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Induction of Skin Papillomas, Carcinomas, and Sarcomas in Mice in Which the Connexin 43 Gene is Heterologously Deleted

2000; Elsevier BV; Volume: 114; Issue: 2 Linguagem: Inglês

10.1046/j.1523-1747.2000.00873.x

1523-1747

Kohji Yamakage, Yasufumi Omori, M.L. Dagli, Marie‐Pierre Cros, Hiroshi Yamasaki,

Nicotinic Acetylcholine Receptors Study

It has been suggested that blocked gap junctional intercellular communication plays a crucial part in multistage carcinogenesis. The mouse skin tumor-promoting phorbol esters are potent inhibitors of gap junctional intercellular communication and this inhibition is considered to be a mechanism by which clonal expansion of ‘‘initiated’’ cells is promoted. We examined whether mice in which the gene for a gap junction protein, connexin 43, is heterozygously deleted are more susceptible to chemical carcinogenesis; connexin 43 is expressed in the basal cell layer and the dermis of the skin. When the back skin was painted with 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol 13-acetate, the incidence and yields of both papillomas and carcinomas were similar in connexin 43+/− and connexin 43+/+ mice; for this experiment, the original mice with C57BL/6 genetic background was crossed with CD1 strain for three generations. Subcutaneous injection of 7,12-dimethylbenz[a]anthracene resulted in induction of fibrosarcomas in connexin 43+/− and connexin 43+/+ mice to a similar extent. All papillomas and carcinomas induced with 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol 13-acetate contained the 7,12-dimethylbenz[a] anthracene-specific mutation in the ras gene (A to T transversion at the 61st codon). About 50% of fibrosarcomas also contained this mutation, but in the Ki-ras gene; there was no difference in the prevalence of this mutation in tumors from connexin 43+/− and connexin 43+/+ mice. None of the tumors examined, however, showed any mutation in the connexin 43 gene. These results suggest that the deletion of one allele of the connexin 43 gene does not significantly contribute to, nor alter, the molecular events involved in skin carcinogenesis. These results are compatible with previous observations that nongenetic disruption of function rather than mutations of connexins, commonly occurs in cancer cells. It has been suggested that blocked gap junctional intercellular communication plays a crucial part in multistage carcinogenesis. The mouse skin tumor-promoting phorbol esters are potent inhibitors of gap junctional intercellular communication and this inhibition is considered to be a mechanism by which clonal expansion of ‘‘initiated’’ cells is promoted. We examined whether mice in which the gene for a gap junction protein, connexin 43, is heterozygously deleted are more susceptible to chemical carcinogenesis; connexin 43 is expressed in the basal cell layer and the dermis of the skin. When the back skin was painted with 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol 13-acetate, the incidence and yields of both papillomas and carcinomas were similar in connexin 43+/− and connexin 43+/+ mice; for this experiment, the original mice with C57BL/6 genetic background was crossed with CD1 strain for three generations. Subcutaneous injection of 7,12-dimethylbenz[a]anthracene resulted in induction of fibrosarcomas in connexin 43+/− and connexin 43+/+ mice to a similar extent. All papillomas and carcinomas induced with 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol 13-acetate contained the 7,12-dimethylbenz[a] anthracene-specific mutation in the ras gene (A to T transversion at the 61st codon). About 50% of fibrosarcomas also contained this mutation, but in the Ki-ras gene; there was no difference in the prevalence of this mutation in tumors from connexin 43+/− and connexin 43+/+ mice. None of the tumors examined, however, showed any mutation in the connexin 43 gene. These results suggest that the deletion of one allele of the connexin 43 gene does not significantly contribute to, nor alter, the molecular events involved in skin carcinogenesis. These results are compatible with previous observations that nongenetic disruption of function rather than mutations of connexins, commonly occurs in cancer cells. connexin 7,12-dimethylbenz[a] anthracene gap junctional intercellular communication Gap junctional intercellular communication (GJIC) is believed to play a key part in the maintenance of homeostasis by keeping the level of second messengers equal among gap junction-connected cells in a given tissue. The gap junction channels are considered to be ‘‘connexons’’ composed of hexamers of proteins called connexins and so far, 13 different connexin genes have been identified (Bruzzone et al., 1996Bruzzone R. White T.W. Paul D.L. Connections with connexins: the molecular basis of direct intercellular signaling.Eur J Biochem. 1996; 238: 1-27Crossref PubMed Scopus (1236) Google Scholar;Goodenough et al., 1996Goodenough D.A. Goliger J.A. Paul D.L. Connexins, connexons, and intercellular communication.Annu Rev Biochem. 1996; 65: 475-502Crossref PubMed Scopus (1080) Google Scholar). Reflecting the importance of GJIC in homeostasis, mutations of connexin genes are associated with various human diseases, such as X-linked Charcot–Marie–Tooth syndrome (Bergoffen et al., 1993Bergoffen J. Scherer S.S. Wang S. et al.Connexin mutations in X-linked Charcot-Marie-Tooth disease.Science. 1993; 262: 2039-2042Crossref PubMed Scopus (972) Google Scholar), hereditary nonsyndromic deafness (Kelsell et al., 1997Kelsell D.P. Dunlop J. Stevens H.P. et al.Connexin 26 mutations in hereditary non-syndromic sensorineural deafness.Nature. 1997; 387: 80-83Crossref PubMed Scopus (1227) Google Scholar;Xia et al., 1998Xia J.H. Liu C.Y. Tang B.S. et al.Mutations in the gene encoding gap junction protein beta-3 associated with autosomal dominant hearing impairment.Nat Genet. 1998; 20: 370-373Crossref PubMed Scopus (384) Google Scholar), and heterotaxia (Britz-Cunningham et al., 1995Britz-Cunningham S.H. Shah M.M. Zuppan C.W. Fletcher W.H. Mutations of the connexin43 gap-junction gene in patients with heart malformations and defects of laterality.N Engl J Med. 1995; 332: 1323-1329Crossref PubMed Scopus (351) Google Scholar). Mutation of the connexin (Cx) 31 gene has also been reported to predispose patients to a skin disease, erythrokeratodermia variabilis (Richard et al., 1998Richard G. Smith L.E. Bailey R.A. et al.Mutations in the human connexin gene GJB3 cause erythrokeratodermia variabilis.Nat Genet. 1998; 20: 366-369Crossref PubMed Scopus (302) Google Scholar). In addition, the essential roles of Cx genes in differentiation and development have been proven in Cx43-deficient (knock-out) and Cx43-transgenic mice (Reaume et al., 1995Reaume A.G. de Sousa P.A. Kulkarni S. et al.Cardiac malformation in neonatal mice lacking connexin43.Science. 1995; 267: 1831-1834Crossref PubMed Scopus (1126) Google Scholar;Ewart et al., 1997Ewart J.L. Cohen M.F. Meyer R.A. et al.Heart and neural tube defects in transgenic mice overexpressing the Cx43 gap junction gene.Development. 1997; 124: 1281-1292PubMed Google Scholar), Cx46-deficient mice (Gong et al., 1997Gong X. Li E. Klier G. et al.Disruption of alpha3 connexin gene leads to proteolysis and cataractogenesis in mice.Cell. 1997; 91: 833-843Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar), Cx37-deficient mice (Simon et al., 1997Simon A.M. Goodneough D.A. Li E. Paul D.L. Female infertility in mice lacking connexin 37.Nature. 1997; 385: 525-529Crossref PubMed Scopus (585) Google Scholar), Cx40-deficient mice (Kirchhoff et al., 1998Kirchhoff S. Nelles E. Hagendorff A. Kruger O. Traub O. Willecke K. Reduced cardiac conduction velocity and predisposition to arrhythmias in connexin40-deficient mice.Curr Biol. 1998; 26: 299-302Abstract Full Text Full Text PDF Scopus (326) Google Scholar), and Cx26-deficient mice (Gabriel et al., 1998Gabriel H.D. Jung D. Butzler C. Temme A. Traub O. Winterhager E. Willecke K. Transplacental uptake of glucose is decreased in embryonic lethal connexin26-deficient mice.J Cell Biol. 1998; 140: 1453-1461Crossref PubMed Scopus (234) Google Scholar), all of which show developmental embryonic death or malfunction of specific tissues. Based on the idea that deviation from normal homeostasis is a key event in carcinogenesis, the role of aberrant GJIC has long been postulated (Yamasaki and Naus, 1996Yamasaki H. Naus C.C. Role of connexin genes in growth control.Carcinogenesis. 1996; 17: 1199-1213Crossref PubMed Scopus (451) Google Scholar;Trosko and Ruch, 1998Trosko J.E. Ruch R.J. Cell-cell communication in carcinogenesis.Front Biosc. 1998; 15: D208-D236Google Scholar). Since the discovery that a human tumor lacked GJIC capacity (Loewenstein and Kanno, 1966Loewenstein W. Kanno Y. Intercellular communication and the control of the tissue growth; lack of communication between cancer cells.Nature. 1966; 209: 1248-1249Crossref PubMed Scopus (326) Google Scholar), this has been confirmed and extended in numerous studies, so that aberrant GJIC is now regarded as a hallmark of cancer cells (Yamasaki and Naus, 1996Yamasaki H. Naus C.C. Role of connexin genes in growth control.Carcinogenesis. 1996; 17: 1199-1213Crossref PubMed Scopus (451) Google Scholar). GJIC in cultured cells as well as in rat liver and mouse skin in vivo is inhibited by promoting agents (Yamasaki, 1996Yamasaki H. Role of disrupted gap junctional intercellular communication in detection and characterization of carcinogens.Mutat Res. 1996; 365: 91-105Crossref PubMed Scopus (81) Google Scholar). Disappearance of gap junctional structures in mouse skin in vivo after treatment with the tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA) has also been reported (Kalimi and Sirsat, 1984Kalimi G.H. Sirsat S.M. Phorbol ester tumor promoter affects the mouse epidermal gap junctions.Cancer Lett. 1984; 22: 343-350Abstract Full Text PDF PubMed Scopus (34) Google Scholar). Cells transformed by chemical carcinogens or oncogenes such as v-ras, v-src, and v-raf display loss of homologous or heterologous GJIC (Enomoto and Yamasaki, 1984Enomoto T. Yamasaki H. Lack of intercellular communication between chemically transformed and surrounding nontransformed BALB/c 3T3 cells.Cancer Res. 1984; 44: 5200-5203PubMed Google Scholar;Azarnia et al., 1988Azarnia R. Reddy S. Kmiecik T.E. Shalloway D. Loewenstein W.R. The cellular src gene product regulates junctional cell-to-cell communication.Science. 1988; 239: 398-401Crossref PubMed Scopus (138) Google Scholar;Bignami et al., 1988Bignami M. Rosa S. Falcone G. Tato F. Katoh F. Yamasaki H. Specific viral oncogenes cause differential effects on cell-to-cell communication, relevant to the suppression of the transformed phenotype by normal cells.Mol Carcinog. 1988; 1: 67-75Crossref PubMed Scopus (59) Google Scholar). When a connexin gene is transfected into transformed or cancerous cells, tumor formation, cell growth, or both are suppressed (Kalimi et al., 1992Kalimi G.H. Hampton L.L. Trosko J.E. Thorgeirsson S.S. Huggett A.C. Homologous and heterologous gap-junctional intercellular communication in v-raf, v-myc, and v-raf/v-myc-transduced rat liver epithelial cell lines.Mol Carcinog. 1992; 5: 301-310Crossref PubMed Scopus (46) Google Scholar;Eghbali et al., 1991Eghbali B. Kessler J.A. Reid L.M. Roy C. Spray D.C. Involvement of gap junctions in tumorigenesis: Transfection of tumor cells with connexin 32 cDNA retards growth in vivo.Proc Natl Acad Sci USA. 1991; 88: 10701-10705Crossref PubMed Scopus (230) Google Scholar;Rose et al., 1993Rose B. Mehta P.P. Loewenstein W.R. Gap-junction protein gene suppresses tumorigenicity.Carcinogenesis. 1993; 14: 1073-1075Crossref PubMed Scopus (171) Google Scholar;Mesnil et al., 1995Mesnil M. Krutovskikh V. Piccoli C. et al.Negative growth control of HeLa cells by connexin genes: Connexin species specificity.Cancer Res. 1995; 55: 629-639PubMed Google Scholar). Inhibition of heterologous GJIC by transfection of anti-sense Cx43 into normal cells enhances tumor formation (Goldberg et al., 1994Goldberg G.S. Martyn K.D. Lau A.F. A connexin 43 antisense vector reduces the ability of normal cells to inhibit the foci formation of transformed cells.Mol Carcinog. 1994; 11: 106-114Crossref PubMed Scopus (62) Google Scholar), and restoration of heterologous GJIC in transformed cells by chemicals (Yamasaki and Katoh, 1988Yamasaki H. Katoh F. Further evidence for the involvement of gap-junctional intercellular communication in induction and maintenance of transformed foci in BALB/c 3T3 cells.Cancer Res. 1988; 48: 3490-3495PubMed Google Scholar) or by transfection of a connexin gene suppresses cell transformation (Esinduy et al., 1995Esinduy C.B. Chang C.C. Trosko J.E. Ruch R.J. In vitro growth inhibition of neoplastically transformed cells by non-transformed cells: Requirement for gap junctional intercellular communication.Carcinogenesis. 1995; 16: 915-921Crossref PubMed Scopus (54) Google Scholar). In some experimental tumors, mutations of connexin genes have been reported (Omori et al., 1996Omori Y. Krutovskikh V. Mironov N. Tsuda H. Yamasaki H. Cx32 gene mutation in a chemically induced rat liver tumour.Carcinogenesis. 1996; 17: 2077-2080Crossref PubMed Scopus (56) Google Scholar;Saito et al., 1997Saito T. Barbin A. Omori Y. Yamasaki H. Connexin 37 mutations in rat hepatic angiosarcomas induced by vinyl chloride.Cancer Res. 1997; 57: 375-377PubMed Google Scholar). These results suggest that intact homologous and/or heterologous GJIC is necessary for normal cell growth control and that connexin genes have tumor-suppressive effects. Recent experiments using Cx32 gene knock-out mice have provided more direct evidence for the involvement of aberrant GJIC in carcinogenesis. Mice lacking the Cx32 gene, one of the major connexins in liver, had 8–25-fold more spontaneous liver tumors per mouse than wild-type mice (Temme et al., 1997Temme A. Buchmann A. Gabriel H.D. Nelles E. Schwarz M. Willecke K. High incidence of spontaneous and chemically induced liver tumors in mice deficient for connexin32.Curr Biol. 1997; 7: 713-716Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). In addition, when such mice were treated with the carcinogen diethylnitrosamine, liver tumors appeared at earlier times and were larger than the spontaneous tumors. In order to determine whether an effect of a specific connexin gene deletion could also clearly be seen in skin carcinogenesis, we employed Cx43-deficient mice, as the Cx43 gene is expressed in the basal layer of the skin as well as in the dermis (Butterweck et al., 1994Butterweck A. Elfgang C. Willecke K. Traube O. Differential expression of the gap junction proteins connexin45–43–31, and -26 in mouse skin.Eur J Cell Biol. 1994; 65: 152-163PubMed Google Scholar). Abnormal expression of Cx43 in skin tumors has already been reported (Budunova et al., 1995Budunova I.V. Carbajal S. Slaga T.J. The expression of gap junctional proteins during different stages of mouse skin carcinogenesis.Carcinogenesis. 1995; 16: 2717-2724Crossref PubMed Scopus (44) Google Scholar;Kamibayashi et al., 1995Kamibayashi Y. Oyamada Y. Mori M. Oyamada M. Aberrant expression of gap junction proteins (connexins) is associated with tumor progression during multistage mouse skin carcinogenesis in vivo.Carcinogenesis. 1995; 16: 1287-1297Crossref PubMed Scopus (94) Google Scholar;Sawey et al., 1996Sawey M.J. Goldschmidt M.H. Risek B. Gilula N.B. Lo S.W. Perturbation in connexin 43 and connexin 26 gap-junction expression in mouse skin hyperplasia and neoplasia.Mol Carcinog. 1996; 17: 49-61Crossref PubMed Scopus (62) Google Scholar). As Cx43–/− mice are not viable, we used Cx43+/− mice; such heterozygous mice can be considered as germ-line carriers of loss of heterozygosity in terms of the Cx43 locus which may be more vulnerable to carcinogenic insults. We induced epidermal tumors as well as dermal tumors in these mice with 7,12-dimethylbenz[a] anthracene (DMBA) and compared the results with those from wild-type control mice. Cx43-deficient mice were originally established byReaume et al., 1995Reaume A.G. de Sousa P.A. Kulkarni S. et al.Cardiac malformation in neonatal mice lacking connexin43.Science. 1995; 267: 1831-1834Crossref PubMed Scopus (1126) Google Scholar and we purchased them from Jackson Laboratories (Paris). As Cx43-nullzygous mice die just after birth due to cardiac malformation (Reaume et al., 1995Reaume A.G. de Sousa P.A. Kulkarni S. et al.Cardiac malformation in neonatal mice lacking connexin43.Science. 1995; 267: 1831-1834Crossref PubMed Scopus (1126) Google Scholar), heterozygous mice were used for breeding. The original genetic background of these mice was C57BL/6, and we crossed them with a CD1 mice to generate heterozygous Cx43-deficient mice with CD1 genetic background, as C57BL/6 are known to be resistant to epidermal carcinogenesis. For identification of the genotype of the mice, a small piece of tail was digested in 0.7 ml of extraction buffer (0.1 M NaCl, 0.05 M Tris–HCl, 0.1 M ethylenediamine tetraacetic acid, 0.49 mg protease K per ml, 1% sodium dodecyl sulfate) at 37°C overnight. After shaking, 0.25 ml of 5 M NaCl was added and mixed well, then samples were centrifuged. About 0.7 ml of the aqueous phase was transferred into a new tube and 0.5 ml of isopropanol was added to precipitate DNA. After centrifuging, DNA was washed with 70% ethanol and dissolved in 0.5 ml of TE buffer. DNA from each mouse was analyzed by PCR with primers for neo genes as described previously (Yamakage et al., 1998Yamakage K. Omori Y. Piccoli C. Yamasaki H. Growth control of 3T3 fibroblast cell line established from connexin 43-deficient mice.Mol Carcinog. 1998; 23: 121-128Crossref PubMed Scopus (14) Google Scholar) For the two-stage carcinogenicity study by skin painting, female Cx43+/+ and Cx43+/− mice were used, which had been crossed with CD1 mice for three generations. We also included female C57BL/6 and CD1 mice as reference controls. The dorsal lumbar region of 7 wk old mice was clipped clean of fur with an electric hair clipper. After clipping, each mouse was painted with 10 μg DMBA (Sigma, St Louis, MO) or acetone (0.1 ml) twice a week for 5 wk followed by painting with 10 μg TPA (Chemsyn Science Lab., Lenexa, Kansas) or acetone (0.1 ml) twice a week for more than 40 wk. Fifteen mice were used in each group; the mice were observed daily and tumor appearance and numbers were recorded weekly. In order to induce fibrosarcomas, 12–15 wk old male mice with C57BL/6 genetic background were subcutaneously injected once with 250 μg DMBA at two dorsal regions. The Cx43 gene was screened for mutations by PCR–single strand conformation polymorphism analysis (Omori et al., 1996Omori Y. Krutovskikh V. Mironov N. Tsuda H. Yamasaki H. Cx32 gene mutation in a chemically induced rat liver tumour.Carcinogenesis. 1996; 17: 2077-2080Crossref PubMed Scopus (56) Google Scholar;Saito et al., 1997Saito T. Barbin A. Omori Y. Yamasaki H. Connexin 37 mutations in rat hepatic angiosarcomas induced by vinyl chloride.Cancer Res. 1997; 57: 375-377PubMed Google Scholar). A reaction mixture composed of sample DNA, PCR buffer, Taq polymerase, primers, dNDP, and [α-33]dCTP was prepared, then PCR amplification of the Cx43 gene was performed in sequential cycles (35 cycles) at 94°C for 1 min, 61°C for 1 min 50 s and 72°C for 1 min 30 s, followed by incubation for 10 min at 72°C. Six pairs of primers were used to cover all exons of the Cx43 gene as shown in Table 1.Table IPairs of primers used to cover all exons of the Cx43 genePrimer 1sense5′-TGTTTTTCTTAGGAGGTGCC-3′Primer 1anti-sense5′-GTTACAGCGAAAGGCAGACT-3′Primer 2sense5′-TTTTCAGAATCCTGCTCCTG-3′Primer 2anti-sense5′-GCTTCTCTTCCTTTCTCATC-3′Primer 3sense5′-ACACTCCTGTACTTGGCTCAC-3′Primer 3anti-sense5′-CGAAGACAGACTTGAAGAGG-3′Primer 4sense5′-TGAGAACCTACATCATCAGC-3′Primer 4anti-sense5′-GATCCTTAACGCCCTTGAAG-3′Primer 5sense5′-GGTGTCTCTCGCTCTGAATA-3′Primer 5anti-sense5′-TTTTGCTCGCTGGCTTGCTT-3′Primer 6sense5′-ATTCCTCCTGCCGCAATTAC-3′Primer 6anti-sense5′-CAGCTTGATGTTCAAGCCTG-3′ Open table in a new tab PCR products were mixed with same volume of F-dye, then denatured for more than 5 min at 94°C, 4 μl of samples were applied to 6% polyacrylamide gel (acrylamide: bisacrylamide, 99:1) with 5% glycerol. After electrophoresis, the gels were dried and exposed to X-ray film (Biomax; Eastma Kodak). The A to T mutation at the 61st codon of the Ki-ras and Ha-ras gene was analysed by PCR–restriction fragment length polymorphism as described previously (Sasaki et al., 1995Sasaki K. Bertrand O. Nakazawa H. Fitzgerald D.J. Mironov N. Yamasaki H. Cell-type-specific ras mutations but not microsatellite instability in chemically induced mouse skin tumors and transformed 3T3 cells.Cancer Res. 1995; 55: 3513-3516PubMed Google Scholar). The primers used for the Ki-ras gene were 5′-GACTCCTACAGGAAACAAGT and 5′-CTATAATGG-TGAATATCTTC and those for the H-ras gene were 5′-GACTCCTAC-CGGAAACAGGT and 5′-AGGAAGCCCTCCCCTGTGCG. The PCR products were digested with XbaI; these digested products were then loaded on to 6% acrylamide gel. An A to T mutation at this codon in both ras genes creates an XbaI digestion site (Bizub et al., 1986Bizub D. Wood A.V. Skalka A.M. Mutagenesis of the Ha-ras oncogene in mouse skin tumors induced by polycyclic aromatic hydrocarbons.Proc Natl Acad Sci USA. 1986; 83: 6048-6052Crossref PubMed Scopus (219) Google Scholar). The skin tumor incidence and yields in various mouse lines after painting with DMBA and/or TPA are summarized in Table 2. As expected, C57BL/6 mice were more resistant to skin carcinogenesis induced by DMBA and TPA. We crossed the Cx43+/− mice of C57BL/6 background with CD1 mice three times to create more susceptible transgenic mice. Theoretically, this will not completely convert the genetic background. As the incidence and yield of papillomas in Cx43+/+ mice were similar to those found in wild-type CD1 mice, however, we conclude that the genetic background of our Cx43+/+ mice was almost completely converted from that of C57BL/6 to CD1 in terms of their susceptibility to epidermal carcinogenesis. There was no significant difference in sensitivity between Cx43+/+ and Cx43+/− mice for skin papilloma induction by DMBA/TPA, but there were more papillomas in Cx43+/+ than Cx43+/− mice after painting with TPA alone. We did not observe any tumors in any mouse line treated with DMBA alone. Some skin papillomas were converted into carcinomas and the rate of such conversion was again similar in Cx43+/+ and Cx43+/− mice (Table 2).Table IIIncidence and yield of skin tumors induced by painting with DMBA and/or TPA on Cx43+/+ and Cx43+/− miceaExperimental details are described in Materials and Methods.No. ot tumorsNo. of mice withGenotype of Cx43InitiationPromotionNo. of mice usedPapillomaCarcinomaPapillomaCarcinoma+/+bOriginal background strain was C57BL. For this experiment, they were crossed with CD-1 mice. The experiments were performed with offsprings after crossing with CD-1 three times.AcetoneAcetone150000AcetoneTPA15261101DMBAAcetone150000DMBATPA154011151+/–bOriginal background strain was C57BL. For this experiment, they were crossed with CD-1 mice. The experiments were performed with offsprings after crossing with CD-1 three times.AcetoneAcetone150000AcetoneTPA1511060DMBAAcetone150000DMBATPA153333153+/+AcetoneAcetone150000(original CD-1)DMBATPA153452152+/+AcetoneAcetone150000(originalC57BL/6)DMBATPA15551141a Experimental details are described in Materials and Methods.b Original background strain was C57BL. For this experiment, they were crossed with CD-1 mice. The experiments were performed with offsprings after crossing with CD-1 three times. Open table in a new tab The time course of skin papilloma induction by DMBA and TPA painting is shown in Figure 1. It is again clear that there was no difference between Cx43+/+ and Cx43+/− mice in the papilloma induction rate. The first fibrosarcoma appeared in Cx43+/− mice 15 wk after subcutaneous injection of DMBA. The appearance of the first fibrosarcoma was 1 wk later in Cx43+/+ mice, but the percentage of mice bearing a tumor reached the same level 2 wk later (Figure 2). As shown in Table 3, the final percentage of mice bearing tumors was very similar between Cx43+/+ and Cx43+/− mice. Although Cx43+/− mice showed only fibrosarcomas, the Cx43+/+ mice had two rhabdomyosarcomas, a liposarcoma and an anaplastic sarcoma (Table 3).Table IIIIncidence of fibrosarcomas in mice subcutaneously injected with 250 μg of DMBAaExperimental details are described in Materials and Methods.Genotype of Cx43InitiationNo. of mice usedNo. of injectionsNo. of fibrosarcomas (%)No. of other malignant tumors (%)+/+Olive oil10200 (0)0 (0)DMBA91810 (55.6)4bTwo rhabdomyosarcomas, one liposarcoma, one anaplastic sarcoma.(22.2)+/–Olive oil9180 (0)0 (0)DMBA7149 (64.3)0 (0)a Experimental details are described in Materials and Methods.b Two rhabdomyosarcomas, one liposarcoma, one anaplastic sarcoma. Open table in a new tab We examined the presence of Cx43 gene mutations in tumors induced by DMBA and/or TPA in various mouse lines. We specifically considered the possibility that the remaining allele of the Cx43 gene in Cx43+/− mice might be a target for DMBA mutagenesis. Examination of mutations by single strand conformation polymorphism analysis of the whole coding region of the Cx43 gene revealed no mutation in papillomas, carcinomas or fibrosarcomas that developed in any line of mice (Table 4).Table IVCx43, Ki-, and Ha-ras mutations in fibrosarcomas, papillomas and carcinomasaMutation analysis was performed as described in Materials and Methods.No. of mutations/No. of tumors analysedTumorGenotype of Cx43Cx43Ki-ras (%)H-ras (%)FibrosarcomabTumors from the experiment presented in Table 3.+/+0/10 (0.0)8/10 (80.0)0/10 (0.0)+/–0/9 (0.0)5/9 (55.6)0/9 (0.0)Total0/19 (0.0)13/19 (68.4)0/19 (0.0)PapillomacTumors from the experiment presented in Table 2.+/+0/16 (0.0)NT6/6 (100.0)+/–0/13 (0.0)NT9/9 (100.0)Total0/29 (0.0)15/15 (100.0)CarcinomacTumors from the experiment presented in Table 2.+/+0/2 (0.0)NT1/1 (100.0)+/–0/1 (0.0)NTNTTotal0/3 (0.0)1/1 (100.0)a Mutation analysis was performed as described in Materials and Methods.b Tumors from the experiment presented in Table 3.c Tumors from the experiment presented in Table 2. Open table in a new tab When skin tumors are induced by DMBA, many of them contain a specific A to T transversion at the 61st codon of the Ha-ras or Ki-ras gene. In papillomas and carcinomas, the Ha-ras gene is usually mutated, whereas the Ki-ras gene is mutated in fibrosarcomas (Bizub et al., 1986Bizub D. Wood A.V. Skalka A.M. Mutagenesis of the Ha-ras oncogene in mouse skin tumors induced by polycyclic aromatic hydrocarbons.Proc Natl Acad Sci USA. 1986; 83: 6048-6052Crossref PubMed Scopus (219) Google Scholar;Sasaki et al., 1995Sasaki K. Bertrand O. Nakazawa H. Fitzgerald D.J. Mironov N. Yamasaki H. Cell-type-specific ras mutations but not microsatellite instability in chemically induced mouse skin tumors and transformed 3T3 cells.Cancer Res. 1995; 55: 3513-3516PubMed Google Scholar). We were interested in whether the absence of one allele of Cx43 gene in Cx43+/− mice would affect the molecular events of DMBA-induced carcinogenesis. PCR/restriction fragment length polymorphism analysis revealed the presence of A to T mutations at the 61st codon of the Ha-ras gene in papillomas and carcinomas. On the other hand, only Ki-ras gene mutations were observed in fibrosarcomas, confirming our earlier results (Figure 3). As summarized in Table 4, the prevalence of Ha-ras and Ki-ras gene mutations in papillomas or carcinomas and fibrosarcomas, respectively, was similar among those that developed in mice with different Cx43 genotypes. These results suggest that the absence of one allele of the Cx43 gene did not affect the molecular mechanisms involved in DMBA-induced carcinogenesis. We have shown here that Cx43+/− mice are not more susceptible than Cx43+/+ mice to skin carcinogenesis after DMBA/TPA painting and to fibrosarcoma production by subcutaneous injection of DMBA. As none of the tumors induced in Cx43+/− mice carried mutations in the remaining allele of the Cx43 gene, it is likely that loss of Cx43 gene function as a result of mutations is not a major pathway of DMBA-induced skin carcinogenesis. This is in line with our previous finding that connexin gene mutations occur rarely in tumors: we detected only one Cx32 mutation in a rat liver tumor and one Cx37 mutation in a rat hemangiosarcoma (Omori et al., 1996Omori Y. Krutovskikh V. Mironov N. Tsuda H. Yamasaki H. Cx32 gene mutation in a chemically induced rat liver tumour.Carcinogenesis. 1996; 17: 2077-2080Crossref PubMed Scopus (56) Google Scholar;Saito et al., 1997Saito T. Barbin A. Omori Y. Yamasaki H. Connexin 37 mutations in rat hepatic angiosarcomas induced by vinyl chloride.Cancer Res. 1997; 57: 375-377PubMed Google Scholar). In fact, we have found no mutations in the Cx32, Cx37, and Cx43 genes in human liver, stomach, lung, and breast cancers, nor in meningiomas (Krutovskikh et al., 1994Krutovskikh V. Mazzoleni G. Mironov N. et al.Altered homologous and heterologous gap junctional intercellular communication in primary human liver tumors associated with aberrant protein localization but not gene mutation of connexin 32.Int J Cancer. 1994; 56: 87-94Crossref PubMed Scopus (179) Google Scholar,Krutovskikh et al., 1996Krutovskikh V. Mironov N. Yamasaki H. Human connexin37 is polymorphic but not mutated in tumors.Carcinogenesis. 1996; 17: 1761-1763Crossref PubMed Scopus (30) Google Scholar;Mironov et al., 1994Mironov N. Aguelon A.-M. Potapova G.I. Omori Y. Gorbunov O.V. Klimenkov A.A. Yamasaki H. Alterations of (CA) n DNA repeats and tumor-suppressor genes in human gastric cancer.Cancer Res. 1994; 54: 41-44PubMed Google Scholar;Sato et al., 1997Sato K. Gratas C. Lampe J. Biernat W. Kleihues P. Yamasaki H. Ohgaki H. Reduced expression of the P2 form of the gap junction protein connexin43 in malignant meningiomas.J Neuropathol Exp Neurol. 1997; 56: 835-839Crossref PubMed Scopus (20) Google Scholar). On the other hand, aberrant expression and/or localization of connexins are often seen in cancerous cells (Trosko and Ruch, 1998Trosko J.E. Ruch R.J. Cell-cell communication in carcinogenesis.Front Biosc. 1998; 15: D208-D236Google Scholar). Indeed, aberrant expression of Cx43 has been observed during mouse skin carcinogenesis (Budunova et al., 1995Budunova I.V. Carbajal S. Slaga T.J. The expression of gap junctional proteins during different stages of mouse skin carcinogenesis.Carcinogenesis. 1995; 16: 2717-2724Crossref PubMed Scopus (44) Google Scholar;Kamibayashi et al., 1995Kamibayashi Y. Oyamada Y. Mori M. Oyamada M. Aberrant expression of gap junction proteins (connexins) is associated with tumor progression during multistage mouse skin carcinogenesis in vivo.Carcinogenesis. 1995; 16: 1287-1297Crossref PubMed Scopus (94) Google Scholar;Sawey et al., 1996Sawey M.J. Goldschmidt M.H. Risek B. Gilula N.B. Lo S.W. Perturbation in connexin 43 and connexin 26 gap-junction expression in mouse skin hyperplasia and neoplasia.Mol Carcinog. 1996; 17: 49-61Crossref PubMed Scopus (62) Google Scholar). Therefore, it is likely that the function of connexins is not generally impaired through mutations, but rather through the level of their expression and as a result of post-translational modifications during carcinogenesis. As Cx43–/− mice do not survive after birth, it was not possible to include them in our experiments. We previously established fibroblast cell lines, however, by the 3T3 method from embryos of such mice and examined their growth properties (Yamakage et al., 1998Yamakage K. Omori Y. Piccoli C. Yamasaki H. Growth control of 3T3 fibroblast cell line established from connexin 43-deficient mice.Mol Carcinog. 1998; 23: 121-128Crossref PubMed Scopus (14) Google Scholar). Our results suggest that there was no significant difference in the immortalization process and in the growth characteristics of 3T3 fibroblasts established from Cx43+/+ and Cx43–/− mice (Yamakage et al., 1998Yamakage K. Omori Y. Piccoli C. Yamasaki H. Growth control of 3T3 fibroblast cell line established from connexin 43-deficient mice.Mol Carcinog. 1998; 23: 121-128Crossref PubMed Scopus (14) Google Scholar). We thus concluded that Cx43 gene itself has weak growth control ability by itself. On the other hand, it has been clearly shown that the Cx43 gene can control growth of already transformed or tumorigenic cells (Rose et al., 1993Rose B. Mehta P.P. Loewenstein W.R. Gap-junction protein gene suppresses tumorigenicity.Carcinogenesis. 1993; 14: 1073-1075Crossref PubMed Scopus (171) Google Scholar;Krutovskikh et al., 1998Krutovskikh V. Yamasaki H. Tsuda H. Asamoto M. Inhibition of intrinsic gap-junction intercellular communication and enhancement of tumorigenicity of the rat bladder carcinoma cell line BC31 by a dominant-negative connexin 43 mutant.Mol Carcinog. 1998; 23: 254-261Crossref PubMed Scopus (52) Google Scholar). It has also been shown that Cx43–/− mouse embryo fibroblasts established under conditions other than the 3T3 method acquired some transformed phenotypes (Martyn et al., 1997Martyn K.D. Kurata W.E. Warn-Cramer B.J. Burt J.M. TenBroek E. Lauf A.F. Immortalized connexin43 knockout cell lines display a subset of biological properties associated with the transformed phenotype.Cell Growth Differ. 1997; 9: 1015-1027Google Scholar). These results suggest that Cx43, and possibly also other connexins, may exert growth control ability when cells are under certain constraints. This is compatible with the idea that GJIC is essential for the maintenance of homeostasis and we propose that GJIC is involved in growth regulation through such a process. Analysis of ras gene mutations in tumors revealed the typical molecular signature of DMBA, i.e., A to T transversion at the 61st codon. We also confirmed that only Ha-ras gene mutations were seen in papillomas and carcinomas, whereas fibrosarcomas contained only Ki-ras gene mutations. These results are consistent with our previous suggestion that DMBA may induce the same mutation in both Ha-ras and Ki-ras genes in both epidermal and dermal cells, but that their recruitment processes are different in these two cell types (Sasaki et al., 1995Sasaki K. Bertrand O. Nakazawa H. Fitzgerald D.J. Mironov N. Yamasaki H. Cell-type-specific ras mutations but not microsatellite instability in chemically induced mouse skin tumors and transformed 3T3 cells.Cancer Res. 1995; 55: 3513-3516PubMed Google Scholar). As both Cx43+/+ and Cx43+/− mice bore tumors with the same pattern of ras gene mutations, loss of one Cx43 gene allele does not seem to influence this recruitment process. This conclusion is also reinforced by the finding that Cx43+/− mice developed fewer papillomas than Cx43+/+ mice when painted by TPA alone; in this experimental group, TPA is likely to promote the growth of ‘‘spontaneously’’ initiated cells. On the other hand, it is difficult to explain why Cx43+/− mice developed fewer papillomas. Most cell types express more than one type of connexin gene. In rodent and human keratinocytes, besides Cx43, Cx26, Cx30.3, Cx31, Cx31.1, Cx37, Cx40, and Cx45 genes are expressed (Salomon et al., 1993Salomon D. Masgrau E. Vischer M. et al.Gap-junction proteins and communication in human epidermis.in: Hall J.E. Zampighi G.A. Davis R.M. Progress in Cell Research. Vol. 3. Elsevier, Amsterdam1993: 225-233Crossref Google Scholar;Willecke et al., 1993Willecke K. Hennermann H. Dahl E. Jungbluth S. The mouse connexin gene family.in: Hall J.E. Zampighi G.A. Davis R.M. Progress in Cell Research. Vol. 3. Elsevier, Amsterdam1993: 33-41Crossref Google Scholar;Butterweck et al., 1994Butterweck A. Elfgang C. Willecke K. Traube O. Differential expression of the gap junction proteins connexin45–43–31, and -26 in mouse skin.Eur J Cell Biol. 1994; 65: 152-163PubMed Google Scholar;Goliger and Paul, 1994Goliger J.A. Paul D.L. Expression of gap junction proteins Cx26, Cx31.1, Cx37 and Cx43 in developing and mature rat epidermis.Dev Dyn. 1994; 200: 1-13Crossref PubMed Scopus (137) Google Scholar). As it has been shown that different connexins form gap junctions which have different conductance and as some different connexins are known to form heteromeric connexons (Stauffer, 1995Stauffer K.A. The gap junction proteins β1-connexin (connexin-32) and β2-connexin (connexin-26) can form heteromeric hemichannels.J Biol Chem. 1995; 270: 6768-6772Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar;Jiang and Goodenough, 1996Jiang J.X. Goodenough D.A. Heteromeric connexons in lens gap junction channels.Proc Natl Acad Sci USA. 1996; 93: 1287-1291Crossref PubMed Scopus (229) Google Scholar), it is possible that the presence of so many different connexin genes is designed to allow the fine-tuning of intercellular communication. On the other hand, it is also possible that multiple connexin genes are there to provide compensatory activity when any connexin is damaged. If this is the case, it is not surprising that Cx43+/− mice are not more prone to carcinogenesis, as other connexins may be able to maintain fairly normal function of keratinocytes and dermal cells. So far, an increased incidence of tumors in mice has been associated only with a deficiency of the Cx32 gene (Temme et al., 1997Temme A. Buchmann A. Gabriel H.D. Nelles E. Schwarz M. Willecke K. High incidence of spontaneous and chemically induced liver tumors in mice deficient for connexin32.Curr Biol. 1997; 7: 713-716Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). In this mouse line, the higher incidence of tumors was limited to the liver (Temme et al., 1997Temme A. Buchmann A. Gabriel H.D. Nelles E. Schwarz M. Willecke K. High incidence of spontaneous and chemically induced liver tumors in mice deficient for connexin32.Curr Biol. 1997; 7: 713-716Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). In the hepatocytes, the Cx26 and Cx32 genes are expressed. In the Cx32-nullzygous mice, there was also a drastic decrease in Cx26 gene expression in the hepatocytes and an increased rate of DNA synthesis in the liver, as measured by bromodeoxyuridine labelling compared with wild-type mice (Temme et al., 1997Temme A. Buchmann A. Gabriel H.D. Nelles E. Schwarz M. Willecke K. High incidence of spontaneous and chemically induced liver tumors in mice deficient for connexin32.Curr Biol. 1997; 7: 713-716Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). This may support the concept outlined above that a deficiency of one connexin is not sufficient to contribute strongly to carcinogenesis, but that a more general reduction in GJIC may affect growth control. We are grateful to Mrs Chantal Déchaux and to Miss Gaëlle Reguer for secretarial and technical assistance, respectively, and to Dr. John Cheney for editing the manuscript. Part of this work was supported by a grant from the US National Cancer Institute (R01-CA40534).