Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Growth-Suppressing Activity of the Transfected Cx26 on BICR-M1Rk Breast Cancer Cell Line

  • Received : 2010.12.28
  • Accepted : 2011.02.18
  • Published : 2011.05.28
Download PDF
⟨ Previous Next ⟩

Abstract

There are accumulating evidences suggesting that connexin (Cx), a gap junction channel-forming protein, acts as a growth suppressor in various cancer cells, and this effect is attributeed to the gap junction-mediated intercellular communication (GJIC). In order to characterize the relationship between the growth-arresting activity of Cx26 and its cytoplasmic localizations after expression, we linked a nuclear export signal (NES) sequence to Cx26 cDNA before transfecting into a rat breast cancer cell line. A confocal fluorescent microscopic observation revealed that the insertion of NES minimized the nuclear expression of Cx26, and increased its cytoplasmic expression, including plasma membrane junctions. Total cell counting and BrdUrd-labeling experiments showed that the growth of the breast cancer cells was inhibited by 74% upon transfection of Cx26-NES, whereas only 9% inhibition was observed with only Cx26 cDNA.

Keywords

References

  1. Bond, S. L., J. F. Bechberger, N. K. Khoo, and C. C. Naus. 1994. Transfection of C6 glioma cells with connexin32: The effects of expression of a nonendogenous gap junction protein. Cell Growth Differ. 5: 179-186.
  2. Bruzzone, R., T. W. White, and D. L. Paul. 1996. Connections with connexins: The molecular basis of direct intercellular signaling. Eur. J. Biochem. 238: 1-27. https://doi.org/10.1111/j.1432-1033.1996.0001q.x
  3. Cascio, M., N. M. Kumar, R. Safarik, and N. B. Gilula. 1995. Physical characterization of gap junction membrane connexons (hemi-channels) isolated from rat liver. J. Biol. Chem. 270: 18643-18648. https://doi.org/10.1074/jbc.270.31.18643
  4. Chen, S. C., D. B. Pelletier, P. Ao, and A. L. Boynton. 1995. Connexin43 reverses the phenotype of transformed cells and alters their expression of cyclin/cyclin-dependent kinases. Cell Growth Differ. 6: 681-690.
  5. Dang, X., B. W. Doble, and E. Kardami. 2003. The carboxy-tail of connexin-43 localizes to the nucleus and inhibits cell growth. Mol. Cell Biochem. 242: 35-38. https://doi.org/10.1023/A:1021152709313
  6. Fu, C. T., J. F. Bechberger, M. A. Ozog, B. Perbal, and C. C. Naus. 2004. CCN3 (NOV) interacts with connexin43 in C6 glioma cells: Possible mechanism of connexin-mediated growth suppression. J. Biol. Chem. 279: 36943-36950. https://doi.org/10.1074/jbc.M403952200
  7. Gellhaus, A., X. Dong, S. Propson, K. Maass, L. Klein-Hitpass, M. Kibschull, et al. 2004. Connexin43 interacts with NOV: A possible mechanism for negative regulation of cell growth in choriocarcinoma cells. J. Biol. Chem. 279: 36931-36941. https://doi.org/10.1074/jbc.M404073200
  8. Giepmans, B. N., I. Verlaan, T. Hengeveld, H. Janssen, J. Calafat, M. M. Falk, and W. H. Moolenaar. 2001. Gap junction protein connexin-43 interacts directly with microtubules. Curr. Biol. 11: 1364-1368. https://doi.org/10.1016/S0960-9822(01)00424-9
  9. Hirschi, K. K., C. E. Xu, T. Tsukamoto, and P. Sager. 1996. Gap junction genes Cx26 and Cx43 individually suppress the cancer phenotype of human mammary carcinoma cells and restore differentiation potential. Cell Growth Differ. 7: 861-870.
  10. Huang, R. P., Y. Fan, M. Z. Hossain, A. Peng, Z. L. Zeung, and A. L. Boynton. 1998. Reversion of the neoplastic phenotype of human glioblastoma cells by connexin 43 (cx43). Cancer Res. 58: 5089-5096.
  11. King, T. J. and J. S. Bertram. 2005. Connexins as targets for cancer chemoprevention and chemotherapy. Biochem. Biophys. Acta 1719: 146-160. https://doi.org/10.1016/j.bbamem.2005.08.012
  12. Kumar, N. M. and N. B. Gilula. 1996. The gap junction communication channel. Cell 84: 381-388. https://doi.org/10.1016/S0092-8674(00)81282-9
  13. Kyo, N., H. Yamamoto, Y. Takeda, K. Ezumi, C. Y. Ngan, M. Terayama, et al. 2008. Overexpression of connexin26 in carcinoma of the pancreas. Oncol. Rep. 19: 627-631.
  14. Lai, C. P., J. F. Bechberger, and C. C. Naus. 2009. Pannexin2 as a novel growth regulator in C6 glioma cells. Oncogene 28: 4402-4408. https://doi.org/10.1038/onc.2009.283
  15. Laird, D. W. and J. P. Revel. 1990. Biochemical and immunochemical analysis of the arrangement of connexin43 in rat heart gap junction membranes. J. Cell Sci. 97: 109-117.
  16. Lau, A. F., M. Y. Kanemitsu, W. E. Kurata, S. Danesh, and A. L. Boynton. 1992. Epidermal growth factor disrupts gapjunctional communication and induces phosphorylation of connexin43 on serine. Mol. Biol. Cell 3: 865-874.
  17. Lee, H. J., I. K. Lee, K. H. Seul, and S. K. Rhee. 2002. Growth inhibition by connexin26 expression in cultured rodent tumor cells. Mol. Cells 14: 136-142.
  18. Mehta, P. P., A. Hotz-Wagenblatt, B. Rose, D. Shalloway, and W. R. Loewenstein. 1991. Incorporation of the gene for a cellcell channel protein into transformed cells leads to normalization of growth. J. Membr. Biol. 124: 207-225. https://doi.org/10.1007/BF01994355
  19. Mesnil, M., V. A. Krutovskikh, C. Piccoli, C. Elfang, O. Traub, K. Willecke, and H. Yamasaki. 1995. Negative growth control of HeLa cells by connexin genes: Connexin species specificity. Cancer Res. 55: 629-639.
  20. Milks, L. C., N. M. Kumar, R. Houghten, N. Unwin, and N. B. Gilula. 1988. Topology of the 32-kd liver gap junction protein determined by site-directed antibody localizations. EMBO J. 7: 2967-2975.
  21. Moorby, C. and M. Patel. 2001. Dual function for connexins: Cx43 regulates growth independently of gap junction formation. Exp. Cell Res. 271: 238-248. https://doi.org/10.1006/excr.2001.5357
  22. Muramatsu, A., M. Iwai, T. Morikawa, S. Tanaka, T. Mori, Y. Harada, and T. Okanoue. 2002. Influence of transfection with connexin 26 gene on malignant potential of human hepatoma cells. Carcinogenesis 23: 351-358. https://doi.org/10.1093/carcin/23.2.351
  23. Princen, F., P. Robe, D. Gros, T. Jarry-Guichard, J. Gielen, M. P. Merville, and V. Bours. 2001. Rat gap junction connexin-30 inhibits proliferation of glioma cell lines. Carcinogenesis 22: 507-513. https://doi.org/10.1093/carcin/22.3.507
  24. Qin, H., Q. Shao, H. Curtis, J. Galipeau, D. J. Belliveau, T. Wang, M. A. Alaoui-Jamali, and D. W. Laird. 2002. Retroviral delivery of connexin genes to human breast tumor cells inhibits in vivo tumor growth by a mechanism that is independent of significant gap junctional intercellular communication. J. Biol. Chem. 277: 29132-29138. https://doi.org/10.1074/jbc.M200797200
  25. Quist, A. P., S. K. Rhee, H. Lin, and R. Lal. 2000. Physiological role of gap-junctional hemichannels. Extracellular calciumdependent isosmotic volume regulation. J. Cell Biol. 148: 1063- 1074. https://doi.org/10.1083/jcb.148.5.1063
  26. Richards, S. A., K. M. Lounsbury, K. L. Carey, and I. G. Macara. 1996. A nuclear export signal is essential for the cytosolic localization of the Ran binding protein, RanBP1. J. Cell Biol. 134: 1157-1168. https://doi.org/10.1083/jcb.134.5.1157
  27. Saez, J. C., V. M. Berthoud, M. C. Branes, A. D. Martinez, and E. C. Beyer. 2003. Plasma membrane channels formed by connexins: Their regulation and functions. Physiol. Rev. 83: 1359-1400.
  28. Statuto, M., C. Audebet, H. Tonoli, S. Selmi-Ruby, B. Rousset, and Y. Munari-Silem. 1997. Restoration of cell-to-cell communication in thyroid cell lines by transfection with and stable expression of the connexin32 gene. Impact on cell proliferation and tissuespecific gene expression. J. Biol. Chem. 272: 24710-24716. https://doi.org/10.1074/jbc.272.39.24710
  29. Thimm, J., A. Mechler, H. Lin, S. K. Rhee, and R. Lal. 2005. Calcium-dependent open/closed conformations and interfacial energy maps of reconstituted hemichannels. J. Biol. Chem. 280: 10646-10654. https://doi.org/10.1074/jbc.M412749200
  30. Toyofuku, T., Y. Akamatsu, H. Zhang, T. Kuzuya, M. Tada, and M. Hori. 2001. c-Src regulates the interaction between connexin- 43 and ZO-1 in cardiac myocytes. J. Biol. Chem. 276: 1780- 1788. https://doi.org/10.1074/jbc.M005826200
  31. Trosko, J. E. and R. J. Ruch. 2002. Gap junctions as targets for cancer chemoprevention and chemotherapy. Curr. Drug Targets 3: 465-482. https://doi.org/10.2174/1389450023347371
  32. Umemura, T., K. Sai-Kato, A. Takagi, R. Hasegawa, and Y. Kurokawa. 1996. Oxidative DNA damage and cell proliferation in the liver cell of B6C3F1 mice exposed to pentachlorophenol in their diet. Fundam. Appl. Toxicol. 30: 285-289. https://doi.org/10.1006/faat.1996.0066
  33. Wen, W., A. T. Harootunian, S. R. Adams, J. Feramisco, R. Y. Tsien, J. L. Meinkoth, and S. S. Taylor. 1995. Heat-stable inhibitors of cAMP-dependent protein kinase carry a nuclear export signal. J. Biol. Chem. 269: 32214-32220.
  34. Willecke, K., J. Eiberger, J. Degen, D. A. Eckardt, A. Romualdi, M. Guldenagel, U. Deutsch, and G. Sohl. 2002. Structural functional diversity of connexin genes in the mouse and human genome. Biol. Chem. 383: 725-737.
  35. Yamasaki, H. 1990. Role of cell-cell communication in tumor suppression. Immunol. Ser. 51: 245-266.
  36. Yamasaki, H. and C. C. G. Naus. 1996. Role of connexin genes in growth control. Carcinogenesis 17: 1199-1213. https://doi.org/10.1093/carcin/17.6.1199
  37. Zhang, J. T. and B. J. Nicholson. 1994. The topological structure of connexin 26 and its distribution compared to connexin 32 in hepatic gap junctions. J. Membr. Biol. 139: 15-29.
  38. Zhang, Y. W., K. Nakayama, and I. Morita. 2003. A novel route for connexin 43 to inhibit cell proliferation: Negative regulation of S-phase kinase-associated protein (Skp 2). Cancer Res. 63: 1623-1630.
  39. Zhu, D., S. Caveney, G. M. Kidder, and C. C. Naus. 1991. Transfection of C6 glioma cells with connexin43 cDNA: Analysis of expression, intercellular coupling, and cell proliferation. Proc. Natl. Acad. Sci. USA 88: 1883-1887. https://doi.org/10.1073/pnas.88.5.1883

Cited by

  1. Construction of recombinant adenovirus vector with connexin 43 gene vol.44, pp.10, 2011, https://doi.org/10.1093/abbs/gms069
  2. Keratitis, ichthyosis, and deafness (KID) syndrome: A review of infectious and neoplastic complications vol.69, pp.1, 2011, https://doi.org/10.1016/j.jaad.2012.12.965
  3. Connexins—Therapeutic Targets in Cancers vol.21, pp.23, 2020, https://doi.org/10.3390/ijms21239119