Abstract
The proinflammatory cytokine tumor necrosis factor-alpha (TNFα) exists naturally in two forms, a 26 kDa transmembrane form (TM-TNFα), and a 17 kDa secretory form (S-TNFα). The biological roles for each of these forms of TNFα in tumor killing have not been completely elucidated. Therefore, in this study, three different recombinant retroviral vectors, wild-type TNFα, solely secretable TNFα mutant, and uncleavable transmembrane TNFα mutant, were constructed by molecular techniques and stably transfected into a murine hepatic carcinoma cell line (H22). TNFα, either secreted in cell culture supernatants by secretable TNFα mutant- or wild-type TNFα-producing tumor cells, or as a treansmembrane form expressed on the cell surface of uncleavable TNFα mutant- or wild-type TNFα-synthesizing tumor cells, was demonstrated to be cytotoxic against the TNF sensitive L929 cell line. The H22 cells transfected with the three different forms of TNFα were shown to kill parental H22 cells in an in vitro cytotoxicity assay [effect/target (E/T) ratio-dependent manner], and their maximal killing rates were ~38–43% at E/T ratio of 5:1. The injection of total 2.5×105 mixed cells containing transfected and parental H22 tumor cells at different ratios into syngeneic mice resulted in the inhibition of tumor growth with a maximal inhibition rates of ~57~72% at E/T ratio of 5:1. A transient weight loss was found in mice bearing solely secretable TNFα mutant producing tumors, whereas no obvious side effects were seen in mice bearing uncleavable TNFα mutant or wild-type TNFα expressing tumors. Finally, we demonstrate that tumors secreting S-TNFα promoted the subsequent infiltration of CD4+ T cells, and to a lesser extent CD8+ T cells, to the tumor site. The TM-TNFα expressing tumors up-regulated Fas (CD95) expression and inhibited the expression of tumor metastasis associated molecule CD44v3. These results suggest that S-TNFα and TM-TNFα kill cancer cells in vivo through different mechanisms of action. We conclude that the non-secreted form of TNFα may be an ideal candidate for cancer gene therapy due to its therapeutic potential and lowered side effect profile.
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References
Kriegler M, Perez C, DeFay K, Albert I, Lu SD (1988) A novel form of TNF/cachectin is a cell surface cytotoxic transmembrane protein: ramifications for the complex physiology of TNF. Cell 53:45
Li X, Fan H (2004) Loss of ectodomain shedding due to mutations in the metalloprotease and cysteine-rich/disintegrin domains of the tumor necrosis factor-alpha converting enzyme (TACE). J Biol Chem 279:27365
Haas E, Grell M, Wajant H, Scheurich P (1999) Continuous autotropic signaling by membrane-expressed tumor necrosis factor. J Biol Chem 274:18107
Peck R, Brockhaus M, JR Frey (1989) Cell surface tumor necrosis factor (TNF) accounts for monocyte- and lymphocyte-mediated killing of TNF-resistant target cells. Cell Immunol 122:1
Mueller C, Corazza N, Trachsel-Loseth S, Eugster HP, Buhler-Jungo M, Brunner T, Imboden MA (1999) Noncleavable transmembrane mouse tumor necrosis factor-alpha (TNFalpha) mediates effects distinct from those of wild-type TNFalpha in vitro and in vivo. J Biol Chem 274:38112
Aversa G, Punnonen J, de Vries JE (1993) The 26-kD transmembrane form of tumor necrosis factor alpha on activated CD4+ T cell clones provides a costimulatory signal for human B cell activation. J Exp Med 177:1575
Birkland TP, Sypek JP, Wyler DJ (1992) Soluble TNF and membrane TNF expressed on CD4+ T lymphocytes differ in their ability to activate macrophage antileishmanial defense. J Leukoc Biol 51:296
Baxevanis CN, Voutsas IF, Tsitsilonis OE, Tsiatas ML, Gritzapis AD, Papamichail M (2000) Compromised anti-tumor responses in tumor necrosis factor-alpha knockout mice. Eur J Immunol 30:1957
Williamson BD, Carswell EA, Rubin BY, Prendergast JS, Old LJ (1983) Human tumor necrosis factor produced by human B-cell lines: synergistic cytotoxic interaction with human interferon. Proc Natl Acad Sci USA 80:5397
Fichtner I, Lemm M, Becker M, Tanneberger S (1990) Determination of antineoplastic activity and toxicity of tumor necrosis factor (TNF) in animal experiments. Correlation to clinical findings. Neoplasma 37:301
Karp SE, Hwu P, Farber A, Restifo NP, Kriegler M, Mule JJ, Rosenberg SA (1992) In vivo activity of tumor necrosis factor (TNF) mutants. Secretory but not membrane-bound TNF mediates the regression of retrovirally transduced murine tumor. J Immunol 149:2076
Marr RA, Addison CL, Snider D, Muller WJ, Gauldie J, Graham FL (1997) Tumour immunotherapy using an adenoviral vector expressing a membrane-bound mutant of murine TNF alpha. Gene Ther 4:1181
Hay H, CJ (1989) Studies on the specificity of the L929 cell bioassay for the measurement of tumour necrosis factor. J Clin Lab Immunol 29:151
Bosselman RA, Hsu RY, Bruszewski J, Hu S, Martin F, Nicolson M (1987) Replication-defective chimeric helper proviruses and factors affecting generation of competent virus: expression of Moloney murine leukemia virus structural genes via the metallothionein promoter. Mol Cell Biol 7:1797
Ji YB, Gao SY, Cheng WP (2005) Effect of Haimiding on the functioning of red cell membrane of FC and H22 tumor-bearing mice. World J Gastroenterol 11:823
Perez C, Albert I, DeFay K, Zachariades N, Gooding L, Kriegler M (1990) A nonsecretable cell surface mutant of tumor necrosis factor (TNF) kills by cell-to-cell contact. Cell 63:251
Decoster E, Vanhaesebroeck B, Vandenabeele P, Grooten J, Fiers W (1995) Generation and biological characterization of membrane-bound, uncleavable murine tumor necrosis factor. J Biol Chem 270:18473
Renz H, Gong JH, Schmidt A, Nain M, Gemsa D (1988) Release of tumor necrosis factor-alpha from macrophages. Enhancement and suppression are dose-dependently regulated by prostaglandin E2 and cyclic nucleotides. J Immunol 141:2388
Hahnfeldt P, Panigrahy D, Folkman J, Hlatky L (1999) Tumor Development under Angiogenic Signaling: a dynamical theory of tumor growth, treatment response, and postvascular dormancy. Cancer Res 59:4770
Mocellin S, Rossi CR, Pilati P, Nitti D (2005) Tumor necrosis factor, cancer and anticancer therapy. Cytokine Growth Factor Rev 16:35
Shi W, Li Z, Gong F (1998) Comparison of the cytocidal effect induced by transmembrane and secreted TNF-alpha. Chin J Microbiol Immunol 18:499
Higuchi M, Aggarwal BB (1994) TNF induces internalization of the p60 receptor and shedding of the p80 receptor. J Immunol 152:3550
Matory YL, Dorfman DM, Wu L, Chen M, Goedegebuure P, Eberlein TJ (1999) Treatment of established tumor is associated with ICAM-1 upregulation and reversed by CD8 depletion in a tumor necrosis factor-alpha gene transfected mouse mammary tumor. Pathobiology 67:186
Nagy T, Glavinas H, Szincsak N, Hunyadi J, Janossy T, Duda E, Vizler C, Juhasz I (2003) Tumor cells expressing membrane-bound tumor necrosis factor activate macrophages and have a compromised growth in immunosuppressed and immunodeficient mice. Cancer Lett 196:49
Matsumura R, Umemiya K, Goto T, Nakazawa T, Ochiai K, Kagami M, Tomioka H, Tanabe E, Sugiyama T, Sueishi M (2000) Interferon gamma and tumor necrosis factor alpha induce Fas expression and anti-Fas mediated apoptosis in a salivary ductal cell line. Clin Exp Rheumatol 18:311
Spanaus KS, Schlapbach R, Fontana A (1998) TNF-alpha and IFN-gamma render microglia sensitive to Fas ligand-induced apoptosis by induction of Fas expression and down-regulation of Bcl-2 and Bcl-xL. Eur J Immunol 28:4398
Lewis M, Tartaglia L, Lee A, Bennett G, Rice G, Wong G, Chen E, Goeddel D (1991) Cloning and expression of cDNAs for two distinct murine tumor necrosis factor receptors demonstrate one receptor is species specific. PNAS 88:2830
Quirk SM, Porter DA, Huber SC, Cowan RG (1998) Potentiation of fas-mediated apoptosis of murine granulosa cells by interferon-{gamma}, tumor necrosis factor-{alpha}, and cycloheximide. Endocrinology 139:4860
Grell M, Zimmermann G, Gottfried E, Chen CM, Grunwald U, Huang DC, Wu Lee YH, Durkop H, Engelmann H, Scheurich P, Wajant H, Strasser A (1999) Induction of cell death by tumour necrosis factor (TNF) receptor 2, CD40 and CD30: a role for TNF-R1 activation by endogenous membrane-anchored TNF. Embo J 18:3034
Hudson DL, Speight PM, Watt FM (1996) Altered expression of CD44 isoforms in squamous-cell carcinomas and cell lines derived from them. Int J Cancer 66:457
Chuang CK, Liao SK (2003) Differential expression of CD44 variant isoforms by cell lines and tissue specimens of transitional cell carcinomas. Anticancer Res 23:4635
Katerinaki E, Evans GS, Lorigan PC, MacNeil S (2003) TNF-alpha increases human melanoma cell invasion and migration in vitro: the role of proteolytic enzymes. Br J Cancer 89:1123
Acknowledgments
The authors wish to thank Mary Reiche for careful review of the manuscript and Professor Xuetao Cao for his generous gift of the PA317 and H22 cell lines. This project was supported by the National Natural Science Foundation of China (30200257), Key Project of Science and Technology from the Education Ministry of China (02136), the Hi-tech Research and Development Program of China (2001AA215431) and National Key Basic Research Program of China from the Ministry of Science and Technology of the People’s Republic of China (2001CB510008), and The Leukemia and Lymphoma Society of the United States of America (6249-05, to M.Z.).
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Li, Q., Li, L., Shi, W. et al. Mechanism of action differences in the antitumor effects of transmembrane and secretory tumor necrosis factor-alpha in vitro and in vivo. Cancer Immunol Immunother 55, 1470–1479 (2006). https://doi.org/10.1007/s00262-006-0150-x
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DOI: https://doi.org/10.1007/s00262-006-0150-x