Although several abnormalities in gap junction (GJ) structure and/or function have been described in neoplasms, the molecular mechanisms responsible for many of the alterations remain unknown. The identification of a family of GJ proteins, termed connexins, prompted this study of connexin32 (Cx32), connexin26 (Cx26) and connexin43 (Cx43) expression during rat hepatocarcinogenesis. Using antibody, cDNA and cRNA probes, we investigated connexin mRNA and protein expression in preneoplastic and neoplastic rat livers. In normal liver, Cx32 is expressed in hepatocytes throughout the hepatic acinus, Cx26 is restricted to periportal hepatocytes, and Cx43 is expressed by mesothelial cells forming Glisson's capsule. Most preneoplastic altered hepatic foci generated by diethylnitrosamine (DEN) initiation and either phenobarbital (PB) or 2,3,7,8-dichlorodibenzo-p-dioxin (TCDD) promotion exhibited decreased Cx32 or increased Cx26 staining. Foci from either protocol failed to display Cx43 immunoreactivity. In the majority of PB-promoted foci, Cx32 immunoreactivity decreased independently of changes in mRNA abundance. Continuous thymidine labeling, following cessation of PB promotion, showed that downregulation of Cx32 staining is reversible in foci that are promoter-dependent for growth, but irreversible in lesions that are promoter-independent for growth. Hepatic neoplasms from rats initiated with DEN and promoted with PB or TCDD also displayed modified connexin expression. While all 24 neoplasms studied were deficient in normal punctate Cx32 and Cx26 staining, altered cellular localization of these proteins was apparent in some tumors. Immunoblotting of crude tissue extracts revealed that neoplasms with disordered Cx32 staining showed immunoreactive bands with altered electrophoretic mobility. These observations show that hepatomas may downregulate Cx32 expression through changes in the primary structure of Cx32 or by post-translational modifications. Northern blotting of total tumor mRNAs failed to demonstrate consistent changes in the abundance of Cx32, Cx26 or Cx43 transcripts. Some tumors expressed steady-state transcripts without observable immunoreactivity, indicating that some hepatomas downregulate connexin immunoreactivity independently of mRNA abundance. Increased levels of Cx43 mRNA and protein were found in several neoplasms, but immunostaining was always localized to nonparenchymal cells. Areas of bile duct proliferation and cholangiomas displayed Cx43 staining, whereas, cholangiocarcinomas were deficient in immunoreactivity. These findings show that alterations in the expression of connexins, by either downregulation or differential induction, represent common modifications during hepatocarcinogenesis. Although our results imply that connexins represent useful markers for the boundary between tumor promotion and progression, preneoplastic and neoplastic rat hepatocytes fail to use a common mechanism to modify connexin expression.

REFERENCES

Asamoto
M.
,
Oyamada
M.
,
El Aoumari
A.
,
Gros
D.
,
Yamasaki
H.
(
1991
).
Molecular mechanisms of TPA-mediated inhibition of gap-junctional intercellular communication: evidence for action on the assembly or function but not the expression of connexin 43 in rat liver epithelial cells.
Mol. Carcinogen
4
,
322
327
Barrio
L. C.
,
Suchyna
T.
,
Bargiello
T.
,
Xu
L. X.
,
Roginski
R. S.
,
Bennett
M. V.
,
Nicholson
B. J.
(
1991
).
Gap junctions formed by connexin 26 and 32 alone and in combination are differently affected by applied voltage.
Proc. Nat. Acad. Sci. USA
88
,
8410
8414
Beer
D. G.
,
Neveu
M. J.
,
Paul
D. L.
,
Rapp
U. R.
,
Pitot
H. C.
(
1988
).
Expression of the c-raf proto-oncogene, gamma-glutamyltranspeptidase and gap junction protein in rat liver neoplasms.
Cancer Res
48
,
1610
1617
Bennett
M. V. L.
,
Barrio
L. C.
,
Bargiello
T. A.
,
Spray
D. C.
,
Hertzberg
E.
,
Sáez
J. C.
(
1991
).
Gap junctions: new tools, new answers, new questions.
Neuron
6
,
305
320
Berthoud
V. M.
,
Iwanij
V.
,
Garcia
A. M.
,
Saez
J. C.
(
1992
).
Connexins and glucagon receptors during development of rat hepatic acinus.
Amer. J. Physiol
263
,
650
–.
Berthoud
V. M.
,
Ledbetter
M. L. S.
,
Hertzberg
E. L.
,
Saez
J. C.
(
1992
).
Connexin43 in MDCK cells: regulation by a tumor-promoting phorbol ester and Ca2+.
Eur. J. Cell Biol
57
,
40
50
Beyer
E. C.
,
Kistler
J.
,
Paul
D. L.
,
Goodenough
D. A.
(
1989
).
Antisera directed against connexin43 peptides reacts with a 43-kD protein localized to gap junctions in myocardium and other tissues.
J. Cell Biol
108
,
595
605
Beyer
E. C.
,
Paul
D. L.
,
Goodenough
D. A.
(
1990
).
Topical review: connexin family gap junctional proteins.
J. Membr. Biol
116
,
187
194
Chomczynski
P.
,
Sacchi
N.
(
1987
).
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol/chloroform extraction.
Anal. Biochem
162
,
156
159
Dragan
Y. P.
,
Rizvi
T.
,
Xu
Y.-H.
,
Hully
J. R.
,
Bawa
N.
,
Campbell
H. A.
,
Maronpot
R. R.
,
Pitot
H. C.
(
1991
).
An initiation-promotion assay in rat liver as a potential complement to the two-year carcinogenesis bioassay.
Fund. Appl. Toxicol
16
,
525
547
Eghbali
B.
,
Kessler
J. A.
,
Reid
L. M.
,
Roy
C.
,
Spray
D. C.
(
1991
).
Involvement of gap junctions in tumorigenesis: transfection of tumor cells with connexin32 cDNA retards growth in vivo.
Proc. Nat. Acad. Sci. USA
88
,
10701
10705
Faris
R. A.
,
Monfils
B. A.
,
Dunsford
H. A.
,
Hixson
D. C.
(
1991
).
Antigenic relationship between oval cells and a subpopulation of hepatic foci, nodules, and carcinomas induced by the ‘resistant hepatocyte’ model.
Cancer Res
51
,
1308
1317
Feldman
D.
,
Swarm
R. L.
,
Becker
J.
(
1981
).
Ultrastructrural study of rat liver and liver neoplasms after long-term treatment with phenobarbital.
Cancer Res
41
,
2151
2162
Fitzgerald
D. J.
,
Mesnil
M.
,
Oyamada
M.
,
Tsuda
H.
,
Ito
N.
,
Yamasaki
H.
(
1989
).
Changes in gap junction protein (connexin 32) gene expression during rat liver carcinogenesis.
J. Cell. Biochem
41
,
97
102
Hendrich
S.
,
Campbell
H. A.
,
Pitot
H. C.
(
1987
).
Quantitative stereological evalulation of four histochemical markers of altered foci in multistage hepatocarcinogenesis in the rat.
Carcinogenesis
8
,
1245
1250
Hertzberg
E. L.
,
Skibbens
R. V.
(
1984
).
A protein homologous to the 27, 000 dalton liver gap junction protein is present in a wide variety of species and tissues.
Cell
39
,
61
69
Jaenicke
R.
(
1987
).
Folding and association of proteins.
Prog. Biophys. Mol. Biol
49
,
117
237
Janssen-Timmen
U.
,
Traub
O.
,
Dermietzel
R.
,
Rabes
H. M.
,
Willecke
K.
(
1986
).
Reduced number of gap junctions in rat hepatocarcinomas detected by monoclonal antibody.
Carcinogenesis
7
,
1475
1482
Jongen
W. M. F.
,
Fitzgerald
D. J.
,
Asamoto
M.
,
Piccoli
C.
,
Slaga
T. J.
,
Gros
D.
,
Takeichi
M.
,
Yamasaki
H.
(
1991
).
Regulation of connexin 43-mediated gap junctional intercellular communication by CA2+in mouse epidermal cells is controlled by E-cadherin.
J. Cell Biol
114
,
545
555
Klaunig
J. E.
(
1991
).
Alterations in intercellular communication during the stage of promotion.
Proc. Soc. Exp. Biol. Med
198
,
688
692
Krutovskikh
V. A.
,
Oyamada
M.
,
Yamasaki
H.
(
1991
).
Sequential changes of gap-junctional intercellular communications during multistage rat liver carcinogenesis: direct measurement of communication in vivo.
Carcinogenesis
12
,
1701
1706
Laemmli
U. K.
(
1970
).
Cleavage of structural proteins during the assembly of the head of Bacteriophage T4.
Nature
227
,
680
685
Larsen
W. J.
(
1983
).
Biological implications of gap junction structure, distribution, and composition: a review.
Tissue & Cell
15
,
645
671
Lee
S. W.
,
Tomasetto
C.
,
Sager
R.
(
1991
).
Positive selection of candidate tumor suppressor genes by subtractive hybridization.
Proc. Nat. Acad. Sci. USA
88
,
2825
2829
Loewenstein
W. R.
(
1979
).
Junctional intercellular communication and the control of growth.
Biochim. Biophys. Acta
560
,
1
65
Loewenstein
W. R.
(
1981
).
Junctional intercellular communicaiton: the cell-to-cell membrane channel.
Physiol. Rev.
61
,
830
913
Loewenstein
W. R.
(
1990
).
Cell-to-cell communication and the control of growth.
Amer. Rev. Respir. Dis
142
,
48
53
MacDonald
C.
(
1982
).
Genetic complementation in hybrid cells dervied from two metabolic co-operation defective mammalian cell lines.
Exp. Cell Res
138
,
303
310
Mehta
P. P.
,
Hotz-Wagenblatt
A.
,
Rose
B.
,
Shalloway
D.
,
Loewenstein
W. R.
(
1991
).
Incorporation of the gene for a cell-cell channel protein into transformed cells leads to normalization of growth.
J. Membr. Biol
124
,
207
225
Mehta
P. P.
,
Loewenstein
W. R.
(
1991
).
Differential regulation of communication by retinoic acid in homologous and heterologous junctions between normal and transformed cells.
J. Cell Biol
113
,
371
379
Miyashita
T.
,
Takeda
A.
,
Iwai
M.
,
Shimazu
T.
(
1992
).
Single administration of hepatotoxic chemicals transiently decreases the gap-junction-protein levels of connexin 32 in rat liver.
Eur. J. Biochem
196
,
37
42
Musil
L. S.
,
Cunningham
B. A.
,
Edelman
G. M.
,
Goodenough
D. A.
(
1990
).
Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and-deficient cell lines.
J. Cell Biol
111
,
2077
2088
Musil
L. S.
,
Goodenough
D. A.
(
1990
).
Gap junctional intercellular communication and the regulation of connexin expression and function.
Curr. Opin. Cell Biol
2
,
875
880
Nakatsukasa
H.
,
Nagy
P.
,
Evarts
R. P.
,
Hsia
C.-C.
,
Marsden
E.
,
Thorgeirsson
S. S.
(
1990
).
Cellular distribution of transforming growth factor-1 and procollagen types I, III, and IV transcripts in carbon tetrachloride-induced rat liver fibrosis.
J. Clin. Invest
85
,
1833
1843
Naus
C. C. G.
,
Elisevich
K.
,
Zhu
D.
,
Belliveau
D. J.
,
Del Maestro
R. F.
(
1992
).
In vivo growth of C6 glioma cells transfected with connexin43 cDNA.
Cancer Res
52
,
4208
4213
Neveu
M. J.
,
Hully
J. R.
,
Paul
D. L.
,
Pitot
H. C.
(
1990
).
Reversible alteration in the expression of the gap junctional protein connexin 32 during tumor promotion in rat liver and its role during cell proliferation.
Cancer Commun
2
,
21
31
Oyamada
M.
,
Krutovskikh
V. A.
,
Mesnil
M.
,
Partensky
C.
,
Berger
F.
,
Yamasaki
H.
(
1990
).
Aberrant expression of gap junction gene in primary human hepatocellular carcinomas: increased expression of cardiac-type gap junction gene connexin 43.
Mol. Carcinogen
3
,
273
278
Paul
D. L.
(
1986
).
Molecular cloning of cDNA from rat liver GJ protein.
J. Cell Biol
103
,
123
134
Peterson
G. L.
(
1977
).
A simplification of the protein assay method of Lowry which is more generally applicable.
Anal. Biochem
83
,
346
356
Rentrop
M.
,
Knapp
B.
,
Winter
H.
,
Schweizer
J.
(
1986
).
Aminoalkylsilane-treated glass slides as support for in situ hybridization of keratin cDNAs to frozen tissue sections under varying fixation and pretreatment conditions.
Histochem. J
18
,
271
276
Robenek
H.
,
Rassat
J.
,
Themann
H.
(
1981
).
A quantitative freeze-fracture analysis of gap and tight junctions in the normal and cholestatic human liver.
Virchows Arch. B Cell Pathol
38
,
39
56
Rosenberg
E.
,
Spray
D. C.
,
Reid
L. M.
(
1992
).
Transcriptional and posttranscriptional control of connexin mRNAs in periportal and pericentral rat hepatocytes.
Eur. J. Cell Biol
59
,
21
26
Saeter
G.
,
Seglen
P. O.
(
1990
).
Cell biology of hepatocarcinogenesis.
Crit. Rev. Oncogen
1
,
437
466
Sakamoto
H.
,
Oyamada
M.
,
Enomoto
K.
,
Mori
M.
(
1992
).
Differential changes in expression of gap junction proteins connexin 26 and 32 during hepatocarcinogenesis in rats.
Jpn J. Cancer Res
83
,
1210
1215
Spray
D. C.
,
Chanson
M.
,
Moreno
A. P.
,
Dermietzel
R.
,
Meda
P.
(
1991
).
Distinctive gap junction channel types connect WB cells, a clonal cell line derived from rat liver.
Amer. J. Physiol
260
,
513
–.
Stutenkemper
R.
,
Geisse
S.
,
Jurgen Schwarz
H.
,
Look
J.
,
Traub
O.
,
Nicholson
B. J.
,
Willecke
K.
(
1992
).
The hepatocyte-specific phenotype of murine liver cells correlates with high expression of connexin32 and connexin26 but very low expression of connexin43.
Exp. Cell Res
201
,
43
54
Swift
J. G.
,
Mukherjee
T. M.
,
Rowland
R.
(
1983
).
Intercellular junctions in hepatocellular carcinoma.
J. Submicrosc. Cytol
15
,
799
810
Traub
O.
,
Druge
P. M.
,
Willecke
K.
(
1983
).
Degradation and resynthesis of gap junction protein in plasma membranes of regenerating liver after partial hepatectomy or cholestasis.
Proc. Nat. Acad. Sci. USA
80
,
755
759
Traub
O.
,
Look
J.
,
Dermietzel
R.
,
Brummer
F.
,
Hulser
D.
,
Willecke
K.
(
1989
).
Comparative characterization of the 21-kD and 26-kD gap junction proteins in murine liver and cultured hepatocytes.
J. Cell. Biol
108
,
1039
1051
Wilgenbus
K. K.
,
Kirkpatrick
C. J.
,
Knuechel
R.
,
Willecke
K.
,
Traub
O.
(
1992
).
Expression of Cx26, Cx32 and Cx43 gap junctional proteins in normal and neoplastic human tissues.
Int. J. Cancer
51
,
522
529
Yamamoto
T.
,
Hertzberg
E. L.
,
Nagy
J. I.
(
1990
).
Epitopes of gap junction proteins localized to neuronal subsurface cisterns.
Brain Res
527
,
135
139
Yamasaki
H.
(
1991
).
Aberrant expression and function of gap junctions during carcinogenesis.
Environ. Health Persp
93
,
191
197
Zhang
J. T.
,
Nicholson
B. J.
(
1989
).
Sequence and tissue distribution of a second protein of hepatic gap junctions, Cx26, as deduced from its cDNA.
J. Cell Biol
109
,
3391
3401
This content is only available via PDF.