Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1980 May;77(5):2964–2968. doi: 10.1073/pnas.77.5.2964

Phorbol esters induce differentiation in human malignant T lymphoblasts

Kohei Nagasawa 1, Tak W Mak 1
PMCID: PMC349527  PMID: 6967217

Abstract

At nanomolar concentrations, phorbol ester, a class of potent tumor promoters, can promote differentiation in the human malignant T-lymphoblastic cell line MOLT-3. The optimal dose for induction, as measured by the increase of the number of cells containing sheep erythrocyte receptors (E-rosette assay), is between 8 and 16 nM 12-O-tetradecanoylphorbol 13-acetate (TPA), although there were significant increases of E-rosette-positive (E+) cells at concentrations as low as 1.6 nM TPA. The induction was linear for 4 days, then it reached a plateau. This induction was independent of the cell densities of the cultures, and the viability of the E+ cells remained high (95-100%) even after 10 days of culture in the presence of the tumor promoters. The E+ cells, when measured with the more stable 2-aminoethylisothiouronium bromide E-rosette assay, indicated that virtually all (75-95%) of the MOLT-3 cells became E+ by 4 days in culture. This induction by TPA was also accompanied by a dramatic drop in the plating efficiencies and a reduction in DNA synthesis. Examination of phorbol and other phrobol esters indicated that the ability to induce these cells correlated well with the tumor-promoting activities of these compounds, because only TPA and to a lesser extent phorbol 12,13,-dibenzoate induced E+ cells, while phorbol and 4α-phorbol 12,13-didecanoate had no effect. Studies of MOLT-3 cells depleted of E+ cells indicated that the induction of E+ cells cannot be explained solely on the basis of enrichment or stimulation of the background E+ cells in MOLT-3 cultures. Finally, we have shown that TPA also affected another differentiation marker, the loss of the enzyme terminal deoxyribonucleotidyl transferase. Terminal transferase activities and percentages of terminal-transferase-positive cells in these cultures were reduced to as low as 1/10th in 4 days in the presence of 16 nM TPA.

Keywords: tumor promoter, T-cell leukemia, terminal differentiation, 12-O-tetradecanoylphorbol 13-acetate, terminal deoxyribonucleotidyl transferase

Full text

PDF
2964

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abb J., Bayliss G. J., Deinhardt F. Lymphocyte activation by the tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA). J Immunol. 1979 May;122(5):1639–1642. [PubMed] [Google Scholar]
  2. Bernstein A., Hunt D. M., Crichley V., Mak T. W. Induction by ouabain of hemoglobin synthesis in cultured Friend erythroleukemic cells. Cell. 1976 Nov;9(3):375–381. doi: 10.1016/0092-8674(76)90082-9. [DOI] [PubMed] [Google Scholar]
  3. Brain P., Gordon J., Willetts W. A. Rosette formation by peripheral lymphocytes. Clin Exp Immunol. 1970 May;6(5):681–688. [PMC free article] [PubMed] [Google Scholar]
  4. Chechik B. E., Gelfand E. W. Leukaemia-associated antigen in serum of patients with acute lymphoblastic leukaemia. Lancet. 1976 Jan 24;1(7952):166–168. doi: 10.1016/s0140-6736(76)91273-3. [DOI] [PubMed] [Google Scholar]
  5. Chechik B. E., Minowada J. Quantitation of human thymus-leukemia-associated antigen in established hematopoietic cell lines by radioimmunoassay. J Natl Cancer Inst. 1979 Sep;63(3):609–614. doi: 10.1093/jnci/63.3.609. [DOI] [PubMed] [Google Scholar]
  6. Collins S. J., Ruscetti F. W., Gallagher R. E., Gallo R. C. Terminal differentiation of human promyelocytic leukemia cells induced by dimethyl sulfoxide and other polar compounds. Proc Natl Acad Sci U S A. 1978 May;75(5):2458–2462. doi: 10.1073/pnas.75.5.2458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Coombs R. R., Gurner B. W., Wilson A. B., Holm G., Lindgren B. Rosette-formation between human lymphocytes and sheep red cells not involving immunoglobulin receptors. Int Arch Allergy Appl Immunol. 1970;39(5-6):658–663. doi: 10.1159/000230390. [DOI] [PubMed] [Google Scholar]
  8. Diamond L., O'Brien T. G., Rovera G. Inhibition of adipose conversion of 3T3 fibroblasts by tumour promoters. Nature. 1977 Sep 15;269(5625):247–249. doi: 10.1038/269247a0. [DOI] [PubMed] [Google Scholar]
  9. Fibach E., Yamasaki H., Weinstein I. B., Marks P. A., Rifkind R. A. Heterogeneity of murine erythroleukemia cells with respect to tumor promoter-mediated inhibition of cell differentiation. Cancer Res. 1978 Nov;38(11 Pt 1):3685–3688. [PubMed] [Google Scholar]
  10. Friend C., Scher W., Holland J. G., Sato T. Hemoglobin synthesis in murine virus-induced leukemic cells in vitro: stimulation of erythroid differentiation by dimethyl sulfoxide. Proc Natl Acad Sci U S A. 1971 Feb;68(2):378–382. doi: 10.1073/pnas.68.2.378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gelfand E. W., Shore A., Green B., Lin M. T., Dosch H. M. The E-rosette assay: a cautionary note. Clin Immunol Immunopathol. 1979 Jan;12(1):119–123. doi: 10.1016/0090-1229(79)90117-x. [DOI] [PubMed] [Google Scholar]
  12. Greaves M., Janossy G. Patterns of gene expression and the cellular origins of human leukaemias. Biochim Biophys Acta. 1978 Oct 27;516(2):193–230. doi: 10.1016/0304-419x(78)90008-2. [DOI] [PubMed] [Google Scholar]
  13. Gusella J. F., Housman D. Induction of erythroid differentiation in vitro by purines and purine analogues. Cell. 1976 Jun;8(2):263–269. doi: 10.1016/0092-8674(76)90010-6. [DOI] [PubMed] [Google Scholar]
  14. Hoffman R., Murnane M. J., Benz E. J., Jr, Prohaska R., Floyd V., Dainiak N., Forget B. G., Furthmayr H. Induction of erythropoietic colonies in a human chronic myelogenous leukemia cell line. Blood. 1979 Nov;54(5):1182–1187. [PubMed] [Google Scholar]
  15. Huberman E., Callaham M. F. Induction of terminal differentiation in human promyelocytic leukemia cells by tumor-promoting agents. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1293–1297. doi: 10.1073/pnas.76.3.1293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kung P. C., Long J. C., McCaffrey R. P., Ratliff R. L., Harrison T. A., Baltimore D. Terminal deoxynucleotidyl transferase in the diagnosis of leukemia and malignant lymphoma. Am J Med. 1978 May;64(5):788–794. doi: 10.1016/0002-9343(78)90518-1. [DOI] [PubMed] [Google Scholar]
  17. Lay W. H., Mendes N. F., Bianco C., Nussenzweig V. Binding of sheep red blood cells to a large population of human lymphocytes. Nature. 1971 Apr 23;230(5295):531–532. doi: 10.1038/230531a0. [DOI] [PubMed] [Google Scholar]
  18. Leder A., Leder P. Butyric acid, a potent inducer of erythroid differentiation in cultured erythroleukemic cells. Cell. 1975 Jul;5(3):319–322. doi: 10.1016/0092-8674(75)90107-5. [DOI] [PubMed] [Google Scholar]
  19. Lotem J., Sachs L. In vivo induction of normal differentiation in myeloid leukemia cells. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3781–3785. doi: 10.1073/pnas.75.8.3781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lotem J., Sachs L. Induction of specific changes in the surface membrane of myeloid leukemic cells by steroid hormones. Int J Cancer. 1975 May 15;15(5):731–740. doi: 10.1002/ijc.2910150504. [DOI] [PubMed] [Google Scholar]
  21. Lotem J., Sachs L. Regulation of normal differentiation in mouse and human myeloid leukemic cells by phorbol esters and the mechanism of tumor promotion. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5158–5162. doi: 10.1073/pnas.76.10.5158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Marks P. A., Rifkind R. A. Erythroleukemic differentiation. Annu Rev Biochem. 1978;47:419–448. doi: 10.1146/annurev.bi.47.070178.002223. [DOI] [PubMed] [Google Scholar]
  23. Mastro A. M., Mueller G. C. Synergistic action of phorbol esters in mitogen-activated bovine lymphocytes. Exp Cell Res. 1974 Sep;88(1):40–46. doi: 10.1016/0014-4827(74)90615-6. [DOI] [PubMed] [Google Scholar]
  24. McCulloch E. A. Abnormal myelopoietic clones in man. J Natl Cancer Inst. 1979 Oct;63(4):883–892. [PubMed] [Google Scholar]
  25. McCulloch E. A. Haemopoiesis in myeloblastic leukaemia. Clin Haematol. 1979 Jun;8(2):501–523. [PubMed] [Google Scholar]
  26. McCulloch E. A., Mak T. W., Price G. B., Till J. E. Organization and communication in populations of normal and leukemic hemopoietic cells. Biochim Biophys Acta. 1974 Dec 31;355(3-4):260–299. doi: 10.1016/0304-419x(74)90013-4. [DOI] [PubMed] [Google Scholar]
  27. Miao R. M., Filedsteel A. H., Fodge D. W. Opposing effects of tumor promoters on erythroid differentiation. Nature. 1978 Jul 20;274(5668):271–272. doi: 10.1038/274271a0. [DOI] [PubMed] [Google Scholar]
  28. Miller E. C. Some current perspectives on chemical carcinogenesis in humans and experimental animals: Presidential Address. Cancer Res. 1978 Jun;38(6):1479–1496. [PubMed] [Google Scholar]
  29. Minowada J., Onuma T., Moore G. E. Rosette-forming human lymphoid cell lines. I. Establishment and evidence for origin of thymus-derived lymphocytes. J Natl Cancer Inst. 1972 Sep;49(3):891–895. [PubMed] [Google Scholar]
  30. Okamura S., Crane F., Messner H. A., Mak T. W. Purification of terminal deoxynucleotidyltransferase by oligonucleotide affinity chromatography. J Biol Chem. 1978 Jun 10;253(11):3765–3767. [PubMed] [Google Scholar]
  31. Pellegrino M. A., Ferrone S., Dierich M. P., Reisfeld R. A. Enhancement of sheep red blood cell human lymphocyte rosette formation by the sulfhydryl compound 2-amino ethylisothiouronium bromide. Clin Immunol Immunopathol. 1975 Jan;3(3):324–333. doi: 10.1016/0090-1229(75)90019-7. [DOI] [PubMed] [Google Scholar]
  32. Pyke K. W., Rawlings G. A., Gelfand E. W. Isolation and characterization of the sheep erythrocyte receptor in man. J Immunol. 1975 Jul;115(1):211–215. [PubMed] [Google Scholar]
  33. Rovera G., O'Brien T. G., Diamond L. Induction of differentiation in human promyelocytic leukemia cells by tumor promoters. Science. 1979 May 25;204(4395):868–870. doi: 10.1126/science.286421. [DOI] [PubMed] [Google Scholar]
  34. Sachs L. Control of normal cell differentiation and the phenotypic reversion of malignancy in myeloid leukaemia. Nature. 1978 Aug 10;274(5671):535–539. doi: 10.1038/274535a0. [DOI] [PubMed] [Google Scholar]
  35. Shohat B., Wolach B., Trainin N. Effect of thymic humoral factor in vitro on human bone marrow and blood cells from B-, T- and null-cell acute lymphatic leukemia. Cell Immunol. 1979 Jul;45(2):255–260. doi: 10.1016/0008-8749(79)90386-1. [DOI] [PubMed] [Google Scholar]
  36. Skinnider L. F. The effect of phorbol myristate acetate on human lymphocytes. An ultrastructural study. Exp Mol Pathol. 1979 Aug;31(1):36–43. doi: 10.1016/0014-4800(79)90005-4. [DOI] [PubMed] [Google Scholar]
  37. Touraine J. L., Hadden J. W., Touraine F., Hadden E. M., Estensen R., Good R. A. Phorbol myristate acetate: a mitogen selective for a T-lymphocyte subpopulation. J Exp Med. 1977 Feb 1;145(2):460–465. doi: 10.1084/jem.145.2.460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wan C. W., Mak T. W. Effect of tetrahydrouridine on the action of 1-beta-D-arabinofuranosylcytosine in synchronized cultures of normal rat kidney cells. Cancer Res. 1979 Oct;39(10):3981–3985. [PubMed] [Google Scholar]
  39. Wang J. L., McClain D. A., Edelman G. M. Modulation of lymphocyte mitogenesis. Proc Natl Acad Sci U S A. 1975 May;72(5):1917–1921. doi: 10.1073/pnas.72.5.1917. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES