corresponds to the total number of neighboring, dye-coupled cells in the three dishes divided by the total number of neighboring cells times 100.

Effects of tumor promoters on GJIC The effects of TPA, butylated hydroxytoluene

(BHT), p,p’-dichlorodiphenyltrichloroethane (DDT), and phenobarbital (PB) on Cl0 cell GJIC were evaluated. The promoters were dissolved in dimethylsulfoxide (DMSO) and added to the Cl0 culture medium. Control cultures were treated with DMSO only. The final DMSO concentration in all cultures was 0.1% (v/v). The cells (90- 100%

confluent) were treated with the compounds for 4 h before assaying GJIC by fluorescent dye micro- injection as described above. Three cultures were used per treatment group. Cell viability was monitored by lactate dehydrogenase release [33].

Immunohistochemical staining of gap junctions Affinity purified polyclonal rabbit antibodies

directed against three gap junction proteins (connexin43. connexin32, and connexin26) were used to stain C 10 cell gap junctions by indirect im- munofluorescence. Peptides corresponding to amino acid residues 314-322 of connexin43 [2], 243-252 of connexin32 [27], and 108-l 17 of connexin26 [47] were synthesized in solid phase, coupled to bovine serum albumin and used to im- munize rabbits as previously described [7.9]. The antipeptide IgGs were subsequently purified by affinity chromatography [7].

Cl0 cells were cultured on sterile glass cover- slips, fixed in cold 5% acetic acid/95% methanol for 1 h, then hydrated in phosphate-buffered saline (PBS). The cells were sequentially incubated with normal goat serum ( 1: 100 in PBS), affinity purified anti-connexin antibody ( 1: 10 in PBS), and affinity purified FITC-goat anti-rabbit IgG ( 1: 10 in PBS) (Jackson Immunoresearch Laboratories, Inc., West Grove, PA) for 1 h each at room tempera- ture, with extensive washing with PBS between in-

cubations. Following the last wash, the coverslips were mounted in Slow-Fade mounting medium (Molecular Probes; Eugene, OR). Cells were view- ed and photographed on a Nikon Diaphot invert- ed microscope using an oil immersion 100x fluorescent objective under epifluorescent illumi- nation.

Statistical analyses

Two-by-two Chi-squared analyses with Yates’ correction [4] were used to statistically compare differences in the frequencies of dye-coupled and non-dye-coupled neighboring cells between treat- ment groups. Analyses were performed by compu- ter using InStat statistical software (GraphPad Software, Inc., San Diego, CA).

Results

Dye-coupling was assayed in non-transformed (ClO) and transformed (E9, 82-132, and PCC4) cells in low and high density cultures (Fig. 1). At both densities, Cl0 cells had the highest levels of dye-coupling (85-900/u) followed by 82-132 cells (40-500/o). E9 cells (1%20%) and PCC4 cells (3-10X). Culture density had no significant effect on dye-coupling levels in any cell line. PCC4 cells tended to detach during culture and only the at- tached cells were microinjected and evaluated for dye-coupling. The morphologies and dye-coupling abilities of the four cell lines are depicted in Fig. 2.

100

f%

s 90

!I? 80

70

b a 60

F 50 .- a 40 3 8 30

, 20 aJ 0" 10

0 I.-i

0 40-50% Confluent

m 90-l 00% Confluent

Cl0 E9 82-132 PCC4

Mouse Lung Cell Line

Fig. 1. Levels of GJlC (dye-coupling) in normal (CIO) and

neoplastic (E9. 82-132. and PCC4) mouse lung epithelial cell

lines, The cells were evaluated for dye-coupling at 40-50X1 and

90-100% confluence (N = 135- 186 cells from three dishes).

13

Fig. 2. Morphology and dye-coupling in Cl0 (A,B), E9 (C.D),

PCC4 (E.F). and 82-132 (G,H) cells. The left panels are phase

contrast images and the right panels are epifluorescence

images.

The effects on GJIC in Cl0 cells of several agents known to be tumor promoters in various

organs were determined. The mouse lung [22,39,45] and liver [30] tumor promoter, BHT; the skin tumor promoter, TPA [36]; and the liver tumor promoter, DDT [44], inhibited dye- coupling in Cl0 cells in dose-related fashion (Fig. 3A-C). The most potent inhibitor was TPA followed by DDT and BHT. The liver tumor pro- moter, PB [16,29,30,42]. had no effect on GJIC in Cl0 cells at doses up to 1 mM (Fig. 3D). Higher doses were cytotoxic (data not shown). Dye- coupling and morphologies of control (DMSO- treated) and tumor promoter-treated Cl0 cells are

14

A B 100~ IOOJ

Butylated Hydroxytoluene (pM)

0.0 2.5 5.0 7.5 10.0

DDT WI

5.0 7.5

TPA (ng/ml)

H lj , , , , 0.00 0.25 0.50 0.75 1.00

Phsnobarbital (mM)

Fig. 3. Effects of the tumor promoters. BHT (AI. TPA (13). DDT (c‘). and PB (D). on dyecoupling in C’IO cells. The cells were treated at 90-100% confluence with non-toxic concentrations of the promoters for 4 h bcforc evaluation of dye-coupling. C’ontrol

cultures (0 concentrations) were treated with DMSO (O.l”i!i. Asterisks indicate >lgmficant decreases (P < 0.05) in dye-couplmg fre-

quencies compared to control cultures (ChI-squared IN with Yates‘ correction; N = I 13-126 cells from three dlshcs).

shown in Fig. 4. Extensive dye-coupling is evident in control and PB-treated cultures whereas little coupling is present in BHT-, DDT-, and TPA- treated cells. TPA induced morphological changes in the cells that were reversible [26] but the other promoters had little effect on Cl0 cell morpholo- gy. The promoters were not cytotoxic at the con- centrations reported in Figs. 3 and 4, i.e. no increased release of lactate dehydrogenase was observed from the promoter-treated cells over the 4-h treatment period (data not shown).

Cl0 cells were stained by indirect immuno- fluorescence for the gap junction proteins.

connexin43, connexin32, and connexin26. Dis- crete, plasma membrane-localized sites of punctate staining were evident on Cl0 cells stained with anti-connexin43 antibody. but no such staining was visible using anti-connexin32 and anti- connexin26 antibodies (Fig. 5).

Discussion

In this report, we have demonstrated that several neoplastic mouse lung cell lines have lower levels of GJIC compared to a related normal cell. This is the first report that neoplastic mouse lung

Fig. 4. Morphology and dye-coupling in control and tumor

promoter-treated Cl0 cells. Cells were exposed to DMSO (0.1%: control cells; A.B). BHT (250 PM; C.D). DDT (10 PM:

E.F), TPA (10 ngiml; G,H) or phenobarbital (I mM; 1.J) for 4

h prior to microinjection and photomicrography; [phase con- trast (A.C,E,G.I) and epifluorescence (B.D.F.H.J) images].

cells have reduced GJIC compared to a non- transformed counterpart. Mouse lung tumors have been examined ultrastructurally, but the presence or absence of gap junctions was not reported

13,151. The association between the loss of GJIC and

transformation we report here for mouse lung cell lines has been well documented in other tissues and cell types [18,21] including human and rat lung [1,13]. The prevalence of this relationship suggests that the reduction of GJIC plays a role in the expression of the neoplastic phenotype. Direct evidence for such a role has recently been provid- ed. When poorly coupled sarcoma [24], glioma

Fig. 5. Indirect immunofluorescent staining of untreated Cl0

cells with antibodies against connexin43 (A). connexin32 (B),

and connexin26 (C). Punctate plasma membrane labeling con-

sistent with gap junctions is evident only in connexin43 im-

munostained cells.

[48], or hepatoma [8] cells were induced to com- municate following transfection of connexin cDNAs, the cells displayed reduced growth rates and/or tumorigenicity. Thus, the increase in GJIC apparently resulted in a more normal phenotype.

A similar relationship between the loss of GJIC,

16

enhanced growth, and neoplastic transformation appears to exist for the lung cell lines of the pre- sent study. The growth rate of highly com- municating Cl0 cells is slower than those of the tumor cells [20]. All three tumor cell lines grow well in soft agar and form metastatic tumors in syngeneic mice; Cl0 cells lack these capabilities 138; Malkinson, unpublished data]. Finally, PCC4

cells, which had the lowest levels of GJIC (Fig. 1). have the highest rate of growth in vitro [20] and are the most morphologically abnormal (Fig. 2).

Many tumor promoters inhibit GJIC in vitro

and in vivo [18,41]. In some studies, the inhibition of GJIC by tumor promoters correlated with the tissue- and species-specificity of the promoters [17,19,31]. In the present study, Cl0 cell GJIC was inhibited by several agents (BHT. TPA, and DDT) which promote tumor development. Of these, only BHT has been reported to have lung tumor pro- moting activity [22,39,45] and to enhance Type II epithelial cell growth [45]. TPA and DDT have not been tested in lung tumor promotion or growth bioassays. However, TPA was found to enhance

tracheal carcinogenesis in rats [40]. PB, which is a liver tumor promoter in rodents [ 16,29,30,42], had no effect on Cl0 cell GJIC and has been reported by several groups to be ineffectual in promoting growth and tumor formation in the mouse lung [22,29,42]. Thus, there are direct correlations be- tween the inhibition of GJIC by tumor promoters in vitro and the enhancement of lung cell growth and tumor formation in vivo.

The mechanism(s) of inhibition of Cl0 cell GJIC by BHT. TPA, and DDT need to be deter- mined. The 4-h treatment period used herein to

test for perturbations in GJIC by TPA and BHT is known to reduce the activity and control of pro- tein kinase C (PKC) [6,23]. Since gap junction per- meability may be regulated by connexin phosphorylation by PKC and other kinases [35], it will be interesting to assess the phosphorylation status of connexin43 in Cl0 cells following treat- ment with the tumor promoters.

Gap junctions in Cl0 cells appeared to be com- prised of connexin43, but not connexin26 or connexin32. Connexin43 is the major connexin ex- pressed by cardiac myocytes [2] but has been detected in lung and other tissues [14].

Connexin26 and connexin32 are the major gap junction proteins expressed in liver [27,47]. Other

connexins expressed in lung tissue include connexin37 [43], connexin40 [ 121, and connexin45 [l 11. We are currently examining in greater detail which connexins are expressed by the lung cell lines and lung tissue. and if connexin expression is reduced by neoplastic transformation and treat- ment with tumor promoters.

Acknowledgements

This study was supported by grants from the National Cancer Institute to RJR (CA57612),

AMM (CA33497). and JET (CA21 104) and from the U.S. Air Force Office of Scientific Research to JET (AFOSR-89-0325).

References

Albright. C.D., Grimley. P.M.. Jones. R.T., Fontana.

J.A.. Keenan, K.P. and Resau. J.H. (1991) Cell-to-cell

communication: a differential response to TGF-fl in nor-

mal and transformed (BEAS-2B) human bronchial

epithelial cells. Carcinogenesis, 12. 1993-1999.

Beyer, E.C.. Paul. D.L. and Goodenough, D.A. ( 1987)

Connexin43: a protein from rat heart homologous to a

gap junction protein from liver. J. Cell Biol.. 105,

262 I-2629.

Brooks. R.E. (1968) Pulmonary adenomas of strain A

mice: An electron microscopic study. J. Natl. Cancer

Inst.. 41. 719-742.

Campbell, R.C. (1974) Statistics for Biologists. Cam-

bridge University Press. Cambridge.

Droms. K.A.. Haley. B.A.. Smith. G.J. and Malkinson.

A.M. (1989) Decreased 8N,-[r”P]GTP photolabelling of

GScv in tumorigenic lung epithelial cell lines: Association

with decreased hormone responsiveness and loss of

contact-inhibition. Exp. Cell. Res., 182. 330-339.

Dwyer. L.D.. Miller. A.K. and Malkinson, A.M. (1992)

Protein kinase C isozymes and calparn concentrations

during the growth of mouse lung epithelral cell Irnes. Mol.

Biol. Cell. 3. 336a.

DuPont. E.. El Aoumari. A.. Roustiau-Severe. S., Briand, J.P. and Gros. D. (1988) Immunological characterizatron of rat cardiac gap junctions: presence of common anti-

genie determinants in heart of other vertebrate species and in various organs. J. Memb. Biol.. 104. Il9-178.

Eghbali, B., Kessler. J.A.. Reid, L.M.. Roy. C. and Spray,

D.C. (1991) Involvement of gap junctions in tumorigcn-

esis: Transfection of tumor cells with connexin32 cDNA

retards growth in viva. Proc. Natl. Acad. Sci.. USA. 8X.

10701-10705.

9

IO

II

12

13

14

I5

16

17

18

I9

20

21

22

El Aoumari, A.E.. Frogamet. C.. Dupont. E., Reggio. H..

Durbec. P., Briand. J.-P.. Boiler. K. and Gras, D. ( 1990)

Conservation of a cytoplasmic carboxy-terminal domain

of connexin 43, a gap junctional protein, in mammalian

heart and brain. J. Memb. Biol., 115, 229-240.

Hartman, T.G. and Rosen, J.D. (1983) Inhibition of meta-

bolic cooperation by cigarette smoke condensate and its

fractions in V79 Chinese hamster libroblasts. Proc. Natl.

Acad. Sci., USA, 80, 5305-5309.

Hennemann, H.. Schwarz. H.-J. and Willecke, K. (1992)

Characterization of gap junction genes expressed in F9

embryonic carcinoma cells: molecular cloning of mouse

connexin31 and-45 cDNAs. Eur. J. Cell Biol., 57, 51-58.

Hennemann. H.. Suchnya. T., Lichtenberg-Frate. H..

Jungbluth, S., Dahl, E.. Schwarz, J., Nicholson, B.J. and

Willecke. K. (1992) Molecular cloning and functional ex-

pression of mouse connexin40, a second gap junction gene

preferentially expressed in lung. J. Cell Biol., 117,

l299- I3 IO.

Heussen, G.A.H. and Alink, G.M. (1992) Inhibition of

gap-junctional intercellular communication by TPA and

airborne particulate matter in primary cultures of rat alve-

olar type II cells. Carcinogenesis, 13. 719-722.

Kadle. R.. Zhang. J.T. and Nicholson. B.J. (1991) Tissue-

specific distribution of differently phosphorylated forms

of Cx43. Mol. Cell. Biol.. I I, 363-369.

Kauffman, S.L.. Alexander. L. and Sass. L. (1979)

Histologic and ultrastructural features of the Clara cell

adenoma of the mouse lung. Lab. Invest.. 40, 708-716.

Klaunig. J.E.. Pereira. M.A., Herren-Freund, S.L.. and

Ruth. R.J. (1986) Effect of phenobarbital on the develop-

ment of liver tumors in juvenile and adult mice. J. Natl.

Cancer Inst., 77. 449-452.

Klaunig, J.E. and Ruth, R.J. (1987) Strain and species ef-

fects on the inhibition of hepatocyte intercellular com-

munication by liver tumor promoters. Cancer Lett., 36,

161-168.

Klaunig, J.E. and Ruth. R.J. (1990) Role of intercellular

communication in nongenotoxic carcinogenesis. Lab. In-

vest.. 62. 135-146.

Klaunig, J.E.. Ruth. R.J. and Lin, E.C. (1989) Effects of

trichloroethylene and its metabolites on mouse and rat

hepatocyte intercellular communication. Toxicol. Appl.

Pharmacol.. 99, 454-465.

Lange-Carter. C.A.. Vuillequez, J.J. and Malkinson.

A.M. (1993) 8-Chloroadenosine mediates R-chloro-cyclic

AMP-induced down-regulation of cyclic AMP-dependent

protein kinase in normal and neoplastic mouse lung

epithelial cells by a cyclic AMP-independent mechanism.

Cancer Res., 53. 393-400.

Loewenstein. W.R. (1979) Junctional intercellular com-

munication and the control of growth. Biochim. Biophys.

Acta, 560. l-65.

Malkinson. A.M. and Beer, D.S. (1984) Pharmacologic

and genetic studies on the modulatory effects of butylated

hydroxytoluene on mouse lung adenoma formation. J. Natl. Cancer Inst.. 73. 925-933.

23

24

25

26

27

28

29

30

31

32

33

34

35

36

17

Malkinson, A.M., Beer. D.S.. Sadler. A.J. and Coffman,

D.S. (1985) A decrease in the protein kinase C-catalyzed

phosphorylation of an endogenous 36 K lung protein following treatment of mice with the tumor modulating

agent. butylated hydroxytoluene. Cancer Res.. 45.

575 l-5756.

Mehta, P.P.. Hotz-Wagenblatt. A.. Rose. B., Shalloway,

D. and Loewenstein. W.R. (1991) Incorporation of the

gene for a cell-cell channel protein into transformed cells

leads to normalization of growth. J. Memb. Biol., 124.

207-225.

Murray, A.W. and Fitzgerald, D.J. (1979) Tumor pro-

moters inhibit metabolic cooperation in co-cultures of epi-

dermal and 3T3 cells. Biochem. Biophys. Res. Commun.,

91. 395-401.

Nicks, K.M., Droms, K.A., Fossli. T.. Smith, G.J. and

Malkinson. A.M. (1989) Altered function of protein kin-

ase C and cyclic AMP adenosine monophosphate-

dependent protein kinase in a cell line derived from a

mouse lung papillary tumor. Cancer Res.. 49, 5191-5198.

Paul, D.L. (1986) Molecular cloning of cDNA for rat liver

gap junction protein. J. Cell Biol., 103, 123-134.

Pan. Y.H. L.. Nuzum. E., Hanson. L.A. and Beer, D.G.

( 1990) Ki-Ras activation and expression in transformed

mouse lung cell lines. Mol. Carcinogen.. 3. 279-286.

Pereira, M.A.. Knutsen, G.L. and Herren-Freund, S.L.

(1985) Effect of subsequent treatment of chloroform or

phenobarbital on the incidence of liver and lung tumors

initiated by ethylnitrosurea in I5 day old mice. Car-

cinogenesis. 6. 203-207.

Peraino, C., Fry, R.J.. Staffeldt, E. and Christopher. J.P.

(1977) Enhancing effects of phenobarbital and butylated

hydroxytoluene on 2-acetylaminofluorene-induced hepa-

tic tumorigenesis in the rat. Food Cosmet. Toxicol.. 15.

93-96.

Ruth. R.J. and Klaunig, J.E. (1986) The effects of tumor

promoters, genotoxic carcinogens, and hepatocytotoxins

on mouse hepatocyte intercellular communication. Cell

Biol. Toxicol., 2. 469-483.

Ruth. R.J. and Klaunig, J.E. (1988) Kinetics of phenobar-

bital inhibition of intercellular communication in mouse

hepatocytes. Cancer Res.. 48. 2519-2523.

Ruth, R.J. and Klaunig, J.E. (1988) Inhibition of mouse

hepatocyte intercellular communication by paraquat-

generated oxygen free radicals. Toxicol. Appl. Phar-

macol.. 94. 427-436.

Rutten. A.A.J.J.L.. Jongen, W.M.F., de Hadn, L.H.J.,

Hendriken. E.G.J. and Korman. J.H. (1988) Effect of reti-

nol and cigarette-smoke condensate on dye-coupled inter-

cellular communication between tracheal epithelial cells.

Carcinogenesis. 9. 3 15-320.

Saez, J.C.. Spray, D.C. and Hertzberg. E.L. (1990) Gap

junctions: Biochemical properties and functional regula- tion under physiological and toxicological conditions. In

Vitro Toxicol., 3. 69-86.

Slaga. T.J. (1984) Overview of tumor promotion in ani-

mals. Environ. Health Perspect.. 50. 3-14.

37 Smith. G.J.. LeMesurier. SM.. De Montfort. M.L. and

Lykke, A.W.J. (1984) Development and characterization

of type 2 pneumocyte-related cell lines from normal adult

mouse lung. Pathology, 16. 401-405.

38 Smith. G.J. and Lykke, A.W.J. (1985) Characterization of

a neoplastic epithelia) cell strain derived by dex-

amethasone treatment of cultured normal mouse type 2

pneumocytes. J. Pathol., 147, 165-172.

39 Thompson. J.A.. Bolton. J.L. and Malkinson. A.M.

(1991) Relationship between the metabolism of butylated

hydroxytoluene (BHT) and lung tumor promotion in

mice. Exp. Lung Res.. 17. 439-453.

40 Topping. D.C. and Nettesheim. P. (1980) Promotion-like

enhancement of tracheal carcinogenesis in rats by 12-0

tetradecanoylphorbol-13-acetate. Cancer Res.. 40. 4352-4355.

41 Trosko. J.E. and Chang. C.C. (1988) Nongenotoxic

mechanisms in carcinogenesis: Role of inhibited intercel-

lular communication. In: Banbury Report 31: Carcino-

genic Risk Assessment: New Directions in the Qualitative

and Quantitative Aspects, pp. 139-170. Editors: R.W.

Hart and E.G. Hoerger. Cold Spring Harbor Laboratory

Press. Cold Spring Harbor, New York.

42 Tsuchiya, E.. Concetti. H.F.. Sugano. H. and Kitagdwa.

T. (1984) Lack of promotion effect of phenobarbital on

pulmonary tumorigenesis in DDY mice initiated by

transplacental exposure to 1-ethyl-I-nitrosourea. Gann,

75. 305-310.

43 Willecke. K.. Heynkes. R., Dahl, E., Stutenkemper, R..

Hennemann. H.. Jungbluth. S.. Suchya, T. and

Nicholson, B.J. (1991) Mouse connexin37: cloning and

functional expression of a gap junction gene highly ex-

pressed in lung. J. Cell Biol., 114. 1049- 1057.

44 Williams, G.M. and Numoto, S. (1984) Promotion of mouse liver neoplasms by the organochlorine pesticides

chlordane and heptachlor in comparison to dichloro-

diphenyltrichloroethane. Carcinogenesis. 5, 1689-1696.

45 Witschi, H.P. Malkinson. A.M. and Thompson, J.A.

(1989) Metabolism and pulmonary toxicity of butylated

hydroxytoluene (BHT). Pharmacol. Ther.. 42, 89-l 13.

46 Yotti, L.P., Chang. C.C., and Trosko. J.E. (1979) Elimi-

nation of metabolic cooperation in Chinese hamster cells

by a tumor promoter. Science. 206, 1089-1091.

47 Zhang. J.-T. and Nicholson. B.J. (1989) Sequence and tis-

sue distribution of a second protein of hepatic gap junc-

tions. Cx26, as deduced from its cDNA. J. Cell Biol., 109. 3391-3401.

48 Zhu. D.. Caveney. S.. Kidder. G.M.. and Naus, C.C.G.

(1991) Transfection of C6 glioma cells with connexin 43

cDNA: Analysis of expression, intercellular coupling. and

cell proliferation. Proc. Nat). Acad. Sci., USA, 88.

1883-1887.