Gap Junctional Intercellular Communication Increases Cytotoxicity and Reduces Resistance to Hydroxyurea


Background: Gap junctions enable small molecules to diffuse between adjacent cells and have been associated with greater cytotoxicity of radiation and anti-cancer drugs. We investigated whether this gap junctional intercellular communication (GJIC) affected the cytotoxicity of the classic ribonucleotide reductase (RR) inhibitor and anti-cancer agent, hydroxyurea (HU). Materials and Methods: We used GJIC-proficient and deficient, connexin 43-expressing WB rat liver epithelial cell lines. We compared HU toxicity by crystal violet assay, effects of the drug on deoxynucleotide pools by HPLC, and ability of GJIC to increase toxicity of HU-resistant cells through a bystander effect in co-culture experiments. Results: GJIC-proficient cells were three- to five-fold more sensitive (IC50 0.1 mM) to HU than GJIC-deficient derivatives (IC50 0.3 - 0.5 mM). This sensitivity depended upon GJIC because treatment of GJIC-proficient cells with the GJIC blocker oleamide decreased HU toxicity by approximately 60% - 80% and restoration of GJIC in GJIC-deficient cells by stable transduction of connexin 32-encoding Gjb1 increased HU toxicity (IC500.1 mM). The effects were not due to connexin expression per se or its localization since all cell lines expressed comparable quantities of connexin 43 that was localized to the plasma membrane. Also HU sensitivity was not related to differential effects on nucleotide metabolism in the cells. Thymidine triphosphate levels increased and deoxyadenosine triphosphate levels decreased similarly (15% - 20%) in GJIC-proficient and deficient cells over 24 h of HU treatment. More importantly, when HU-resistant cells were co-cultured with sensitive cells, the resistant cells were killed only when GJIC was present. Conclusion: The data suggest that GJIC enhances cytotoxicity and decreases resistance to HU. These results may be important clinically if GJIC can be enhanced in drug-resistant cells.

Share and Cite:

Ruch, R. , Boucher, P. , Gentry, B. and Shewach, D. (2014) Gap Junctional Intercellular Communication Increases Cytotoxicity and Reduces Resistance to Hydroxyurea. Journal of Cancer Therapy, 5, 1190-1202. doi: 10.4236/jct.2014.513121.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Evans, W.H. and Martin, P.E. (2002) Gap Junctions: Structure and Function (Review). Molecular Membrane Biology, 19, 121-136.
[2] Sohl, G. and Willecke, K. (2003) An Update on Connexin Genes and Their Nomenclature in Mouse and Man. Cell Communication and Adhesion, 10, 173-180.
[3] Dbouk, H.A., Mroue, R.M., El-Sabban, M.E. and Talhouk, R.S. (2009) Connexins: A Myriad of Functions Extending beyond Assembly of Gap Junction Channels. Cell Communication and Signaling, 7, 1-17.
[4] Herve, J.C., Bourmyster, N., Sarrouilhe, D. and Duffy, H.S. (2007) Gap Junctional Complexes: From Partners to Functions. Progress in Biophysics & Molecular Biology, 94, 29-65.
[5] Decrock, E., Vinken, M., De Vuyst, E., Krysko, D.V., D’Herde, K., Vanhaecke, T., Vandenabeele, P., Rogiers, V. and Leybaert, L. (2009) Connexin-Related Signaling in Cell Death: To Live or Let Die? Cell Death and Differentiation, 16, 151-163.
[6] Nocentini, G. (2006) Ribonucleotide Reductase Inhibitors: New Strategies for Cancer Chemotherapy. Critical Reviews in Oncology and Hematology, 22, 89-126.
[7] Navarra, P. and Preziosi, P. (1999) Hydroxyurea: New Insights on an Old Drug. Critical Reviews in Oncology and Hematology, 29, 249-255.
[8] Guan, X., Cravatt, B.F., Ehring, G.R., Hall, J.E., Boger, D.L., Lerner, R.A. and Gilula, N.B. (1997) The Sleep-Inducing Lipid Oleamide Deconvolutes Gap Junction Communication and Calcium Wave Transmission in Glial Cells. Journal of Cell Biology, 139, 1785-1792.
[9] Esinduy, C.B., Chang, C.C., Trosko, J.E. and Ruch, R.J. (1995) In Vitro Growth Inhibition of Neoplastically Transformed Cells by Non-Transformed Cells: Requirement for Gap Junctional Intercellular Communication. Carcinogenesis, 16, 915-921.
[10] Boucher, P.D., Ostruszka, L.J. and Shewach, D.S. (2000) Synergistic Enhancement of Herpes Simplex Virus Thymidine Kinase/Ganciclovir-Mediated Cytoxicity by Hydroxyurea. Cancer Research, 60, 1631-1636.
[11] El-Fouly, M.H., Trosko, J.E. and Chang, C.C. (1987) Scrape-Loading and Dye Transfer: A Rapid and Simple Technique to Study Gap Junctional Intercellular Communication. Experimental Cell Research, 168, 422-430.
[12] Nielsen, M., Ruch, R.J. and Vang, O. (2000) Resveratrol Reverses Tumor-Promoter-Induced Inhibition of Gap-Junctional Intercellular Communication. Biochemical and Biophysical Research Communications, 275, 804-809.
[13] Guan, X.J. and Ruch, R.J. (1996) Gap Junction Endocytosis and Lysosomal Degradation of Connexin 43-P2 in WBF344 Rat Liver Epithelial Cells Treated with DDT and Lindane. Carcinogenesis, 17, 1791-1798.
[14] Mehta, P.P., Lokeshwar, B.L., Schiller, P.C., Bendix, M.V., Ostenson, R.C., Howard, G.A. and Roos, B.A. (1996) Gap-Junctional Communication in Normal and Neoplastic Prostate Epithelial Cells and Its Regulation by cAMP. Molecular Carcinogenesis, 15, 18-32.<18::AID-MC4>3.0.CO;2-O
[15] Tsao, M.S., Smith, J.D., Nelson, K.G. and Grisham, J.W. (1984) A Diploid Epithelial Cell Line from Normal Adult Rat Liver with Phenotypic Properties of “Oval” Cells. Experimental Cell Research, 154, 38-52.
[16] Oh, S.Y., Dupont, E., Madhukar, B.V., Briand, J.P., Chang, C.C., Beyer, E. and Trosko, J.E. (1993) Characterization of Gap Junctional Communication-Deficient Mutants of a Rat Liver Epithelial Cell Line. European Journal of Cell Biology, 60, 250-255.
[17] Rae, R.S., Mehta, P.P., Chang, C.C., Trosko, J.E. and Ruch, R.J. (1998) Neoplastic Phenotype of Gap-Junctional Intercellular Communication-Deficient WB Rat Liver Epithelial Cells and Its Reversal by Forced Expression of Connexin 32. Molecular Carcinogenesis, 22, 120-127.<120::AID-MC7>3.0.CO;2-Q
[18] Jiang, R., Zhang, J.L., Satoh, Y. and Sairenji, T. (2004) Mechanism for Induction of Hydroxyurea Resistance and Loss of Latent EBV Genome in Hydroxyurea-Treated Burkitt’s Lymphoma Cell Line Raji. Journal of Medical Virology, 73, 589-595.
[19] Yen, Y., Grill, S.P., Dutschman, G.E., Chang, C.N., Zhou, B.S. and Cheng, Y.C. (1994) Characterization of a Hydroxyurea-Resistant Human KB Cell Line with Supersensitivity to 6-Thioguanine. Cancer Research, 54, 3686-3691.
[20] Lin, J.H., Weigel, H., Cotrina, M.L., Liu, S., Bueno, E., Hansen, A.J., Hansen, T.W., Goldman, S. and Nedergaard, M. (1998) Gap-Junction-Mediated Propagation and Amplification of Cell Injury. Nature Neuroscience, 1, 494-500.
[21] Hamada, N., Matsumoto, H., Hara, T. and Kobayashi, Y. (2007) Intercellular and Intracellular Signaling Pathways Mediating Ionizing Radiation-Induced Bystander Effects. Journal of Radiation Research, 48, 87-95.
[22] Hei, T.K., Zhou, H., Chai, Y., Ponnaiya, B. and Ivanov, V.N. (2011) Radiation Induced Non-Targeted Response: Mechanism and Potential Clinical Implications. Current Molecular Pharmacology, 4, 96-105.
[23] Nakase, T., Fushiki, S. and Naus, C.C. (2003) Astrocytic Gap Junctions Composed of Connexin 43 Reduce Apoptotic Neuronal Damage in Cerebral Ischemia. Stroke, 34, 1987-1993.
[24] King, S.B. (2003) The Nitric Oxide Producing Reactions of Hydroxyurea. Current Medicinal Chemistry, 10, 437-452.
[25] Mennecier, G., Derangeon, M., Coronas, V., Hervé, J.C. and Mesnil, M. (2008) Aberrant Expression and Localization of Connexin 43 and Connexin 30 in a Rat Glioma Cell Line. Molecular Carcinogenesis, 47, 391-401.
[26] Yu, S.C., Xiao, H.L., Jiang, X.F., Wang, Q.L., Li, Y., Yang, X.J., Ping, Y.F., Duan, J.J., Jiang, J.Y., Ye, X.Z., Xu, S.L., Xin, Y.H., Yao, X.H., Chen, J.H., Chu, W.H., Sun, W., Wang, B., Wang, J.M., Zhang, X. and Bian, X.W. (2012) Connexin 43 Reverses Malignant Phenotypes of Glioma Stem Cells by Modulating E-Cadherin. Stem Cells, 30, 108-120.
[27] Trosko, J.E. and Ruch, R.J. (2002) Gap Junctions as Targets for Cancer Prevention and Chemotherapy. Current Drug Targets, 3, 465-482.

Copyright © 2021 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.