Effects of Okadaic Acid, Retinoic Acid, and Phorbol Myristate Acetate Tumor Promoter on Oncogene Expression


The effect of okadaic acid (OA) on proto-oncogene protein expression of c-neu, c-myc, v-rasH, EGFR, and phosphotyrosine-containing phosphoproteins (P-Tyr) was investigated in rapidly growing (RG) normal human keratinocytes (NHK) and in SV-40 virally-transformed keratinocytes (SVK) cultured in a growth factor supplemented serum-free medium as assessed by indirect immunofluorescence microscopy. P-Tyr positively stains cell surface antigens (cytoplasm) diffusely at monopolar sites in RG NHK cultures. OA-treatment intensifies cytoplasmic P-Tyr staining at localized monopolar intercellular focal adhesion (IFA) sites with reduced cytoplasmic staining. P-Tyr expression was predominate at IFA sites with little cytoplasmic staining in RG SVK cultures. OA-treatment increased monopolar P-Tyr staining and cytoplasmic staining. OA-treatment in RG NHK cultures intensified cytoplasmic staining of c-myc and EGFR (epidermal growth factor receptor) expression. OA-treatment in RG NHK and SVK cultures intensified c-neu staining at monopolar IFA sites and intensified c-neu staining at both cytoplasmic and bipolar IFA sites in RG SVK cells. OA was especially cytotoxic for SVK cells. RA treatment decreased c-neu expression in RG NHK cultures while TPA treatment has a lesser effect on both cytoplasmic and IFA sites. RA treatment also decreased P-Tyr staining in both NHK and SVK cells. Again, TPA had a lesser inhibitory effect on P-Tyr staining pattern. RA-treatment had a similar effect on P-Tyr staining of RG cultures of a mouse fibroblast cell line. These results confirm the generality of OA, RA and TPA on the regulation of oncogene expression in both normal and malignantly transformed keratinocytes.

Share and Cite:

Wille, J. and Park, J. (2014) Effects of Okadaic Acid, Retinoic Acid, and Phorbol Myristate Acetate Tumor Promoter on Oncogene Expression. Journal of Cancer Therapy, 5, 591-604. doi: 10.4236/jct.2014.56068.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Croce, C.M. (2008) Oncogenes and Cancer. The New England Journal of Medicine, 358, 502-511. http://dx.doi.org/10.1056/NEJMra072367
[2] Butel, J.S. and Lednicky, J.A. (1999) Cell and Molecular Biology of Simian Virus 40: Implications for Human Infections and Disease. Journal of the National Cancer Institute, 91, 119-134.
[3] Yaffe, M.B. (2002) Phosphotyrosine-Binding Domains in Signal Transduction. Nature Reviews Molecular Cell Biology, 3, 177-186. http://dx.doi.org/10.1038/nrm759
[4] Varani, J., Zeigler, M., Dame, M.K., Kang, S., Fisher, G.J., Voorhees, J.J., Stoll, S.W. and Elder, J.T. (2001) Heparin-Binding Epidermal-Growth-Factor-Like Growth Factor Activation of Keratinocyte ErbB Receptors Mediates Epidermal Hyperplasia, a Prominent Side-Effect of Retinoid Therapy. Journal of Investigative Dermatology, 117, 1335-1341. http://dx.doi.org/10.1046/j.0022-202x.2001.01564.x
[5] De Potter, I.Y., Poumay, Y., Squillace, K.A. and Pittelkow, M.R. (2001) Human EGF Receptor (HER) Family and Heregulin Members Are Differentially Expressed in Epidermal Keratinocytes and Modulate Differentiation. Experimental Cell Research, 271, 315-328. http://dx.doi.org/10.1006/excr.2001.5390
[6] Lewis, D.A., Zweig, B., Hurwitz, S.A. and Spandau, D.F. (2003) Inhibition of erbB Receptor Family Members Protects HaCaT Keratinocytes from Ultraviolet-B-Induced Apoptosis. Journal of Investigative Dermatology, 120, 483-488. http://dx.doi.org/10.1046/j.1523-1747.2003.12060.x
[7] Montemurro, F. and Scaltriti, M. (2014) Biomarkers of Drugs Targeting HER-Family Signalling in Cancer. The Journal of Pathology, 232, 219-229. http://dx.doi.org/10.1002/path.4269
[8] Reiss, M., Dibble, C.L. and Narayanan, R. (1989) Transcriptional Activation of the c-myc Proto-Oncogene in Murine Keratinocytes Enhances the Response to Epidermal Growth Factor. Journal of Investigative Dermatology, 93, 136-141. http://dx.doi.org/10.1111/1523-1747.ep12277384
[9] Wille, J.J. (1989) Malignant Transformation of Normal Human Keratinocytes by SV-40 Virus. The Annals of the New York Academy of Sciences, 567, 307-310.
[10] Wilke, M.S., Hsu, B.M., Wille Jr., J.J., Pittelkow, M.R. and Scott, R.E. (1988) Biologic Mechanisms for the Regulation of Normal Human Keratinocyte Proliferation and Differentiation. American Journal of Pathology, 131, 171-181.
[11] Suganuma, M., Fujiki, H., Suguri, H., Yoshizawa, S., Hirota, M., Nakayasu, M., Ojika, M., Wakamatsu, K., Yamada, K. and Sugimura, T. (1988) Okadaic Acid: An Additional Non-Phorbol-12-Tetradecanoate-13-Acetate-Type Tumor Promoter. Proceedings of the National Academy of Sciences, 85, 1768-1771. http://dx.doi.org/10.1073/pnas.85.6.1768
[12] Fujiki, H., Suganuma, M., Yoshizawa, S., Nishiwaki, S., Winyar, B. and Sugimura, T. (1991) Mechanisms of Action of Okadaic Acid Class Tumor Promoters on Mouse Skin. Environmental Health Perspectives, 93, 211-214. http://dx.doi.org/10.1289/ehp.9193211
[13] Sakai, R., Ikeda, I., Kitani, H., Fujiki, H., Takaku, F., Rapp, U., Sugimura, T. and Nagao, M. (1989) Flat Reversion by Okadaic Acid of raf and ret-II Transformants. Proceedings of the National Academy of Sciences, 86, 9946-9950. http://dx.doi.org/10.1073/pnas.86.24.9946
[14] Garcia, A., Cayla, X., Guergnon, J., Dessauge, F., Hospital, V., Rebollo, M.P., Fleischer, A. and Rebollo, A. (2003) Serine/Threonine Protein Phosphatases PP1 and PP2A Are Key Players in Apoptosis. Biochimie, 85, 721-726. http://dx.doi.org/10.1016/j.biochi.2003.09.004
[15] Boudreau, R.T. and Hoskin, D.W. (2005) The Use of Okadaic Acid to Elucidate the Intracellular Role(s) of Protein Phosphatase 2A: Lessons from the Mast Cell Model System. International Immunopharmacology, 5, 1507-1518. http://dx.doi.org/10.1016/j.intimp.2005.05.007
[16] Valdiglesias, V., Laffon, B., Pásaro, E. and Méndez, J. (2011) Okadaic Acid Induces Morphological Changes, Apoptosis and Cell Cycle Alterations in Different Human Cell Types. Journal of Environmental Monitoring, 13, 1831-1840. http://dx.doi.org/10.1039/c0em00771d
[17] Matias, W.G., Traore, A., Bonini, M., Sanni, A. and Creppy, E.E. (1999) Oxygen Reactive Radicals Production in Cell Culture by Okadaic Acid and Their Implication in Protein Synthesis Inhibition. Human & Experimental Toxicology, 18, 634-639. http://dx.doi.org/10.1191/096032799678839473
[18] Haystead, T.A., Sim, A.T.R., Carling, D., Honnor, R.C., Tsukitani, Y., et al. (1989) Effects of the Tumour Promoter Okadaic Acid on Intracellular Protein Phosphorylation and Metabolism. Nature, 337, 78-81. http://dx.doi.org/10.1038/337078a0
[19] Haneji, T., Hirashima, K., Teramachi, J. and Morimoto, H. (2013) Okadaic Acid Activates the PKR Pathway and Induces Apoptosis through PKR Stimulation in MG63 Osteoblast-Like Cells. International Journal of Oncology, 42, 1904-1910.
[20] Zhang, M.L., Tao, Y., Zhou, W.Q., Ma, P.C., Cao, Y.P., et al. (2014) All-Trans Retinoic Acid Induces Cell-Cycle Arrest in Human Cutaneous Squamous Carcinoma Cells by Inhibiting the Mitogen-Activated Protein Kinase-Activated Protein 1 Pathway. Clinical and Experimental Dermatology, 39, 354-360. http://dx.doi.org/10.1111/ced.12227
[21] Vahlquist, A., Lee, J.B., Micha?lsson, G. and Rollman, O. (1982) Vitamin A in Human Skin: II Concentrations of Carotene, Retinol and Dehydroretinol in Various Components of Normal Skin. Journal of Investigative Dermatology, 79, 94-97. http://dx.doi.org/10.1111/1523-1747.ep12500033
[22] Lotan, R. and Nicolson, G.L. (1977) Inhibitory Effects of Retinoic Acid or Retinyl Acetate on the Growth of Untransformed, Transformed, and Tumor Cells in Vitro. JNCI Journal of the National Cancer Institute, 59, 1717-1722.
[23] Wille, J.J.B.J., Park, J.Y. and Triggs, G. (2002) Retinoid Stimulation of Autocrine Growth Factor Controlled Keratinocyte Proliferation. Wound Healing Society, Baltimore.
[24] Wille, J.J. and Chopra, D.P. (1988) Reversal by Retinoids of Keratinization Induced by Benzo[alpha]pyrene in Normal Hamster Tracheal Explants: Comparison with the Assay Involving Organ Culture of Tracheas from Vitamin A-Deficient Hamsters. Cancer Letters, 40, 235-246.
[25] Verma, A.K., Rice, H.M., Shapas, B.G., and Boutwell, R.K. (1978) Inhibition of 12-O-tetradecanoylphorbol-13-acetate-Induced Ornithine Decarboxylase Activity in Mouse Epidermis by Vitamin A Analogs (Retinoids). Cancer Research, 38, 793-801.
[26] Verma, A.K., Shapas, B.G., Rice, H.M. and Boutwell, R.K. (1979) Correlation of the Inhibition by Retinoids of Tumor Promoter-Induced Mouse Epidermal Ornithine Decarboxylase Activity and of Skin Tumor Promotion. Cancer Research, 39, 419-425.
[27] Cope, F.O. and Wille, J.J. (1989) Retinoid Receptor Antisense DNAs Inhibit Alkaline Phosphatase Induction and Clonogenicity in Malignant Keratinocytes. Proceedings of the National Academy of Sciences of the United States of America, 86, 5590-5594. http://dx.doi.org/10.1073/pnas.86.14.5590
[28] Iizuka, H., Sakai, H. and Tamura, T. (1989) Effects of the Tumor Promoter, Phorbol 12-Myristate, 13-Acetate, on the Epidermal Adenylate Cyclase System: Evidence for Adenylate Cyclase-Regulation by Protein Kinase C. Journal of Investigative Dermatology, 93, 387-391.
[29] Wille Jr., J.J., Pittelkow, M.R. and Scott, R.E. (1985) Normal and Transformed Human Prokeratinocytes Express Divergent Effects of a Tumor Promoter on Cell Cycle-Mediated Control of Proliferation and Differentiation. Carcinogenesis, 6, 1181-1187. http://dx.doi.org/10.1093/carcin/6.8.1181
[30] Wille, J. (2004) Effect of Okadiac Acid on Oncogene Expression in Normal and Transformed Human Keratinocytes. Proceedings of the American Association for Cancer Research, 27-31 March 2004, Orlando, Abstract #: 2778.
[31] Wille Jr., J.J., Pittelkow, M.R., Shipley, G.D. and Scott, R.E. (1984) Integrated Control of Growth and Differentiation of Normal Human Prokeratinocytes Cultured in Serum-Free Medium: Clonal Analyses, Growth Kinetics, and Cell Cycle Studies. Journal of Cellular Physiology, 121, 31-44. http://dx.doi.org/10.1002/jcp.1041210106
[32] Maguire Jr., H.C., Jaworsky, C., Cohen, J.A., Hellman, M., Weiner, D.B. and Greene, M.I. (1989) Distribution of Neu (C-erbB-2) Protein in Human Skin. Journal of Investigative Dermatology, 92, 786-790. http://dx.doi.org/10.1111/1523-1747.ep12696796
[33] Taylor, R.B., Duffus, W.P.H., Raff, M.C. and de Petris, S. (1971) Redistribution and Pinocytosis of Lymphocyte Surface Immunoglobulin Molecules Induced by Anti-Immunoglobulin Antibody. Nature New Biology, 233, 225-229. http://dx.doi.org/10.1038/newbio233225a0
[34] Wille, J. and Park, J.Y. (2012) Retinoid and Ethanol-Sensitive Benzo[Alpha]pyrene Induction of Cytochrome P-450 in Human Keratinocytes. Journal of Cancer Therapy, 3, 1080-1085.

Copyright © 2022 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.