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W2014850908 abstract "How tumor cells develop resistance to apoptosis induced by cytokines and chemotherapeutic agents is incompletely understood. In the present report, we investigated apoptosis induction by tumor necrosis factor (TNF) in two human T cell lines, Jurkat and HuT-78. While TNF inhibited the growth of Jurkat cells and activated caspase-3, it had no effect on HuT-78 cells. It was further found that HuT-78 cells constitutively expressed the nuclear transcription factor NF-κB. TNF activated NF-κB in Jurkat cells but not in HuT-78 cells. HuT-78 cells were also resistant to NF-κB activation induced by phorbol ester, H2O2, ceramide, endotoxin, and interleukin-1. Despite the presence of preactivated NF-κB, HuT-78 cells also expressed high levels of IκB-α, the inhibitory subunit of NF-κB and, unlike Jurkat cells, were resistant to TNF-induced degradation of IκB-α. Its half-life in HuT-78 cells was 12 h as opposed to 45 min in Jurkat cells. Antibodies against TNF blocked the constitutive activation of NF-κB and proliferation of HuT-78 cells but had no significant effect on Jurkat cells, suggesting an autocrine role for TNF. The antioxidant pyrrolidine dithiocarbamate also suppressed constitutive NF-κB activation and it reversed the cell's sensitivity to TNF-induced cytotoxicity and activation of caspase-3. Overall, these results suggest that constitutive activation of NF-κB, TNF, and prooxidant pathway in certain T cell lymphomas causes resistance to apoptosis, and this can be reversed by antioxidants. How tumor cells develop resistance to apoptosis induced by cytokines and chemotherapeutic agents is incompletely understood. In the present report, we investigated apoptosis induction by tumor necrosis factor (TNF) in two human T cell lines, Jurkat and HuT-78. While TNF inhibited the growth of Jurkat cells and activated caspase-3, it had no effect on HuT-78 cells. It was further found that HuT-78 cells constitutively expressed the nuclear transcription factor NF-κB. TNF activated NF-κB in Jurkat cells but not in HuT-78 cells. HuT-78 cells were also resistant to NF-κB activation induced by phorbol ester, H2O2, ceramide, endotoxin, and interleukin-1. Despite the presence of preactivated NF-κB, HuT-78 cells also expressed high levels of IκB-α, the inhibitory subunit of NF-κB and, unlike Jurkat cells, were resistant to TNF-induced degradation of IκB-α. Its half-life in HuT-78 cells was 12 h as opposed to 45 min in Jurkat cells. Antibodies against TNF blocked the constitutive activation of NF-κB and proliferation of HuT-78 cells but had no significant effect on Jurkat cells, suggesting an autocrine role for TNF. The antioxidant pyrrolidine dithiocarbamate also suppressed constitutive NF-κB activation and it reversed the cell's sensitivity to TNF-induced cytotoxicity and activation of caspase-3. Overall, these results suggest that constitutive activation of NF-κB, TNF, and prooxidant pathway in certain T cell lymphomas causes resistance to apoptosis, and this can be reversed by antioxidants. Development of resistance to apoptosis induction by cytokines and chemotherapeutic agents is one of the major problems in cancer therapy (1Baldini N. Nat. Med. 1997; 3: 378-380Crossref PubMed Scopus (53) Google Scholar). Overcoming this resistance has been largely unsuccessful, because the mechanism of development of resistance is not understood. Multiple drug resistance (MDR) 1The abbreviations used are: MDR, multiple drug resistance; TNF, tumor necrosis factor; PMA, phorbol 12-myristate 13 acetate; EMSA, electrophoretic mobility shift assay; ROS, reactive oxygen species; PDTC, pyrrolidine dithiocarbamate; PAGE, polyacrylamide gel electrophoresis; MTT, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; IL, interleukin; CAT, chloramphenicol acetyltransferase; PARP, poly(ADP-ribose) polymerase.1The abbreviations used are: MDR, multiple drug resistance; TNF, tumor necrosis factor; PMA, phorbol 12-myristate 13 acetate; EMSA, electrophoretic mobility shift assay; ROS, reactive oxygen species; PDTC, pyrrolidine dithiocarbamate; PAGE, polyacrylamide gel electrophoresis; MTT, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; IL, interleukin; CAT, chloramphenicol acetyltransferase; PARP, poly(ADP-ribose) polymerase.P-glycoprotein, Bcl-2, inactivation of p53 and related proteins, glutathione S-transferase, protein kinase C, MDR-related proteins, transglutaminase, and heat shock proteins (e.g. hsp 27) all play a role (2Bellamy W.T. Dalton W.S. Adv. Clin. Chem. 1994; 31: 1-61Crossref PubMed Scopus (62) Google Scholar, 3Broxterman H.J. Giaccone G. Lankelma J. Curr. Opin. Oncol. 1995; 7: 532-540Crossref PubMed Scopus (80) Google Scholar, 4Reed J.C. Curr. Opin. Oncol. 1995; 7: 541-546Crossref PubMed Scopus (486) Google Scholar, 5Harrison D.J. J. Pathol. 1995; 175: 7-12Crossref PubMed Scopus (77) Google Scholar). Besides these factors, an activated form of the nuclear transcription factor NF-κB has recently been implicated in development of resistance to tumor necrosis factor (TNF) (6Beg A.A. Baltimore D. Science. 1996; 274: 782-784Crossref PubMed Scopus (2925) Google Scholar, 7Van Antwerp D.J. Martin S.J. Kafri T. Green D.R. Verma I.M. Science. 1996; 274: 787-789Crossref PubMed Scopus (2440) Google Scholar, 8Wang C.Y. Mayo M.W. Baldwin Jr., A.S. Science. 1996; 274: 784-787Crossref PubMed Scopus (2499) Google Scholar).Under normal conditions, NF-κB is present in the cytoplasm in its inactive state as a heterotrimer consisting of p50, p65, and IκB-α (9Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4581) Google Scholar). When activated IκB-α undergoes ubiquitination, phosphorylation, and degradation, and the p50-p65 complex is released to be translocated to the nucleus where it causes gene activation. The activation of NF-κB is initiated by a wide variety of stress stimuli, which themselves cause apoptosis. Among these are TNF, IL-1, x-rays, γ-radiation, phorbol ester, ceramide, endotoxin, calcium ionophores, and H2O2 (9Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4581) Google Scholar, 10Brach M.A. Gruss H.J. Kaisho T. Asano Y. Hirano T. Herrmann F. J. Biol. Chem. 1993; 268: 8466-8472Abstract Full Text PDF PubMed Google Scholar). Interestingly, several chemotherapeutic drugs such as the anthracyclines doxorubicin and daunorubicin (11Boland M.P. Foster S.J. O'Neill L.A.J. J. Biol. Chem. 1997; 272 (,): 12952-12960Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 12Das K.C. White C.W. J. Biol. Chem. 1997; 272: 14914-14920Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar), taxol, the vinca alkaloids vinblastine and vincristine (12Das K.C. White C.W. J. Biol. Chem. 1997; 272: 14914-14920Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar), camptothecin (13Piret B. Piette J. Nucleic Acids Res. 1996; 24: 4242-4248Crossref PubMed Scopus (119) Google Scholar) and etoposide (14Perez, C., Vilaboa, N. E., Garcia-Bermejo, L., de Blas, E., Creighton, A. M., and Aller, P. (1997) J. Cell Sci.110 (pt 3),337–343Google Scholar) also cause NF-κB activation. Several genes whose proteins are involved in tumor promotion and metastasis, such as ICAM-1, VCAM-1, ELAM-1, cyclooxygenase-2, and matrix metalloprotease-9, are regulated by NF-κB (15Collins T. Read M.A. Neish A.S. Whitley M.Z. Thanos D. Maniatis T. FASEB J. 1995; 9: 899-909Crossref PubMed Scopus (1559) Google Scholar, 16Roshak A.K. Jackson J.R. McGough K. Chabot-Fletcher M. Mochan E. Marshal L.A. J. Biol. Chem. 1996; 271: 31496-31501Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 17Huhtala P. Tuuttila A. Chow L.T. Lohi J. Keski-Oja J. Tryggvason K. J. Biol. Chem. 1991; 266: 16485-16490Abstract Full Text PDF PubMed Google Scholar).A progressive activation of constitutive NF-κB has recently been correlated with progression of breast cancer, melanoma, and juvenile myelomonocytic leukemia (18Nakshatri H. Bhat-Nakshatri P. Martin D.A. Goulet Jr., R.J. Sledge Jr., G.W. Mol. Cell. Biol. 1997; 17: 3629-3639Crossref PubMed Google Scholar, 19Raziuddin A. Court D. Sarkar F.H. Liu Y.-L. Kung H.F Raziuddin R. J. Biol. Chem. 1997; 272: 15715-15720Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 20Shattuck-Brandt R.L. Richmond A. Cancer Res. 1997; 57: 3032-3039PubMed Google Scholar, 21Kochetkova M. Iversen P.O. Lopez A.F. Shannon M.F. J. Clin. Invest. 1997; 99: 3000-3008Crossref PubMed Scopus (47) Google Scholar). How NF-κB is constitutively activated in some tumor cells and what role it plays in induction of resistance to apoptosis is not clear. In the present study, we show that in contrast to acute T cell leukemic Jurkat cells, cutaneous T cell lymphoma HuT-78 cells were resistant to the apoptotic effects of TNF and constitutively expressed high levels of activated form of NF-κB and of IκB-α simultaneously. The latter was resistant to TNF-induced IκB-α degradation and had a very long half-life. Neutralizing anti-TNF antibodies down-regulated the constitutive NF-κB activation and induced apoptosis in HuT-78 but not in Jurkat cells. Pyrrolidine dithiocarbamate (PDTC), a quencher of reactive oxygen intermediates, also inhibited constitutively activated NF-κB and rendered HuT-78 cells susceptible to TNF-induced killing.DISCUSSIONIn the present study we investigated the mechanism by which tumor cells develop resistance to apoptosis induced by TNF. Two human T cell lines, Jurkat and HuT-78, were examined. While TNF inhibited the growth of Jurkat cells and activated caspase-3, it had no effect on HuT-78 cells; constitutive activation of NF-κB was demonstrated in HuT-78 cells and may account for this difference in growth and apoptosis induction. TNF and various other stimuli activated NF-κB in Jurkat cells but not in HuT-78 cells. Despite preactivated NF-κB, HuT-78 cells also expressed high levels of IκB-α, which unlike Jurkat cells, were resistant to TNF-induced degradation. The half-life of the IκB-α in HuT-78 cells is much longer than Jurkat cells. Antibodies against TNF down-regulated the constitutive activation of NF-κB and proliferation of HuT-78 cells, suggesting an autocrine role for TNF. The antioxidant PDTC also suppressed constitutive NF-κB activation and reversed sensitivity to TNF for cytotoxicity and caspase-3 activation. These results suggest that constitutive NF-κB activation causes resistance to apoptosis through generation of ROS and TNF.Our results show that HuT-78 cells express activated NF-κB and IκB-α simultaneously. This is not too surprising as the synthesis of IκB-α requires NF-κB activation (29Chiao P.J. Miyamoto S. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 28-32Crossref PubMed Scopus (390) Google Scholar), but why IκB-α fails to inhibit constitutively activated Nκ-kB, however, is not clear. Our results show that IκB-α in HuT-78 is degraded very slowly compared with Jurkat cells. In WEHI-3 cells, which were also shown to express constitutively activated NF-κB, the rate of degradation of IκB-α is faster than normal (30Miyamoto S. Chiao P.J. Verma I.M. Mol. Cell. Biol. 1994; 14: 3276-3282Crossref PubMed Google Scholar). Previously, we have shown that IκB-α phosphorylated at Tyr-42 is refractory to TNF-induced degradation (31Singh S. Darnay B.G. Aggarwal B.B. J. Biol. Chem. 1996; 271: 31049-31054Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Thus, it is possible that IκB-α from HuT-78 cells is phosphorylated at Tyr-42. Phosphotyrosine blots, however, did not reveal any tyrosine phosphorylation (data not shown). A lack of retarded mobility of IκB-α from HuT-78 cells on SDS-PAGE gels also indicated a lack of phosphorylation. It is possible that the p50-p65 heterocomplex is mutated so that it can no longer bind to endogenous IκB-α. However, it is unlikely because we found that the p50-p65 heterocomplex can bind both exogenously added IκB-α, can bind to the NF-κB binding site in the DNA, and is supershifted by the antibodies.NF-κB activation in HuT-78 cells was not only refractile to TNF, the constitutive DNA binding activity in these cells remained unchanged by a host of other NF-κB activators, including lipopolysaccharide, H2O2, ceramide, IL-1, and phorbol 12-myristate 13-acetate. Western blot analysis indicated that besides a nuclear pool there is a cytoplasmic pool of p50-p65 in HuT-78 cells. Coprecipitation followed by Western blot analysis results indicated that the cytoplasmic pool of p50-p65 subunits in HuT-78 cells exists in complex with IκB-α. The absence of further activation by various stimuli suggest the lack of nuclear translocation of p50-p65 from the cytoplasm, perhaps because of decreased proteolytic activity of the enzyme required to degrade IκB-α, as noted earlier.Our results indicate that NF-κB activation and apoptosis are linked, but how constitutive activation of NF-κB prevents apoptosis of HuT-78 cells is not clear. Several genes that are known to down-regulate apoptosis are regulated by NF-κB activation, including the zinc finger protein A20 (32Opipari Jr., A.W. Hu H.M. Yabkowitz R. Dixit V.M. J. Biol. Chem. 1992; 267: 12424-12427Abstract Full Text PDF PubMed Google Scholar), manganese superoxide dismutase (33Perera C.S. St. Clair D.K. Mc Clain C.J. Arch. Biochem. Biophys. 1995; 323: 471-476Crossref PubMed Scopus (55) Google Scholar), and cIAP2 (cellular inhibitor for apoptosis) (34Chu Z.-L. McKinsey T.A. Liu L. Gentery J. Malim M.H. Ballard D.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10057-10062Crossref PubMed Scopus (820) Google Scholar). It is possible that these genes are constitutively expressed in HuT-78 cells, thus leading to inhibition of apoptosis. Our results are consistent with reports that mice that lack the NF-κB p65 gene die early in embryonic development from massive cellular death of hepatic parenchyma (35Beg A. Sha W. Bronson R. Ghosh S. Nature. 1995; 37: 167-170Crossref Scopus (1625) Google Scholar). The antiapoptotic role of NF-κB was also demonstrated from the observation that embryonic fibroblast from IκB knockout mice are resistance to TNF (6Beg A.A. Baltimore D. Science. 1996; 274: 782-784Crossref PubMed Scopus (2925) Google Scholar). Similarly, transfection of a dominant negative form of IκB-α cDNA prevented the TNF-induced apoptosis of cells (7Van Antwerp D.J. Martin S.J. Kafri T. Green D.R. Verma I.M. Science. 1996; 274: 787-789Crossref PubMed Scopus (2440) Google Scholar, 8Wang C.Y. Mayo M.W. Baldwin Jr., A.S. Science. 1996; 274: 784-787Crossref PubMed Scopus (2499) Google Scholar).Our results show that pretreatment of HuT-78 cells with PDTC inhibited constitutive NF-κB activation and sensitized the cells to TNF-induced apoptosis, thus suggesting a critical role of ROS. PDTC is a potent inhibitor of inducible NF-κB activation (28Schreck R. Meier B. Mannel D.N. Droge W. Baeuerle P.A. J. Exp. Med. 1992; 175: 1181-1194Crossref PubMed Scopus (1442) Google Scholar, 36Ziegler-Heitbrock H.W. Sternsdorf T. Liese J. Belohardsky B. Weber C. Wedel A. Schreck R. Baeuerle P.A. J. Immunol. 1993; 151: 6986-6993PubMed Google Scholar) in most cells. It displays antioxidant property both by metal chelating and by acting as a radical scavenger (37Wolfe J.T. Ross D. Cohen G.M. FEBS Lett. 1994; 352: 58-62Crossref PubMed Scopus (175) Google Scholar, 38Baboir B.M. Cerutti P.A. Fridovich I. McCord J.M. Oxy-radicals in Molecular Biology and Pathology. Alan R. Liss, Inc., New York1988: 39-48Google Scholar). Generation of ROS has been proposed as an important mechanism to mediate the apoptotic and gene regulatory effects of TNF (39Schulze-Oshoff K. Beyaert R. Vandevoorde V. Haegeman G. Fiers W. EMBO J. 1993; 12: 3095-3104Crossref PubMed Scopus (548) Google Scholar, 40Goossens V. Grooten J. Fiers W. J. Biol. Chem. 1996; 271: 192-196Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Our results, however, indicate ROS is also involved in protection of cells from apoptosis by activating NF-κB.Besides PDTC, treatment of cells with anti-TNF antibodies also down-regulated NF-κB and inhibited cell growth. These results, which are consistent with previous report (41O'Connell A.M. Cleere R. Long A. O'Neill L.A.J. Kelleher D. J. Biol. Chem. 1995; 270: 7399-7404Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), suggest an autocrine role for TNF in induction of resistance. In addition, studies employing a TNF expression vector and an antisense TNF mRNA expression vector transfected into TNF-sensitive and -insensitive cells clearly demonstrated that endogenously made TNF is protective against TNF-induced cytotoxicity (42Himeno T. Watanabe N. Yamauchi N. Maeda M. Tsuji Y. Okamoto T. Neda H. Niitsu Y. Cancer Res. 1990; 50: 4941-4945PubMed Google Scholar). These observations further confirm the inhibitory role of NF-κB in apoptosis. Additionally, our study demonstrates the autocrine growth-promoting role of TNF through the generation of reactive oxygen intermediates. Inhibitors of both nuclear factor-κB and activator protein-1 activation have been shown to block the neoplastic transformation response (43Li J.J. Westergaard C. Ghosh P. Colburn N.H. Cancer Res. 1997; 57: 3569-3576PubMed Google Scholar). In summary, our study demonstrates the role of constitutively expressed NF-κB in the induction of resistance in HuT-78 cells through generation of TNF and ROS. Development of resistance to apoptosis induction by cytokines and chemotherapeutic agents is one of the major problems in cancer therapy (1Baldini N. Nat. Med. 1997; 3: 378-380Crossref PubMed Scopus (53) Google Scholar). Overcoming this resistance has been largely unsuccessful, because the mechanism of development of resistance is not understood. Multiple drug resistance (MDR) 1The abbreviations used are: MDR, multiple drug resistance; TNF, tumor necrosis factor; PMA, phorbol 12-myristate 13 acetate; EMSA, electrophoretic mobility shift assay; ROS, reactive oxygen species; PDTC, pyrrolidine dithiocarbamate; PAGE, polyacrylamide gel electrophoresis; MTT, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; IL, interleukin; CAT, chloramphenicol acetyltransferase; PARP, poly(ADP-ribose) polymerase.1The abbreviations used are: MDR, multiple drug resistance; TNF, tumor necrosis factor; PMA, phorbol 12-myristate 13 acetate; EMSA, electrophoretic mobility shift assay; ROS, reactive oxygen species; PDTC, pyrrolidine dithiocarbamate; PAGE, polyacrylamide gel electrophoresis; MTT, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; IL, interleukin; CAT, chloramphenicol acetyltransferase; PARP, poly(ADP-ribose) polymerase.P-glycoprotein, Bcl-2, inactivation of p53 and related proteins, glutathione S-transferase, protein kinase C, MDR-related proteins, transglutaminase, and heat shock proteins (e.g. hsp 27) all play a role (2Bellamy W.T. Dalton W.S. Adv. Clin. Chem. 1994; 31: 1-61Crossref PubMed Scopus (62) Google Scholar, 3Broxterman H.J. Giaccone G. Lankelma J. Curr. Opin. Oncol. 1995; 7: 532-540Crossref PubMed Scopus (80) Google Scholar, 4Reed J.C. Curr. Opin. Oncol. 1995; 7: 541-546Crossref PubMed Scopus (486) Google Scholar, 5Harrison D.J. J. Pathol. 1995; 175: 7-12Crossref PubMed Scopus (77) Google Scholar). Besides these factors, an activated form of the nuclear transcription factor NF-κB has recently been implicated in development of resistance to tumor necrosis factor (TNF) (6Beg A.A. Baltimore D. Science. 1996; 274: 782-784Crossref PubMed Scopus (2925) Google Scholar, 7Van Antwerp D.J. Martin S.J. Kafri T. Green D.R. Verma I.M. Science. 1996; 274: 787-789Crossref PubMed Scopus (2440) Google Scholar, 8Wang C.Y. Mayo M.W. Baldwin Jr., A.S. Science. 1996; 274: 784-787Crossref PubMed Scopus (2499) Google Scholar). Under normal conditions, NF-κB is present in the cytoplasm in its inactive state as a heterotrimer consisting of p50, p65, and IκB-α (9Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4581) Google Scholar). When activated IκB-α undergoes ubiquitination, phosphorylation, and degradation, and the p50-p65 complex is released to be translocated to the nucleus where it causes gene activation. The activation of NF-κB is initiated by a wide variety of stress stimuli, which themselves cause apoptosis. Among these are TNF, IL-1, x-rays, γ-radiation, phorbol ester, ceramide, endotoxin, calcium ionophores, and H2O2 (9Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4581) Google Scholar, 10Brach M.A. Gruss H.J. Kaisho T. Asano Y. Hirano T. Herrmann F. J. Biol. Chem. 1993; 268: 8466-8472Abstract Full Text PDF PubMed Google Scholar). Interestingly, several chemotherapeutic drugs such as the anthracyclines doxorubicin and daunorubicin (11Boland M.P. Foster S.J. O'Neill L.A.J. J. Biol. Chem. 1997; 272 (,): 12952-12960Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 12Das K.C. White C.W. J. Biol. Chem. 1997; 272: 14914-14920Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar), taxol, the vinca alkaloids vinblastine and vincristine (12Das K.C. White C.W. J. Biol. Chem. 1997; 272: 14914-14920Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar), camptothecin (13Piret B. Piette J. Nucleic Acids Res. 1996; 24: 4242-4248Crossref PubMed Scopus (119) Google Scholar) and etoposide (14Perez, C., Vilaboa, N. E., Garcia-Bermejo, L., de Blas, E., Creighton, A. M., and Aller, P. (1997) J. Cell Sci.110 (pt 3),337–343Google Scholar) also cause NF-κB activation. Several genes whose proteins are involved in tumor promotion and metastasis, such as ICAM-1, VCAM-1, ELAM-1, cyclooxygenase-2, and matrix metalloprotease-9, are regulated by NF-κB (15Collins T. Read M.A. Neish A.S. Whitley M.Z. Thanos D. Maniatis T. FASEB J. 1995; 9: 899-909Crossref PubMed Scopus (1559) Google Scholar, 16Roshak A.K. Jackson J.R. McGough K. Chabot-Fletcher M. Mochan E. Marshal L.A. J. Biol. Chem. 1996; 271: 31496-31501Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 17Huhtala P. Tuuttila A. Chow L.T. Lohi J. Keski-Oja J. Tryggvason K. J. Biol. Chem. 1991; 266: 16485-16490Abstract Full Text PDF PubMed Google Scholar). A progressive activation of constitutive NF-κB has recently been correlated with progression of breast cancer, melanoma, and juvenile myelomonocytic leukemia (18Nakshatri H. Bhat-Nakshatri P. Martin D.A. Goulet Jr., R.J. Sledge Jr., G.W. Mol. Cell. Biol. 1997; 17: 3629-3639Crossref PubMed Google Scholar, 19Raziuddin A. Court D. Sarkar F.H. Liu Y.-L. Kung H.F Raziuddin R. J. Biol. Chem. 1997; 272: 15715-15720Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 20Shattuck-Brandt R.L. Richmond A. Cancer Res. 1997; 57: 3032-3039PubMed Google Scholar, 21Kochetkova M. Iversen P.O. Lopez A.F. Shannon M.F. J. Clin. Invest. 1997; 99: 3000-3008Crossref PubMed Scopus (47) Google Scholar). How NF-κB is constitutively activated in some tumor cells and what role it plays in induction of resistance to apoptosis is not clear. In the present study, we show that in contrast to acute T cell leukemic Jurkat cells, cutaneous T cell lymphoma HuT-78 cells were resistant to the apoptotic effects of TNF and constitutively expressed high levels of activated form of NF-κB and of IκB-α simultaneously. The latter was resistant to TNF-induced IκB-α degradation and had a very long half-life. Neutralizing anti-TNF antibodies down-regulated the constitutive NF-κB activation and induced apoptosis in HuT-78 but not in Jurkat cells. Pyrrolidine dithiocarbamate (PDTC), a quencher of reactive oxygen intermediates, also inhibited constitutively activated NF-κB and rendered HuT-78 cells susceptible to TNF-induced killing. DISCUSSIONIn the present study we investigated the mechanism by which tumor cells develop resistance to apoptosis induced by TNF. Two human T cell lines, Jurkat and HuT-78, were examined. While TNF inhibited the growth of Jurkat cells and activated caspase-3, it had no effect on HuT-78 cells; constitutive activation of NF-κB was demonstrated in HuT-78 cells and may account for this difference in growth and apoptosis induction. TNF and various other stimuli activated NF-κB in Jurkat cells but not in HuT-78 cells. Despite preactivated NF-κB, HuT-78 cells also expressed high levels of IκB-α, which unlike Jurkat cells, were resistant to TNF-induced degradation. The half-life of the IκB-α in HuT-78 cells is much longer than Jurkat cells. Antibodies against TNF down-regulated the constitutive activation of NF-κB and proliferation of HuT-78 cells, suggesting an autocrine role for TNF. The antioxidant PDTC also suppressed constitutive NF-κB activation and reversed sensitivity to TNF for cytotoxicity and caspase-3 activation. These results suggest that constitutive NF-κB activation causes resistance to apoptosis through generation of ROS and TNF.Our results show that HuT-78 cells express activated NF-κB and IκB-α simultaneously. This is not too surprising as the synthesis of IκB-α requires NF-κB activation (29Chiao P.J. Miyamoto S. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 28-32Crossref PubMed Scopus (390) Google Scholar), but why IκB-α fails to inhibit constitutively activated Nκ-kB, however, is not clear. Our results show that IκB-α in HuT-78 is degraded very slowly compared with Jurkat cells. In WEHI-3 cells, which were also shown to express constitutively activated NF-κB, the rate of degradation of IκB-α is faster than normal (30Miyamoto S. Chiao P.J. Verma I.M. Mol. Cell. Biol. 1994; 14: 3276-3282Crossref PubMed Google Scholar). Previously, we have shown that IκB-α phosphorylated at Tyr-42 is refractory to TNF-induced degradation (31Singh S. Darnay B.G. Aggarwal B.B. J. Biol. Chem. 1996; 271: 31049-31054Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Thus, it is possible that IκB-α from HuT-78 cells is phosphorylated at Tyr-42. Phosphotyrosine blots, however, did not reveal any tyrosine phosphorylation (data not shown). A lack of retarded mobility of IκB-α from HuT-78 cells on SDS-PAGE gels also indicated a lack of phosphorylation. It is possible that the p50-p65 heterocomplex is mutated so that it can no longer bind to endogenous IκB-α. However, it is unlikely because we found that the p50-p65 heterocomplex can bind both exogenously added IκB-α, can bind to the NF-κB binding site in the DNA, and is supershifted by the antibodies.NF-κB activation in HuT-78 cells was not only refractile to TNF, the constitutive DNA binding activity in these cells remained unchanged by a host of other NF-κB activators, including lipopolysaccharide, H2O2, ceramide, IL-1, and phorbol 12-myristate 13-acetate. Western blot analysis indicated that besides a nuclear pool there is a cytoplasmic pool of p50-p65 in HuT-78 cells. Coprecipitation followed by Western blot analysis results indicated that the cytoplasmic pool of p50-p65 subunits in HuT-78 cells exists in complex with IκB-α. The absence of further activation by various stimuli suggest the lack of nuclear translocation of p50-p65 from the cytoplasm, perhaps because of decreased proteolytic activity of the enzyme required to degrade IκB-α, as noted earlier.Our results indicate that NF-κB activation and apoptosis are linked, but how constitutive activation of NF-κB prevents apoptosis of HuT-78 cells is not clear. Several genes that are known to down-regulate apoptosis are regulated by NF-κB activation, including the zinc finger protein A20 (32Opipari Jr., A.W. Hu H.M. Yabkowitz R. Dixit V.M. J. Biol. Chem. 1992; 267: 12424-12427Abstract Full Text PDF PubMed Google Scholar), manganese superoxide dismutase (33Perera C.S. St. Clair D.K. Mc Clain C.J. Arch. Biochem. Biophys. 1995; 323: 471-476Crossref PubMed Scopus (55) Google Scholar), and cIAP2 (cellular inhibitor for apoptosis) (34Chu Z.-L. McKinsey T.A. Liu L. Gentery J. Malim M.H. Ballard D.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10057-10062Crossref PubMed Scopus (820) Google Scholar). It is possible that these genes are constitutively expressed in HuT-78 cells, thus leading to inhibition of apoptosis. Our results are consistent with reports that mice that lack the NF-κB p65 gene die early in embryonic development from massive cellular death of hepatic parenchyma (35Beg A. Sha W. Bronson R. Ghosh S. Nature. 1995; 37: 167-170Crossref Scopus (1625) Google Scholar). The antiapoptotic role of NF-κB was also demonstrated from the observation that embryonic fibroblast from IκB knockout mice are resistance to TNF (6Beg A.A. Baltimore D. Science. 1996; 274: 782-784Crossref PubMed Scopus (2925) Google Scholar). Similarly, transfection of a dominant negative form of IκB-α cDNA prevented the TNF-induced apoptosis of cells (7Van Antwerp D.J. Martin S.J. Kafri T. Green D.R. Verma I.M. Science. 1996; 274: 787-789Crossref PubMed Scopus (2440) Google Scholar, 8Wang C.Y. Mayo M.W. Baldwin Jr., A.S. Science. 1996; 274: 784-787Crossref PubMed Scopus (2499) Google Scholar).Our results show that pretreatment of HuT-78 cells with PDTC inhibited constitutive NF-κB activation and sensitized the cells to TNF-induced apoptosis, thus suggesting a critical role of ROS. PDTC is a potent inhibitor of inducible NF-κB activation (28Schreck R. Meier B. Mannel D.N. Droge W. Baeuerle P.A. J. Exp. Med. 1992; 175: 1181-1194Crossref PubMed Scopus (1442) Google Scholar, 36Ziegler-Heitbrock H.W. Sternsdorf T. Liese J. Belohardsky B. Weber C. Wedel A. Schreck R. Baeuerle P.A. J. Immunol. 1993; 151: 6986-6993PubMed Google Scholar) in most cells. It displays antioxidant property both by metal chelating and by acting as a radical scavenger (37Wolfe J.T. Ross D. Cohen G.M. FEBS Lett. 1994; 352: 58-62Crossref PubMed Scopus (175) Google Scholar, 38Baboir B.M. Cerutti P.A. Fridovich I. McCord J.M. Oxy-radicals in Molecular Biology and Pathology. Alan R. Liss, Inc., New York1988: 39-48Google Scholar). Generation of ROS has been proposed as an important mechanism to mediate the apoptotic and gene regulatory effects of TNF (39Schulze-Oshoff K. Beyaert R. Vandevoorde V. Haegeman G. Fiers W. EMBO J. 1993; 12: 3095-3104Crossref PubMed Scopus (548) Google Scholar, 40Goossens V. Grooten J. Fiers W. J. Biol. Chem. 1996; 271: 192-196Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Our results, however, indicate ROS is also involved in protection of cells from apoptosis by activating NF-κB.Besides PDTC, treatment of cells with anti-TNF antibodies also down-regulated NF-κB and inhibited cell growth. These results, which are consistent with previous report (41O'Connell A.M. Cleere R. Long A. O'Neill L.A.J. Kelleher D. J. Biol. Chem. 1995; 270: 7399-7404Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), suggest an autocrine role for TNF in induction of resistance. In addition, studies employing a TNF expression vector and an antisense TNF mRNA expression vector transfected into TNF-sensitive and -insensitive cells clearly demonstrated that endogenously made TNF is protective against TNF-induced cytotoxicity (42Himeno T. Watanabe N. Yamauchi N. Maeda M. Tsuji Y. Okamoto T. Neda H. Niitsu Y. Cancer Res. 1990; 50: 4941-4945PubMed Google Scholar). These observations further confirm the inhibitory role of NF-κB in apoptosis. Additionally, our study demonstrates the autocrine growth-promoting role of TNF through the generation of reactive oxygen intermediates. Inhibitors of both nuclear factor-κB and activator protein-1 activation have been shown to block the neoplastic transformation response (43Li J.J. Westergaard C. Ghosh P. Colburn N.H. Cancer Res. 1997; 57: 3569-3576PubMed Google Scholar). In summary, our study demonstrates the role of constitutively expressed NF-κB in the induction of resistance in HuT-78 cells through generation of TNF and ROS. In the present study we investigated the mechanism by which tumor cells develop resistance to apoptosis induced by TNF. Two human T cell lines, Jurkat and HuT-78, were examined. While TNF inhibited the growth of Jurkat cells and activated caspase-3, it had no effect on HuT-78 cells; constitutive activation of NF-κB was demonstrated in HuT-78 cells and may account for this difference in growth and apoptosis induction. TNF and various other stimuli activated NF-κB in Jurkat cells but not in HuT-78 cells. Despite preactivated NF-κB, HuT-78 cells also expressed high levels of IκB-α, which unlike Jurkat cells, were resistant to TNF-induced degradation. The half-life of the IκB-α in HuT-78 cells is much longer than Jurkat cells. Antibodies against TNF down-regulated the constitutive activation of NF-κB and proliferation of HuT-78 cells, suggesting an autocrine role for TNF. The antioxidant PDTC also suppressed constitutive NF-κB activation and reversed sensitivity to TNF for cytotoxicity and caspase-3 activation. These results suggest that constitutive NF-κB activation causes resistance to apoptosis through generation of ROS and TNF. Our results show that HuT-78 cells express activated NF-κB and IκB-α simultaneously. This is not too surprising as the synthesis of IκB-α requires NF-κB activation (29Chiao P.J. Miyamoto S. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 28-32Crossref PubMed Scopus (390) Google Scholar), but why IκB-α fails to inhibit constitutively activated Nκ-kB, however, is not clear. Our results show that IκB-α in HuT-78 is degraded very slowly compared with Jurkat cells. In WEHI-3 cells, which were also shown to express constitutively activated NF-κB, the rate of degradation of IκB-α is faster than normal (30Miyamoto S. Chiao P.J. Verma I.M. Mol. Cell. Biol. 1994; 14: 3276-3282Crossref PubMed Google Scholar). Previously, we have shown that IκB-α phosphorylated at Tyr-42 is refractory to TNF-induced degradation (31Singh S. Darnay B.G. Aggarwal B.B. J. Biol. Chem. 1996; 271: 31049-31054Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Thus, it is possible that IκB-α from HuT-78 cells is phosphorylated at Tyr-42. Phosphotyrosine blots, however, did not reveal any tyrosine phosphorylation (data not shown). A lack of retarded mobility of IκB-α from HuT-78 cells on SDS-PAGE gels also indicated a lack of phosphorylation. It is possible that the p50-p65 heterocomplex is mutated so that it can no longer bind to endogenous IκB-α. However, it is unlikely because we found that the p50-p65 heterocomplex can bind both exogenously added IκB-α, can bind to the NF-κB binding site in the DNA, and is supershifted by the antibodies. NF-κB activation in HuT-78 cells was not only refractile to TNF, the constitutive DNA binding activity in these cells remained unchanged by a host of other NF-κB activators, including lipopolysaccharide, H2O2, ceramide, IL-1, and phorbol 12-myristate 13-acetate. Western blot analysis indicated that besides a nuclear pool there is a cytoplasmic pool of p50-p65 in HuT-78 cells. Coprecipitation followed by Western blot analysis results indicated that the cytoplasmic pool of p50-p65 subunits in HuT-78 cells exists in complex with IκB-α. The absence of further activation by various stimuli suggest the lack of nuclear translocation of p50-p65 from the cytoplasm, perhaps because of decreased proteolytic activity of the enzyme required to degrade IκB-α, as noted earlier. Our results indicate that NF-κB activation and apoptosis are linked, but how constitutive activation of NF-κB prevents apoptosis of HuT-78 cells is not clear. Several genes that are known to down-regulate apoptosis are regulated by NF-κB activation, including the zinc finger protein A20 (32Opipari Jr., A.W. Hu H.M. Yabkowitz R. Dixit V.M. J. Biol. Chem. 1992; 267: 12424-12427Abstract Full Text PDF PubMed Google Scholar), manganese superoxide dismutase (33Perera C.S. St. Clair D.K. Mc Clain C.J. Arch. Biochem. Biophys. 1995; 323: 471-476Crossref PubMed Scopus (55) Google Scholar), and cIAP2 (cellular inhibitor for apoptosis) (34Chu Z.-L. McKinsey T.A. Liu L. Gentery J. Malim M.H. Ballard D.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10057-10062Crossref PubMed Scopus (820) Google Scholar). It is possible that these genes are constitutively expressed in HuT-78 cells, thus leading to inhibition of apoptosis. Our results are consistent with reports that mice that lack the NF-κB p65 gene die early in embryonic development from massive cellular death of hepatic parenchyma (35Beg A. Sha W. Bronson R. Ghosh S. Nature. 1995; 37: 167-170Crossref Scopus (1625) Google Scholar). The antiapoptotic role of NF-κB was also demonstrated from the observation that embryonic fibroblast from IκB knockout mice are resistance to TNF (6Beg A.A. Baltimore D. Science. 1996; 274: 782-784Crossref PubMed Scopus (2925) Google Scholar). Similarly, transfection of a dominant negative form of IκB-α cDNA prevented the TNF-induced apoptosis of cells (7Van Antwerp D.J. Martin S.J. Kafri T. Green D.R. Verma I.M. Science. 1996; 274: 787-789Crossref PubMed Scopus (2440) Google Scholar, 8Wang C.Y. Mayo M.W. Baldwin Jr., A.S. Science. 1996; 274: 784-787Crossref PubMed Scopus (2499) Google Scholar). Our results show that pretreatment of HuT-78 cells with PDTC inhibited constitutive NF-κB activation and sensitized the cells to TNF-induced apoptosis, thus suggesting a critical role of ROS. PDTC is a potent inhibitor of inducible NF-κB activation (28Schreck R. Meier B. Mannel D.N. Droge W. Baeuerle P.A. J. Exp. Med. 1992; 175: 1181-1194Crossref PubMed Scopus (1442) Google Scholar, 36Ziegler-Heitbrock H.W. Sternsdorf T. Liese J. Belohardsky B. Weber C. Wedel A. Schreck R. Baeuerle P.A. J. Immunol. 1993; 151: 6986-6993PubMed Google Scholar) in most cells. It displays antioxidant property both by metal chelating and by acting as a radical scavenger (37Wolfe J.T. Ross D. Cohen G.M. FEBS Lett. 1994; 352: 58-62Crossref PubMed Scopus (175) Google Scholar, 38Baboir B.M. Cerutti P.A. Fridovich I. McCord J.M. Oxy-radicals in Molecular Biology and Pathology. Alan R. Liss, Inc., New York1988: 39-48Google Scholar). Generation of ROS has been proposed as an important mechanism to mediate the apoptotic and gene regulatory effects of TNF (39Schulze-Oshoff K. Beyaert R. Vandevoorde V. Haegeman G. Fiers W. EMBO J. 1993; 12: 3095-3104Crossref PubMed Scopus (548) Google Scholar, 40Goossens V. Grooten J. Fiers W. J. Biol. Chem. 1996; 271: 192-196Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Our results, however, indicate ROS is also involved in protection of cells from apoptosis by activating NF-κB. Besides PDTC, treatment of cells with anti-TNF antibodies also down-regulated NF-κB and inhibited cell growth. These results, which are consistent with previous report (41O'Connell A.M. Cleere R. Long A. O'Neill L.A.J. Kelleher D. J. Biol. Chem. 1995; 270: 7399-7404Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), suggest an autocrine role for TNF in induction of resistance. In addition, studies employing a TNF expression vector and an antisense TNF mRNA expression vector transfected into TNF-sensitive and -insensitive cells clearly demonstrated that endogenously made TNF is protective against TNF-induced cytotoxicity (42Himeno T. Watanabe N. Yamauchi N. Maeda M. Tsuji Y. Okamoto T. Neda H. Niitsu Y. Cancer Res. 1990; 50: 4941-4945PubMed Google Scholar). These observations further confirm the inhibitory role of NF-κB in apoptosis. Additionally, our study demonstrates the autocrine growth-promoting role of TNF through the generation of reactive oxygen intermediates. Inhibitors of both nuclear factor-κB and activator protein-1 activation have been shown to block the neoplastic transformation response (43Li J.J. Westergaard C. Ghosh P. Colburn N.H. Cancer Res. 1997; 57: 3569-3576PubMed Google Scholar). In summary, our study demonstrates the role of constitutively expressed NF-κB in the induction of resistance in HuT-78 cells through generation of TNF and ROS. We thank Dr. Sunil Manna for performing NF-κB-CAT reporter assay." @default.
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W2014850908 title "Constitutive Activation of NF-κB Causes Resistance to Apoptosis in Human Cutaneous T Cell Lymphoma HuT-78 Cells" @default.
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