Abstract
Prothymosin α (proTα) is a 109 amino acid long polypeptide presenting distinct immunoenhancing activity in vitro and in vivo. Recent reports suggest that in apoptotic cells, proTα is cleaved by caspases at its carboxy(C)-terminus generating potentially bioactive fragments. In this study, we identified the peptide segment of proTα presenting maximum immunomodulatory activity. Calf thymus proTα was trypsinised, and the five fragments produced (spanning residues 1–14, 21–30, 31–87, 89–102 and 103–109) were tested for their ability to stimulate healthy donor- and cancer patient-derived peripheral blood mononuclear cell (PBMC) proliferation in autologous mixed lymphocyte reaction (AMLR), natural killer and lymphokine-activated killer cell activity, intracellular production of perforin, upregulation of adhesion molecules and CD25 expression. ProTα(89–102) and proTα(103–109) significantly fortified healthy donor-lymphocytes’ immune responses to levels comparable to those induced by intact proTα. These effects were more pronounced in cancer patients, where peptides proTα(89–102) and proTα(103–109) partly, however significantly, restored the depressed AMLR and cytolytic ability of PBMC, by simulating the biological activity exerted by intact proTα. ProTα(1–14), proTα(21–30) and proTα(31–87) marginally upregulated lymphocyte activation. This is the first report showing that proTα’s immunomodulating activity can be substituted by its C-terminal peptide(s). Whether generation and externalization of such immunoactive proTα fragments occurs in vivo, needs further investigation. However, if these peptides can trigger immune responses, they may eventually be used therapeutically to improve some PBMC functions of cancer patients.
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Abbreviations
- AMLR:
-
Autologous mixed lymphocyte reaction
- BRM:
-
Biologic response modifiers
- IFN:
-
Interferon
- IL-2:
-
Interleukin-2
- IL-2R:
-
Interleukin-2 receptor
- LAK cells:
-
Lymphokine-activated killer cells
- NK cells:
-
Natural killer cells
- NLS:
-
Nuclear localization signal
- PBMC:
-
Peripheral blood mononuclear cells
- PMN:
-
Polymorphonuclear cells
- proTα:
-
Prothymosin α
- Τα1 :
-
Thymosin α1
References
Haritos AA, Goodall GJ, Horecker BL (1984) Prothymosin α: isolation and properties of the major immunoreactive form of thymosin α1 in rat thymus. Proc Natl Acad Sci USA 81:1008–1011
Haritos AA (1987) α-Thymosins: relationships in structure, distribution, and function. Isozymes Curr Top Biol Med Res 14:123–152
Eschenfeldt WH, Berger SL (1986) The human prothymosin α gene is polymorphic and induced upon growth stimulation: evidence using a cloned cDNA. Proc Natl Acad Sci USA 83:9403–9407
Pineiro A, Cordero OJ, Nogueira M (2000) Fifteen years of prothymosin alpha: contradictory past and new horizons. Peptides 21:1433–1446
Tsitsiloni OE, Stiakakis J, Koutselinis A, Gogas J, Markopoulos C, Yialouris P, Bekris S, Panoussopoulos D, Kiortsis V, Voelter W, Haritos AA (1993) Expression of α-thymosins in human tissues in normal and abnormal growth. Proc Natl Acad Sci USA 90:9504–9507
Eilers M, Schirm S, Bishop JM (1991) The MYC protein activates transcription of the α-prothymosin gene. EMBO J 10:133–141
Perez-Estevez A, Diaz-Jullien C, Covelo G, Salgueiro MT, Freire M (1997) A 180-kDa protein kinase seems to be responsible for the phosphorylation of prothymosin α observed in proliferating cells. J Biol Chem 272:10506–10513
Manrow RE, Sburlati AR, Hanover JA, Berger SL (1991) Nuclear targeting of prothymosin α. J Biol Chem 266:3916–3924
Diaz-Jullien C, Perez-Estevez A, Covelo G, Freire M (1996) Prothymosin α binds histones in vitro and shows activity in nucleosome assembly assay. Biochim Biophys Acta 1296:219–227
Karetsou Z, Kretsovali A, Murphy C, Tsolas O, Papamarcaki T (2002) Prothymosin α interacts with the CREB-binding protein and potentiates transcription. EMBO Rep 3:361–366
Trumbore MW, Wang RH, Enkemann SA, Berger SL (1997) Prothymosin α in vivo contains phosphorylated glutamic acid residues. J Biol Chem 272:26394–26404
Cotter MA, Robertson ES (2000) Modulation of histone acetyltransferase activity through interaction of Epstein-Barr nuclear antigen 3C with prothymosin alpha. Mol Cell Biol 20:5722–5735
Jiang X, Kim HE, Shu H, Zhao Y, Zhang H, Kofron J, Donnelly J, Burns D, Ng SC, Rosenberg S, Wang X (2003) Distinctive roles of PHAP proteins and prothymosin-α in a death regulatory pathway. Science 299:223–226
Segade F, Gomez-Marquez J (1999) Prothymosin α. Int J Biochem Cell Biol 31:1243–1248
Baxevanis CN, Frillingos S, Seferiadis K, Reclos GJ, Arsenis P, Katsiyiannis A, Anastasopoulos E, Tsolas O, Papamichail M (1990) Enhancement of human T lymphocyte function by prothymosin α: increased production of interleukin-2 and expression of interleukin-2 receptors in normal human peripheral blood T lymphocytes. Immunopharmacol Immunotoxicol 12:595–617
Garbin F, Eckert K, Immenschuh P, Kreuser ED, Maurer HR (1997) Prothymosin α1 effects, in vitro, on the antitumor activity and cytokine production of blood monocytes from colorectal tumor patients. Int J Immunopharmacol 19:323–332
Baxevanis CN, Τhanos D, Reclos GJ, Anastasopoulos E, Tsokos GC, Papamatheakis J, Papamichail M (1992) Prothymosin α enhances human and murine MHC class II surface antigen expression and messenger RNA accumulation. J Immunol 148:1979–1984
Voutsas IF, Baxevanis CN, Gritzapis AD, Missitzis I, Stathopoulos GP, Archodakis G, Banis C, Voelter W, Papamichail M (2000) Synergy between interleukin-2 and prothymosin α for the increased generation of cytotoxic T lymphocytes against autologous human carcinomas. Cancer Immunol Immunother 49:449–458
Baxevanis CN, Gritzapis AD, Spanakos G, Tsitsilonis OE, Papamichail M (1995) Induction of tumor-specific T lymphocyte responses in vivo by prothymosin α. Cancer Immunol Immunother 40:410–418
Heidecke H, Eckert K, Schulze-Forster K (1997) Prothymosin α1 effects in vitro on chemotaxis, cytotoxicity and oxidative response of neutrophils from melanoma, colorectal and breast tumor patients. Int J Immunopharmacol 19:413–420
Enkemann SA, Wang RH, Trumbore MW, Berger SL (2000) Functional discontinuities in prothymosin α caused by caspase cleavage in apoptotic cells. J Cell Physiol 182:256–268
Evstafieva AG, Belov GA, Kalkum M, Chichkova NV, Bogdanov AA, Agol VI, Vartapetian AB (2000) Prothymosin α fragmentation in apoptosis. FEBS Lett 467:150–154
Baxevanis CN, Spanakos G, Voutsas IF, Gritzapis AD, Tsitsilonis OE, Mamalaki A, Papamichail M (1999) Increased generation of autologous tumor-reactive lymphocytes by anti-CD3 monoclonal antibody and prothymosin α. Cancer Immunol Immunother 48:71–84
Baxevanis CN, Voutsas JF, Tsitsilonis OE, Gritzapis AD, Sotiriadou R, Papamichail M (2000) Tumor-specific CD4+ T lymphocytes from cancer patients are required for optimal induction of cytotoxic T cells against the autologous tumor. J Immunol 164:3902–3912
Raziuddin S, Abu-Eshy S, Sheikka A (1994) Peripheral T-cell lymphomas. Immunoregulatory cytokine (interleukin-2, interleukin-4, and interferon-γ) abnormalities and autologous mixed lymphocyte reaction. Cancer 74:2843–2849
Goldstein AL, Badamchian M (2004) Thymosins: chemistry and biological properties in health and disease. Expert Opin Biol Ther 4:559–573
Cordero OJ, Sarandeses C, Lopez-Rodriguez JL, Nogueira M (1995) The presence and cytotoxicity of CD16+ CD2- subset from PBL and NK cells in long-term IL-2 cultures enhanced by prothymosin-α. Immunopharmacology 29:215–223
Eckert K, Gruenberg E, Immenschuh P, Gabrin F, Kreuser ED, Maurer HR (1997) Interleukin-2-activated killer cell activity in colorectal tumor patients: evaluation of in vitro effects by prothymosin α1. J Cancer Res Clin Oncol 123:420–428
Eckert K, Schmitt M, Gabrin F, Wahn U, Maurer HR (1994) Thymosin α1 effects, in vitro, on lymphokine-activated killer cells from patients with primary immunodeficiencies: preliminary results. Int J Immunopharmacol 16:1019–1025
Garbin F, Eckert K, Buttner P, Garbe C, Maurer HR (1994) Prothymosin α augments deficient antitumor activity of monocytes from melanoma patients in vitro. Anticancer Res 14:2405–2411
Cerwenka A, Lanier LL (2001) Natural killer cells, viruses and cancer. Nat Rev Immunol 1:41–49
Baxevanis CN, Reclos GJ, Panneerselvam C, Papamichail M (1988) Enhancement of human T lymphocyte functions by prothymosin α. I. Augmentation of mixed lymphocyte culture reactions and soluble protein-induced proliferative responses. Immunopharmacology 15:73–84
Baxevanis CN, Reclos GJ, Papamichail M (1993) Prothymosin α restores depressed allogeneic cell-mediated lympholysis and natural-killer-cell activity in patients with cancer. Int J Cancer 53:264–268
Eschenfeldt WH, Manrow RE, Krug MS, Berger SL (1989) Isolation and partial sequencing of the human prothymosin α gene family. Evidence against export of the gene products. J Biol Chem 264:7546–7555
Evstafieva AG, Belov GA, Rubtsov YP, Kalkum M, Joseph B, Chichkova NV, Sukhacheva EA, Bogdanov AA, Pettersson RF, Agol VI, Vartapetian AB (2003) Apoptosis-related fragmentation, translocation, and properties of human prothymosin alpha. Exp Cell Res 284:211–223
Cordero OJ, Sarandeses C, Nogueira M (1994) Prothymosin α receptors on peripheral blood mononuclear cells. FEBS Lett 341:23–27
Cordero OJ, Sarandeses CS, Lopez JL, Cancio E, Regueiro BJ, Nogueira M (1991) Prothymosin α enhances interleukin 2 receptor expression in normal human T-lymphocytes. Int J Immunopharmacol 13:1059–1065
Lopez-Rodriguez JL, Cordero OJ, Sarandeses CS, Vinuela J, Nogueira M (1994) Interleukin-2 killer cells: in vitro evaluation of combination with prothymosin α. Lymphokine Cytokine Res 13:175–182
Crump WL, Owen-Schaub LB, Grimm EA (1989) Synergy of human recombinant interleukin 1 with interleukin 2 in the generation of lymphokine-activated killer cells. Cancer Res 49:149–153
Gruenberg E, Eckert K, Maurer R (1997) Prothymosin α1 enhances the interleukin-2 activated killer cell adhesion to and immunotoxicity against docetaxel-treated HT-29 colon carcinoma cells in vitro. Int J Thymol 5:415–423
Pross HF, Lotzova E (1993) Role of natural killer cells in cancer. Nat Immun 12:279–292
Odman-Ghazi SO, Hatcher F, Whalen MM (2003) Expression of functionally relevant cell surface markers in dibutyltin-exposed human natural killer cells. Chem Biol Interact 146:1–18
Rose DM, Cardarelli PM, Cobb RR, Ginsberg MH (2000) Soluble VCAM-1 binding to α4 integrins is cell-type specific and activation dependent and is disrupted during apoptosis in T cells. Blood 95:602–609
Sarandeses CS, Covello G, Diaz-Jullien C, Freire M (2003) Prothymosin α is processed to thymosin α1 and thymosin α11 by a lysosomal asparaginyl endopeptidase. J Biol Chem 278:13286–13293
Shiau AL, Lin PR, Chang MY, Wu CL (2001) Retrovirus-mediated transfer of prothymosin gene inhibits tumor growth and prolongs survival in murine bladder cancer. Gene Ther 8:1609–1617
Shakulov VR, Vorobjev IA, Rubtsov YP, Chichkova NV, Vartapetian AB (2000) Interaction of yeast importin α with the NLS of prothymosin α is insufficient to trigger nuclear uptake of cargos. Biochem Biophys Res Commun 274:548–552
Romani L, Bistoni F, Gaziano R, Bozza S, Montagnoli C, Perruccio K, Pitzurra L, Bellocchio S, Velardi A, Rasi G, Di Francesco P, Garaci E (2004) Thymosin α1 activates dendritic cells for antifungal Th1 resistance through toll-like receptor signaling. Blood 103:4232–4239
Matzinger P (2002) The danger model: a renewed sense of self. Science 296:301–305
Acknowledgements
Supported in part by Grants 7285/02 from the GSRT, Greece (to OET and MN) and IKYDA 61/2003 from the Hellenic State Scholarships Foundation and the Deutscher Akademischer Austausch Dienst (to OET and WV). We thank Dr. N. Cacoullos for critically reviewing the manuscript and Dr. C. N. Baxevanis for help with FACS analysis. OET and WV wish to dedicate this report to the memory of Athanassios (Nassos) A. Haritos on his eleventh death anniversary.
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Skopeliti, M., Voutsas, I.F., Klimentzou, P. et al. The immunologically active site of prothymosin α is located at the carboxy-terminus of the polypeptide. Evaluation of its in vitro effects in cancer patients. Cancer Immunol Immunother 55, 1247–1257 (2006). https://doi.org/10.1007/s00262-005-0108-4
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DOI: https://doi.org/10.1007/s00262-005-0108-4