- Email: [email protected]
Expression patterns of connexin 26 and connexin 43 mRNA in canine benign and malignant mammary tumours
The Veterinary Journal The Veterinary Journal 172 (2006) 178–180 www.elsevier.com/locate/tvjl
Short communication
Expression patterns of connexin 26 and connexin 43 mRNA in canine benign and malignant mammary tumours Haruka Gotoh a, Kayono Harada a, Kazuyuki Suzuki a, Shizu Hashimoto b, Hozumi Yamamura b, Tsuneo Sato c, Keiko Fukumoto d, Hiromi Hagiwara d, Tatsuya Ishida d, Kazuhiko Yamada d, Ryuji Asano a, Tomohiro Yano d,* a
Laboratory of Veterinary Pharmacology, Department of Veterinary Medicine, Nihon University College of Bioresource Sciences, Fujisawa, Kanagawa 252-8510, Japan b Kitagawa Animal Hospital, Itabashi, Tokyo 174-0072, Japan c Laboratory of Veterinary Pathology, Department of Veterinary Medicine, Nihon University College of Bioresource Sciences, Fujisawa, Kanagawa 252-8510, Japan d Department of Food Science Research for Health, National Institute of Health and Nutrition, Shinjuku, Tokyo 162-8636, Japan Accepted 17 February 2005
Abstract The expression patterns of connexin (Cx) genes, encoding gap junctional proteins, are tissue- and cell-specific and, as their expression is mostly suppressed during carcinogenic processes, they are appropriate for monitoring tumour development. In this study, using reverse transcriptase-coupled polymerase chain reaction (RT-PCR), the expression of Cx mRNAs was examined in seven normal canine mammary glands and in 31 mammary gland tumour samples. Cx26 and Cx43 gene expression was studied in all normal tissues using specific Cx26 and Cx43 primers. When the expression patterns of Cx26 and Cx43 genes were analyzed in several types of canine mammary gland tumours, it was noted that it was the loss of Cx26 expression rather than the occurrence of Cx43 expression that was associated with malignancy. These results suggest that Cx26 plays an important role in tumourigenesis of canine mammary gland. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Canine; Connexin; Mammary gland tumour; RT-PCR
Homeostasis is one of the most important factors in maintaining tissue function and its disruption often results in organ dysfunction and can give rise to cancer (Mesnil and Yamasaki, 1993). The silencing at an early stage of carcinogenesis of tumour suppressor genes that maintain cellular homeostasis can lead directly to cancer development. Of these, the connexin (Cx) genes, encoding gap junction (GJ) proteins, are frequently silenced in the early stages of the carcinogenic process, and prefer-
*
Corresponding author. Tel.: +81 3 3203 8063; fax: +81 3 3205 6549. E-mail address: [email protected] (T. Yano).
1090-0233/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2005.02.023
entially exert a tumour-suppressive effect on the normal progenitor cell in which the particular Cx gene is naturally expressed (Yano et al., 2001, 2003). Thus, the determination of the Cx gene specifically expressed in a cancer progenitor cell may provide a useful prognostic, preventive and therapeutic target. While the expression of the Cx genes and their tumour suppressive effects have been investigated extensively in human breast tumours, there have been no reports on the status of the Cx genes in canine mammary gland tumours (Locke, 1998; Lee et al., 1992). Mammary and other tumours are clinically important in companion animals (Natsuyama et al., 2001) and the study
H. Gotoh et al. / The Veterinary Journal 172 (2006) 178–180
of the expression profiles of tumour suppressor genes in cancerous tissues has become necessary in order to regulate tumour development. The expression patterns of the Cx genes are tissue-specific, which may offer a clue in the search to establish their roles in canine mammary tumour suppression. As it has been reported that Cx26 and Cx43 are mainly expressed in normal human breast tissues (Pozzi et al., 1995), we selected these two Cx genes to assess any tumour-suppressive effects in canine mammary gland tumours. Seven normal mammary gland tissue samples and 31 mammary gland tumours were obtained from tissues resected surgically at Kitagawa Animal Hospital. For histological examination, part of each tumour sample was fixed in 10% formalin and embedded in paraffin. Thin sections were then prepared and stained with haematoxylin–eosin. The remainder was frozen for RNA extraction by liquid nitrogen. Of the 31 mammary gland tumours, nine were adenomas, six were benign mixed tumours, eleven were adenocarcinomas, and five were malignant mixed tumours. Total RNA extraction from canine normal mammary glands and mammary gland tumours was carried out using RNeasy Lipid Tissue Mini Kit (Qiagen) according to the manufacturerÕs instructions. The concentration and purity of the RNA were determined spectrophotometrically by measuring the absorbance at wavelengths of 260 and 280 nm. Canine cDNA was synthesized using Superscript Reverse Transcriptase (Invitrogen) and poly(dT) oligonucleotide (12– 18mer, Pharmacia). Canine Cx26, Cx43 and GAPDH cDNA fragments were amplified by PCR using the following primers: Cx26, 5 0 -TGTGTCTACCCCTGCTCTCC-3 0 (forward); 5 0 -TTCGATACGGACTTTCTGGC-3 0 (reverse); Cx43, 5 0 -GTCTCCTCCTGGGTACAAG-3 0 (forward); 5 0 CGAGGTCGGCTGCTGGCT-3 0 (reverse), and (GAPDH, 5 0 -AAGGCTGAGAACGGGAAACT-3 0 (forward); 5 0 -GGAGGCATTGCTGACAATCT-3 0 (reverse). GAPDH was used as an internal standard. The conditions for amplification were initial denaturation at 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 55 °C for 1 min, 72 °C for 1 min, and a final extension at 72 C for 5 min. The PCR products were electrophoresed on 2% agarose gels and stained with Gelstar Nucleic Acid Gel Stain (Takara). A wide-range DNA ladder (Takara) was used as a marker for sizing the products. In order to specifically amplify Cx26 and Cx43 cDNA fragments from samples having lower levels in each mRNA, canine cDNA was synthesized using each reverse primer for Cx26 and Cx43 instead of poly(dT) oligonucleotide. Other PCR procedures were performed as mentioned above. To confirm the sequences of the PCR products, some were cloned with TA Cloning Kit (Novagen) and sequenced with the dye-terminator method (Applied Biosystems).
179
To test for differences among groups, data were analyzed by the v2 test followed by the post hoc Bonferroni test. For all analyses, values of P < 0.05 were considered significant. From a report on the expression patterns of normal human breast tissues (Monaghan et al., 1996), it was assumed that Cx26 and Cx43 genes would be mainly expressed in normal canine mammary gland tissues. However, we could not detect the expression of Cx26 and Cx43 genes in normal tissues by RT-PCR in cDNA synthesis using poly(dT) oligonucleotide (Fig. 1). To enhance the sensitivity and specificity of RT-PCR, we synthesized canine cDNA using reverse primers for Cx26 and Cx43 and subsequently performed PCR using the synthesized cDNA. As shown in Figure 1, we detected the expression of Cx26 and Cx43 genes in normal tissues, even if the expression could not be detected in samples by RT-PCR in the case of cDNA synthesis using poly(dT) oligonucleotide. These results suggest that the RT-PCR using the cDNA synthesized by reverse primers for Cx26 and Cx43 is a useful method to detect the expressions of the two genes in canine mammary glands with relatively high sensitivity. Using the established RT-PCR, we next examined the expression patterns of the Cx26 and Cx43 genes in the tumours. In all of the benign tumours (nine adenomas and six benign mixed tumours), the expressions of Cx26 and C43 genes were observed (Table 1). In the malignant tumours, the loss of Cx26 gene expression was observed more frequently than that of Cx43 gene (Table 1), that is 1/5 samples (20%) of the malignant mixed tumours and 4/11 samples (36.4%) of the adenocarcinomas expressed the Cx26 gene whereas 4/5 samples (80%) of the malignant mixed tumours and 10/11
Fig. 1. RT-PCR analysis for Cx26 and Cx43 in canine normal mammary glands. In GAPDH-26, Cx26-1 and Cx26-2, and in GAPDH-43, Cx43-1 and Cx43-2, RT-PCRs were performed using the same samples. In GAPDH-26, GAPDH-43, Cx26-1, and Cx43-1, cDNAs were synthesized using RT and poly(dT) oligonucleotide (12– 18mer, Pharmacia). In Cx26-2 and Cx43-2, cDNAs were synthesized using each reverse primer for Cx26 and Cx43 instead of poly(dT) oligonucleotide. The sizes of the PCR products were 272 bp in GAPDH, 138 bp in Cx26, and 284 bp in Cx43. The sequence of the PCR products completely coincided with those reported previously (data not shown).
180
H. Gotoh et al. / The Veterinary Journal 172 (2006) 178–180
Table 1 Summary of mRNA expression of Cx26 and Cx43 in canine mammary gland tumours Case
Cx26 expression
Cx43 expression
Normal tissue Benign mixed tumour Adenoma Malignant mixed tumour Adenocarcinoma
7/7 (100%) 6/6 (100%) 9/9 (100%) 1/5 (20.0%)* 4/11 (36.4%)*
7/7 (100%) 6/6 (100%) 9/9 (100%) 4/5 (80.0%) 10/11 (90.9%)
* Significant difference from normal tissue, benign mixed tumour and adenoma.
Acknowledgements This study was supported by a research grant from Fuji Foundation for Protein Research, a research grant on Health Sciences Focusing on Drug Innovation from Japan Health Sciences Foundation (KH21012) and a grant-in-aid for Science Research from the Ministry of Education, Culture and Sciences of Japan (16580259) to T.Y.
References (90.9%) of the adenocarcinomas demonstrated Cx43 gene expression. The differences in Cx26 gene expression but not Cx43 gene expression between normal mammary gland tissues and malignant mammary gland tumours were statistically significant indicating that the silencing of Cx26 rather than the occurrence of Cx43 expression is related to malignancy in canine mammary gland tumours. Although several Cx genes have been detected in normal human breast tissue, the expressed subtypes of Cx differ among cells. Weak Cx26 expression has been detected in ductal epithelial cells, from which breast cancers arise (Jamieson et al., 1998). On the other hand, Cx43 is strongly expressed, but only in myoepithelial cells, which cannot be the origin of the cancers (Laird et al., 1999). In some human breast cancer cell lines, the expression of the Cx26 gene is not detected (Lee et al., 1992), and re-expression of the Cx gene in one of the breast cancer cell lines attenuated its malignant phenotype (Momiyama et al., 2003). These reports signify that Cx26 acts as a tumour suppressor gene in human breast cancers. Compared to human breast cancers, canine mammary gland tumours have more complicated morphological features, because the origin of the tumours is from myoepithelial cells in addition to epithelial cells (Brodey et al., 1983). In canines, malignant mixed tumours arise from both myoepithelial cells and epithelial cells in mammary glands (Misdrop et al., 1973). However, in canine neoplastic mammary gland tumours, the silencing of the Cx26 gene is closely related to tumour development. From these data, it appears that Cx26 rather than Cx43 acts as a tumour suppressor gene in canine mammary gland tumours. However, in order to completely establish Cx26 as a tumour suppressor gene in canine mammary glands, it will be necessary to show that the re-expression of the Cx26 gene is definitely associated with negative growth control of the tumours. If we can establish Cx26 as the tumour suppressor gene, Cx26 may be a target molecular to prevent and cure mammary gland tumours as well as human breast cancers (Tan et al., 2002).
Brodey, R.S., Goldschmidt, M.A., Roszel, J.R., 1983. Canine mammary gland neoplasms. Journal of American Animal Hospital Associations 19, 61–90. Jamieson, S., Going, J.J., DÕArcy, R., George, W.D., 1998. Expression of gap junction proteins connexin 26 and connexin 43 in normal human breast and in breast tumours. Journal of Pathology 84, 37– 43. Laird, D.W., Fistouris, P., Batist, G., Alpert, L., Huynh, H.T., Carystinos, G.D., Alaoui-Jamali, M.A., 1999. Deficiency of connexin 43 gap junctions is an independent marker for breast tumours. Cancer Research 59, 4104–4110. Lee, S.W., Tomasetto, C., Paul, D., Keymarsi, K., Sager, R., 1992. Transcriptional downregulation of gap-junction proteins blocks junctional communication in human mammary tumour cell lines. Journal of Cell Biology 118, 1213–1221. Locke, D., 1998. Gap junctions in normal and neoplastic mammary glands. Journal of Pathology 186, 343–349. Natsuyama, S., Nakamura, M., Yonezawa, K., Shimada, T., Ohashi, Y., Takamori, Y., Kubo, K., 2001. Expression patterns of the erbB subfamily mRNA in canine benign and malignant mammary tumours. Journal of Veterinary Medical Science 63, 949–954. Mesnil, M., Yamasaki, H., 1993. Cell–cell communication and growth control of normal and cancer cells: evidence and hypothesis. Molecular Carcinogenesis 7, 14–17. Misdrop, W., Cotchin, E., Hampe, J.F., Jabara, A.G., Von Sanderleben, J., 1973. Canine malignant mammary tumours. III. Special types of carcinomas, malignant mixed tumours. Veterinary Pathology 10, 241–256. Momiyama, M., Omori, Y., Ishizuka, Y., Nishikawa, Y., Tokairin, T., Ogawa, J., Enomoto, K., 2003. Connexin26-mediated gap junctional communication reverses the malignant phenotype of MCF-7 breast cancer cells. Cancer Sciences 95, 501–507. Monaghan, P., Clarke, C., Persinghe, N.P., Moss, D.W., Chen, X.Y., Evans, W.H., 1996. Gap junction distribution and connexin expression in human breast. Experimental Cell Research 223, 29– 38. Pozzi, A., Risek, B., Kiang, D.T., Gilula, N.B., Kumar, N.M., 1995. Analysis of multiple gap junction gene products in the rodent and human mammary gland. Experimental Cell Research 220, 212–219. Tan, L.I., Bianco, T., Dobrovic, A., 2002. Variable promoter region CpG island methylation of the putative tumour suppressor gene connexin 26 in breast cancer. Carcinogenesis 23, 231–236. Yano, T., Hernandez-Blazuqueze, F., Omori, Y., Yamasaki, H., 2001. Reduction of malignant phenotype of HepG2 cell is associated with the expression of connexin 26 but not connexin 32. Carcinogenesis 22, 1593–1600. Yano, T., Ito, F., Satoh, H., Hagiwara, K., Nakazawa, H., Toma, H., Yamasaki, H., 2003. Tumour-suppressive effect of connexin 32 in renal cell carcinoma from haemodialysis patients. Kidney International 63, 381.
Short communication
Expression patterns of connexin 26 and connexin 43 mRNA in canine benign and malignant mammary tumours Haruka Gotoh a, Kayono Harada a, Kazuyuki Suzuki a, Shizu Hashimoto b, Hozumi Yamamura b, Tsuneo Sato c, Keiko Fukumoto d, Hiromi Hagiwara d, Tatsuya Ishida d, Kazuhiko Yamada d, Ryuji Asano a, Tomohiro Yano d,* a
Laboratory of Veterinary Pharmacology, Department of Veterinary Medicine, Nihon University College of Bioresource Sciences, Fujisawa, Kanagawa 252-8510, Japan b Kitagawa Animal Hospital, Itabashi, Tokyo 174-0072, Japan c Laboratory of Veterinary Pathology, Department of Veterinary Medicine, Nihon University College of Bioresource Sciences, Fujisawa, Kanagawa 252-8510, Japan d Department of Food Science Research for Health, National Institute of Health and Nutrition, Shinjuku, Tokyo 162-8636, Japan Accepted 17 February 2005
Abstract The expression patterns of connexin (Cx) genes, encoding gap junctional proteins, are tissue- and cell-specific and, as their expression is mostly suppressed during carcinogenic processes, they are appropriate for monitoring tumour development. In this study, using reverse transcriptase-coupled polymerase chain reaction (RT-PCR), the expression of Cx mRNAs was examined in seven normal canine mammary glands and in 31 mammary gland tumour samples. Cx26 and Cx43 gene expression was studied in all normal tissues using specific Cx26 and Cx43 primers. When the expression patterns of Cx26 and Cx43 genes were analyzed in several types of canine mammary gland tumours, it was noted that it was the loss of Cx26 expression rather than the occurrence of Cx43 expression that was associated with malignancy. These results suggest that Cx26 plays an important role in tumourigenesis of canine mammary gland. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Canine; Connexin; Mammary gland tumour; RT-PCR
Homeostasis is one of the most important factors in maintaining tissue function and its disruption often results in organ dysfunction and can give rise to cancer (Mesnil and Yamasaki, 1993). The silencing at an early stage of carcinogenesis of tumour suppressor genes that maintain cellular homeostasis can lead directly to cancer development. Of these, the connexin (Cx) genes, encoding gap junction (GJ) proteins, are frequently silenced in the early stages of the carcinogenic process, and prefer-
*
Corresponding author. Tel.: +81 3 3203 8063; fax: +81 3 3205 6549. E-mail address: [email protected] (T. Yano).
1090-0233/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2005.02.023
entially exert a tumour-suppressive effect on the normal progenitor cell in which the particular Cx gene is naturally expressed (Yano et al., 2001, 2003). Thus, the determination of the Cx gene specifically expressed in a cancer progenitor cell may provide a useful prognostic, preventive and therapeutic target. While the expression of the Cx genes and their tumour suppressive effects have been investigated extensively in human breast tumours, there have been no reports on the status of the Cx genes in canine mammary gland tumours (Locke, 1998; Lee et al., 1992). Mammary and other tumours are clinically important in companion animals (Natsuyama et al., 2001) and the study
H. Gotoh et al. / The Veterinary Journal 172 (2006) 178–180
of the expression profiles of tumour suppressor genes in cancerous tissues has become necessary in order to regulate tumour development. The expression patterns of the Cx genes are tissue-specific, which may offer a clue in the search to establish their roles in canine mammary tumour suppression. As it has been reported that Cx26 and Cx43 are mainly expressed in normal human breast tissues (Pozzi et al., 1995), we selected these two Cx genes to assess any tumour-suppressive effects in canine mammary gland tumours. Seven normal mammary gland tissue samples and 31 mammary gland tumours were obtained from tissues resected surgically at Kitagawa Animal Hospital. For histological examination, part of each tumour sample was fixed in 10% formalin and embedded in paraffin. Thin sections were then prepared and stained with haematoxylin–eosin. The remainder was frozen for RNA extraction by liquid nitrogen. Of the 31 mammary gland tumours, nine were adenomas, six were benign mixed tumours, eleven were adenocarcinomas, and five were malignant mixed tumours. Total RNA extraction from canine normal mammary glands and mammary gland tumours was carried out using RNeasy Lipid Tissue Mini Kit (Qiagen) according to the manufacturerÕs instructions. The concentration and purity of the RNA were determined spectrophotometrically by measuring the absorbance at wavelengths of 260 and 280 nm. Canine cDNA was synthesized using Superscript Reverse Transcriptase (Invitrogen) and poly(dT) oligonucleotide (12– 18mer, Pharmacia). Canine Cx26, Cx43 and GAPDH cDNA fragments were amplified by PCR using the following primers: Cx26, 5 0 -TGTGTCTACCCCTGCTCTCC-3 0 (forward); 5 0 -TTCGATACGGACTTTCTGGC-3 0 (reverse); Cx43, 5 0 -GTCTCCTCCTGGGTACAAG-3 0 (forward); 5 0 CGAGGTCGGCTGCTGGCT-3 0 (reverse), and (GAPDH, 5 0 -AAGGCTGAGAACGGGAAACT-3 0 (forward); 5 0 -GGAGGCATTGCTGACAATCT-3 0 (reverse). GAPDH was used as an internal standard. The conditions for amplification were initial denaturation at 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 55 °C for 1 min, 72 °C for 1 min, and a final extension at 72 C for 5 min. The PCR products were electrophoresed on 2% agarose gels and stained with Gelstar Nucleic Acid Gel Stain (Takara). A wide-range DNA ladder (Takara) was used as a marker for sizing the products. In order to specifically amplify Cx26 and Cx43 cDNA fragments from samples having lower levels in each mRNA, canine cDNA was synthesized using each reverse primer for Cx26 and Cx43 instead of poly(dT) oligonucleotide. Other PCR procedures were performed as mentioned above. To confirm the sequences of the PCR products, some were cloned with TA Cloning Kit (Novagen) and sequenced with the dye-terminator method (Applied Biosystems).
179
To test for differences among groups, data were analyzed by the v2 test followed by the post hoc Bonferroni test. For all analyses, values of P < 0.05 were considered significant. From a report on the expression patterns of normal human breast tissues (Monaghan et al., 1996), it was assumed that Cx26 and Cx43 genes would be mainly expressed in normal canine mammary gland tissues. However, we could not detect the expression of Cx26 and Cx43 genes in normal tissues by RT-PCR in cDNA synthesis using poly(dT) oligonucleotide (Fig. 1). To enhance the sensitivity and specificity of RT-PCR, we synthesized canine cDNA using reverse primers for Cx26 and Cx43 and subsequently performed PCR using the synthesized cDNA. As shown in Figure 1, we detected the expression of Cx26 and Cx43 genes in normal tissues, even if the expression could not be detected in samples by RT-PCR in the case of cDNA synthesis using poly(dT) oligonucleotide. These results suggest that the RT-PCR using the cDNA synthesized by reverse primers for Cx26 and Cx43 is a useful method to detect the expressions of the two genes in canine mammary glands with relatively high sensitivity. Using the established RT-PCR, we next examined the expression patterns of the Cx26 and Cx43 genes in the tumours. In all of the benign tumours (nine adenomas and six benign mixed tumours), the expressions of Cx26 and C43 genes were observed (Table 1). In the malignant tumours, the loss of Cx26 gene expression was observed more frequently than that of Cx43 gene (Table 1), that is 1/5 samples (20%) of the malignant mixed tumours and 4/11 samples (36.4%) of the adenocarcinomas expressed the Cx26 gene whereas 4/5 samples (80%) of the malignant mixed tumours and 10/11
Fig. 1. RT-PCR analysis for Cx26 and Cx43 in canine normal mammary glands. In GAPDH-26, Cx26-1 and Cx26-2, and in GAPDH-43, Cx43-1 and Cx43-2, RT-PCRs were performed using the same samples. In GAPDH-26, GAPDH-43, Cx26-1, and Cx43-1, cDNAs were synthesized using RT and poly(dT) oligonucleotide (12– 18mer, Pharmacia). In Cx26-2 and Cx43-2, cDNAs were synthesized using each reverse primer for Cx26 and Cx43 instead of poly(dT) oligonucleotide. The sizes of the PCR products were 272 bp in GAPDH, 138 bp in Cx26, and 284 bp in Cx43. The sequence of the PCR products completely coincided with those reported previously (data not shown).
180
H. Gotoh et al. / The Veterinary Journal 172 (2006) 178–180
Table 1 Summary of mRNA expression of Cx26 and Cx43 in canine mammary gland tumours Case
Cx26 expression
Cx43 expression
Normal tissue Benign mixed tumour Adenoma Malignant mixed tumour Adenocarcinoma
7/7 (100%) 6/6 (100%) 9/9 (100%) 1/5 (20.0%)* 4/11 (36.4%)*
7/7 (100%) 6/6 (100%) 9/9 (100%) 4/5 (80.0%) 10/11 (90.9%)
* Significant difference from normal tissue, benign mixed tumour and adenoma.
Acknowledgements This study was supported by a research grant from Fuji Foundation for Protein Research, a research grant on Health Sciences Focusing on Drug Innovation from Japan Health Sciences Foundation (KH21012) and a grant-in-aid for Science Research from the Ministry of Education, Culture and Sciences of Japan (16580259) to T.Y.
References (90.9%) of the adenocarcinomas demonstrated Cx43 gene expression. The differences in Cx26 gene expression but not Cx43 gene expression between normal mammary gland tissues and malignant mammary gland tumours were statistically significant indicating that the silencing of Cx26 rather than the occurrence of Cx43 expression is related to malignancy in canine mammary gland tumours. Although several Cx genes have been detected in normal human breast tissue, the expressed subtypes of Cx differ among cells. Weak Cx26 expression has been detected in ductal epithelial cells, from which breast cancers arise (Jamieson et al., 1998). On the other hand, Cx43 is strongly expressed, but only in myoepithelial cells, which cannot be the origin of the cancers (Laird et al., 1999). In some human breast cancer cell lines, the expression of the Cx26 gene is not detected (Lee et al., 1992), and re-expression of the Cx gene in one of the breast cancer cell lines attenuated its malignant phenotype (Momiyama et al., 2003). These reports signify that Cx26 acts as a tumour suppressor gene in human breast cancers. Compared to human breast cancers, canine mammary gland tumours have more complicated morphological features, because the origin of the tumours is from myoepithelial cells in addition to epithelial cells (Brodey et al., 1983). In canines, malignant mixed tumours arise from both myoepithelial cells and epithelial cells in mammary glands (Misdrop et al., 1973). However, in canine neoplastic mammary gland tumours, the silencing of the Cx26 gene is closely related to tumour development. From these data, it appears that Cx26 rather than Cx43 acts as a tumour suppressor gene in canine mammary gland tumours. However, in order to completely establish Cx26 as a tumour suppressor gene in canine mammary glands, it will be necessary to show that the re-expression of the Cx26 gene is definitely associated with negative growth control of the tumours. If we can establish Cx26 as the tumour suppressor gene, Cx26 may be a target molecular to prevent and cure mammary gland tumours as well as human breast cancers (Tan et al., 2002).
Brodey, R.S., Goldschmidt, M.A., Roszel, J.R., 1983. Canine mammary gland neoplasms. Journal of American Animal Hospital Associations 19, 61–90. Jamieson, S., Going, J.J., DÕArcy, R., George, W.D., 1998. Expression of gap junction proteins connexin 26 and connexin 43 in normal human breast and in breast tumours. Journal of Pathology 84, 37– 43. Laird, D.W., Fistouris, P., Batist, G., Alpert, L., Huynh, H.T., Carystinos, G.D., Alaoui-Jamali, M.A., 1999. Deficiency of connexin 43 gap junctions is an independent marker for breast tumours. Cancer Research 59, 4104–4110. Lee, S.W., Tomasetto, C., Paul, D., Keymarsi, K., Sager, R., 1992. Transcriptional downregulation of gap-junction proteins blocks junctional communication in human mammary tumour cell lines. Journal of Cell Biology 118, 1213–1221. Locke, D., 1998. Gap junctions in normal and neoplastic mammary glands. Journal of Pathology 186, 343–349. Natsuyama, S., Nakamura, M., Yonezawa, K., Shimada, T., Ohashi, Y., Takamori, Y., Kubo, K., 2001. Expression patterns of the erbB subfamily mRNA in canine benign and malignant mammary tumours. Journal of Veterinary Medical Science 63, 949–954. Mesnil, M., Yamasaki, H., 1993. Cell–cell communication and growth control of normal and cancer cells: evidence and hypothesis. Molecular Carcinogenesis 7, 14–17. Misdrop, W., Cotchin, E., Hampe, J.F., Jabara, A.G., Von Sanderleben, J., 1973. Canine malignant mammary tumours. III. Special types of carcinomas, malignant mixed tumours. Veterinary Pathology 10, 241–256. Momiyama, M., Omori, Y., Ishizuka, Y., Nishikawa, Y., Tokairin, T., Ogawa, J., Enomoto, K., 2003. Connexin26-mediated gap junctional communication reverses the malignant phenotype of MCF-7 breast cancer cells. Cancer Sciences 95, 501–507. Monaghan, P., Clarke, C., Persinghe, N.P., Moss, D.W., Chen, X.Y., Evans, W.H., 1996. Gap junction distribution and connexin expression in human breast. Experimental Cell Research 223, 29– 38. Pozzi, A., Risek, B., Kiang, D.T., Gilula, N.B., Kumar, N.M., 1995. Analysis of multiple gap junction gene products in the rodent and human mammary gland. Experimental Cell Research 220, 212–219. Tan, L.I., Bianco, T., Dobrovic, A., 2002. Variable promoter region CpG island methylation of the putative tumour suppressor gene connexin 26 in breast cancer. Carcinogenesis 23, 231–236. Yano, T., Hernandez-Blazuqueze, F., Omori, Y., Yamasaki, H., 2001. Reduction of malignant phenotype of HepG2 cell is associated with the expression of connexin 26 but not connexin 32. Carcinogenesis 22, 1593–1600. Yano, T., Ito, F., Satoh, H., Hagiwara, K., Nakazawa, H., Toma, H., Yamasaki, H., 2003. Tumour-suppressive effect of connexin 32 in renal cell carcinoma from haemodialysis patients. Kidney International 63, 381.