- Short report
- Open Access
Human T-cell leukemia virus type 2 Tax protein induces interleukin 2-independent growth in a T-cell line
Retrovirologyvolume 3, Article number: 88 (2006)
While human T-cell leukemia virus type 1 (HTLV-1) is a causative agent of adult T-cell leukemia, HTLV type 2 (HTLV-2) is not associated with this malignancy. Accumulating evidence suggests that Tax, a transforming protein of HTLV-1 or HTLV-2, plays a crucial role in the distinctive pathogenesis of these two infections. We herein examined whether Tax2 by itself has a growth promoting activity in a mouse T-cell line CTLL-2, and compared the activity with that of Tax1.
We found that Tax2 converts the cell growth of CTLL-2 from an interleukin(IL)-2-dependent growth into an independent one. Cyclosporine A, an inhibitor of transcription factor NFAT, inhibited the growth of two out of four Tax2-transformed CTLL-2 cells, but it had little effect on two Tax1-transformed cells. While the HTLV-2-transformed human T-cell lines produce a significant amount of IL-2, Tax2-transformed CTLL-2 cells only produced a minimal amount of IL-2. These results thus suggest that NFAT-inducible gene(s) other than IL-2 play a role in the cell growth of Tax2-transformed CTLL-2 cells.
These results show that HTLV-2 Tax2 by itself has a growth promoting activity toward a T-cell line CTLL-2, and the CTLL-2 assay used in this study may therefore be a useful tool for comparing the activity of Tax2 with that of Tax1 in T-cells, thereby elucidating the mechanism of HTLV-1 specific leukemogenesis.
Human T-cell leukemia virus type 1 (HTLV-1) and HTLV type 2 (HTLV-2) are a family of retroviruses, which share around a 70% nucleotide identity and similar biological properties [1–6]. For instance, both HTLV-1 and HTLV-2 can efficiently transform primary human T-cells in vitro and establish a life-long persistent infection in humans [7–9]. The clinical outcomes of these two infections are, however, significantly distinctive. While HTLV-1 is etiologically associated with adult T-cell leukemia (ATL), HTLV-2 is associated with only a few cases of variant hairy cell leukemia [5, 10–12].
HTLV-1 and HTLV-2 encode a transforming protein Tax1 and Tax2, respectively, which are essential for the transformation of primary human T-cells in vitro [13–16]. Accumulating evidence suggests that Tax1 is a factor responsible for the high-oncogenic activity of HTLV-1 relative to HTLV-2 [4, 5]. Tax1 and Tax2 have more than 75 % amino acid identities, and they also exhibit strikingly similar functions in infected cells [17, 18]. For instance, Tax1 and Tax2 induce the expression of a number of cellular genes through several transcription factor binding sites, such as NF-κB, CREB/ATF, SRF, and AP-1 [4, 19–25]. These Tax-inducible cellular genes play a critical role in the persistent infection in host T-cells, including the transformation of human T-cells [24, 25], but they alone can not explain the pathogenic differences between HTLV-1 and HTLV-2, since the potencies of these functions are equivalent. On the other hand, recent results identified several differences between Tax1 and Tax2, which are likely to be factors that are responsible for the pathogenic difference of two infections [4, 5, 26–35]. Therefore, a comparative analysis of Tax1 and Tax2 is a promising approach to identify a key process responsible for HTLV-1 specific leukemogenesis.
We previously showed that Tax1 transforms a mouse T-cell line CTLL-2 from an interleukin(IL)-2-dependent growth to an IL-2-independent one, whereas Tax2 can not do so [32, 36]. We herein reexamined the transforming activity of Tax2 in CTLL-2 using a lentivirus vector for the transduction of the tax gene which is much more efficient than the electroporation method used in a previous experiment. Lentiviruses encoding Tax1 or Tax2 were produced in 293T cells, and these viruses were then infected to CTLL-2 cells in a medium containing IL-2. At 48 hours after infection, the infected cells were cultured without IL-2 in a 96 well plate. Four weeks later, the number of wells containing outgrowing cells was counted by light microscopy. Unlike the previous study, Tax2 transduced with a lentivirus induced the IL-2-independent growth of CTLL-2 cells (Figure 1). A Western blotting analysis using Tax1 and Tax2 antibodies showed that all four Tax2-transformed cell lines expressed Tax2 protein but not Tax1 (Figure 2), thus confirming that the tax2-virus induced the transformation. Like Tax1, these Tax2-transformed CTLL-2 cells continuously grow in the absence of IL-2 for at least three months (data not shown). These results showed that Tax2 therefore induced the IL-2-independent growth of CTLL-2 cells.
We previously showed that Tax2 activates IL-2 transcription through the transcription factor NFAT, and such produced IL-2 in some HTLV-2-transformed T-cell lines stimulates their cell growth . To examine whether NFAT plays a role in the Tax2-mediated IL-2-independent growth of CTLL-2, we cultured Tax2-transformed cells with cyclosporine A (CsA), a specific inhibitor of NFAT pathway (Figure 3). Two out of four Tax2-transformed cells showed reduced cell growth in the presence of CsA, while the other two showed little response to CsA treatment. On the other hand, parental CTLL-2 and two Tax1-transformed cells did not show CsA-mediated growth inhibition. These results show that the activation of NFAT by Tax2 stimulates the cell growth of some Tax2-transformed cells, but not Tax1-transformed ones. A real-time polymerase chain reaction with IL-2-specific primers showed that Tax2-transformed CTLL-2 cells minimally expressed IL-2 mRNA, whereas EL-4 T-cell line treated with phorbol myristate acetate and ionomycin produced a significant amount of IL-2 mRNA (Figure 4). We consistently detected IL-2 protein in the culture supernatant of the EL-4 cells treated with the same mitogens, but not those of the Tax2-transformed cell lines (data not shown). These results suggest that NFAT-inducible gene(s) other than IL-2 are thus involved in the Tax2-mediated growth promotion of CTLL-2 cells.
Tax2 has been shown to be essential for HTLV-2-mediated transformation of human T-cells . It, however, remains to be elucidated whether Tax2 by itself has a growth promoting activity toward T-cells like Tax1 . We herein showed that Tax2 can reproducibly convert a mouse T-cell line from an IL-2-dependent growth into an independent one. These results demonstrate that Tax2 by itself without any other viral proteins has a growth promoting activity in T-cells, thus suggesting that this growth promoting activity of Tax2 contributes to HTLV-2-mediated T-cell transformation. Since at least two functions, apoptosis inhibition and cell cycle promotion are both required for CTLL-2 to grow in the absence of IL-2, Tax2 can therefore replace these two functions in CTLL-2.
CsA inhibited the growth of two out of four Tax2-transformed CTLL-2 cells (Figure 3), indicating that NFAT-inducible genes are involved in IL-2-independent growth of these Tax2-transformed cells. These results are consistent with the previous results that CsA inhibited cell growth of some but not all HTLV-2-transformed human T-cell lines . There are at least two explanations for the distinct responses of the Tax2-transformed cells to CsA. Tax2 may have two distinctive activities to induce IL-2-independent growth of CTLL-2 cells. Alternatively, some parental CTLL-2 cells may have genetic or epigenetic change(s) conferring resistance to CsA in Tax2-transformed CTLL-2 cells. In contrast to Tax2, the cell growth of Tax1-transformed cells was little affected by CsA. This finding is also consistent with the result that Tax1 minimally activates NFAT, and thus CsA can not inhibit the cell growth of any HTLV-1-transformed T-cell lines .
Unlike the HTLV-2-transformed human T-cell lines sensitive to CsA-mediated growth inhibition, Tax2-transformed CsA-sensitive cells expressed a small amount of IL-2 mRNA (Figure 4). Since there are several NFAT inducible cytokines which promote T-cell growth, such as IL-4 and IL-21, these results indicated that the NFAT-inducible gene(s) other than IL-2 positively regulate the cell growth of the Tax2-transformed cells, thus suggesting that HTLV-2-transformed human T-cells may also utilize multiple NFAT-inducible T-cell growth promoting factors for their growth.
Accumulating evidence suggests that Tax plays a crucial role in the distinctive pathogenesis between HTLV-1 and HTLV-2 [4, 5, 26, 28, 29, 32, 34]. Therefore, further comparative studies of the Tax1 and Tax2 functions in T-cells are expected to advance our understanding of HTLV-1 leukemogenesis. The CTLL-2 assay used in this study is therefore considered to be a useful tool for examining the functions of Tax2 and Tax1 in T-cells, thereby elucidating the mechanism of HTLV-1 specific leukemogenesis.
Seiki M, Hattori S, Hirayama Y, Yoshida M: Human adult T-cell leukemia virus: complete nucleotide sequence of the provirus genome integrated in leukemia cell DNA. Proc Natl Acad Sci USA. 1983, 80: 3618-3622. 10.1073/pnas.80.12.3618.
Chen IS, McLaughlin J, Gasson JC, Clark SC, Golde DW: Molecular characterization of genome of a novel human T-cell leukaemia virus. Nature. 1983, 305: 502-505. 10.1038/305502a0.
Azran I, Schavinsky-Khrapunsky Y, Aboud M: Role of Tax protein in human T-cell leukemia virus type-I leukemogenicity. Retrovirology. 2004, 1: 20-10.1186/1742-4690-1-20.
Hall WW, Fujii M: Deregulation of cell-signaling pathways in HTLV-1 infection. Oncogene. 2005, 24: 5965-5975. 10.1038/sj.onc.1208975.
Feuer G, Green PL: Comparative biology of human T-cell lymphotropic virus type 1 (HTLV-1) and HTLV-2. Oncogene. 2005, 24: 5996-6004. 10.1038/sj.onc.1208971.
Matsuoka M: Human T-cell leukemia virus type I (HTLV-I) infection and the onset of adult T-cell leukemia (ATL). Retrovirology. 2005, 2: 27-10.1186/1742-4690-2-27.
Miyoshi I, Kubonishi I, Yoshimoto S, Akagi T, Ohtsuki Y, Shiraishi Y, Nagata K, Hinuma Y: Type C virus particles in a cord T-cell line derived by co-cultivating normal human cord leukocytes and human leukaemic T cells. Nature. 1981, 294: 770-771. 10.1038/294770a0.
Yamamoto N, Okada M, Koyanagi Y, Kannagi M, Hinuma Y: Transformation of human leukocytes by cocultivation with an adult T cell leukemia virus producer cell line. Science. 1982, 217: 737-739. 10.1126/science.6980467.
Chen IS, Quan SG, Golde DW: Human T-cell leukemia virus type II transforms normal human lymphocytes. Proc Natl Acad Sci USA. 1983, 80: 7006-7009. 10.1073/pnas.80.22.7006.
Uchiyama T, Yodoi J, Sagawa K, Takatsuki K, Uchino H: Adult T-cell leukemia: clinical and hematologic features of 16 cases. Blood. 1977, 50: 481-492.
Takatsuki K: Discovery of adult T-cell leukemia. Retrovirology. 2005, 2: 16-10.1186/1742-4690-2-16.
Gallo RC: The discovery of the first human retrovirus: HTLV-1 and HTLV-2. Retrovirology. 2005, 2: 17-10.1186/1742-4690-2-17.
Grassmann R, Berchtold S, Radant I, Alt M, Fleckenstein B, Sodroski JG, Haseltine WA, Ramstedt U: Role of human T-cell leukemia virus type 1 X region proteins in immortalization of primary human lymphocytes in culture. J Virol. 1992, 66: 4570-4575.
Akagi T, Shimotohno K: Proliferative response of Tax1-transduced primary human T cells to anti-CD3 antibody stimulation by an interleukin-2-independent pathway. J Virol. 1993, 67: 1211-1217.
Ross TM, Pettiford SM, Green PL: The tax gene of human T-cell leukemia virus type 2 is essential for transformation of human T lymphocytes. J Virol. 1996, 70: 5194-5202.
Robek MD, Ratner L: Immortalization of CD4(+) and CD8(+) T lymphocytes by human T-cell leukemia virus type 1 Tax mutants expressed in a functional molecular clone. J Virol. 1999, 73: 4856-4865.
Slamon DJ, Shimotohno K, Cline MJ, Golde DW, Chen IS: Identification of the putative transforming protein of the human T-cell leukemia viruses HTLV-I and HTLV-II. Science. 1984, 226: 61-65. 10.1126/science.6089351.
Seiki M, Inoue J, Takeda T, Yoshida M: Direct evidence that p40x of human T-cell leukemia virus type I is a trans-acting transcriptional activator. Embo J. 1986, 5: 561-565.
Cross SL, Feinberg MB, Wolf JB, Holbrook NJ, Wong-Staal F, Leonard WJ: Regulation of the human interleukin-2 receptor alpha chain promoter: activation of a nonfunctional promoter by the transactivator gene of HTLV-I. Cell. 1987, 49: 47-56. 10.1016/0092-8674(87)90754-9.
Maruyama M, Shibuya H, Harada H, Hatakeyama M, Seiki M, Fujita T, Inoue J, Yoshida M, Taniguchi T: Evidence for aberrant activation of the interleukin-2 autocrine loop by HTLV-1-encoded p40x and T3/Ti complex triggering. Cell. 1987, 48: 343-350. 10.1016/0092-8674(87)90437-5.
Zhao LJ, Giam CZ: Human T-cell lymphotropic virus type I (HTLV-I) transcriptional activator, Tax, enhances CREB binding to HTLV-I 21-base-pair repeats by protein-protein interaction. Proc Natl Acad Sci USA. 1992, 89: 7070-7074. 10.1073/pnas.89.15.7070.
Fujii M, Tsuchiya H, Chuhjo T, Akizawa T, Seiki M: Interaction of HTLV-1 Tax1 with p67SRF causes the aberrant induction of cellular immediate early genes through CArG boxes. Genes Dev. 1992, 6: 2066-2076.
Iwai K, Mori N, Oie M, Yamamoto N, Fujii M: Human T-cell leukemia virus type 1 tax protein activates transcription through AP-1 site by inducing DNA binding activity in T cells. Virology. 2001, 279: 38-46. 10.1006/viro.2000.0669.
Sun SC, Yamaoka S: Activation of NF-kappaB by HTLV-I and implications for cell transformation. Oncogene. 2005, 24: 5952-5964. 10.1038/sj.onc.1208969.
Grassmann R, Aboud M, Jeang KT: Molecular mechanisms of cellular transformation by HTLV-1 Tax. Oncogene. 2005, 24: 5976-5985. 10.1038/sj.onc.1208978.
Semmes OJ, Majone F, Cantemir C, Turchetto L, Hjelle B, Jeang KT: HTLV-I and HTLV-II Tax: differences in induction of micronuclei in cells and transcriptional activation of viral LTRs. Virology. 1996, 217: 373-379. 10.1006/viro.1996.0126.
Tanaka Y, Hayashi M, Takagi S, Yoshie O: Differential transactivation of the intercellular adhesion molecule 1 gene promoter by Tax1 and Tax2 of human T-cell leukemia viruses. J Virol. 1996, 70: 8508-8517.
Endo K, Hirata A, Iwai K, Sakurai M, Fukushi M, Oie M, Higuchi M, Hall WW, Gejyo F, Fujii M: Human T-cell leukemia virus type 2 (HTLV-2) Tax protein transforms a rat fibroblast cell line but less efficiently than HTLV-1 Tax. J Virol. 2002, 76: 2648-2653. 10.1128/JVI.76.6.2648-2653.2002.
Hirata A, Higuchi M, Niinuma A, Ohashi M, Fukushi M, Oie M, Akiyama T, Tanaka Y, Gejyo F, Fujii M: PDZ domain-binding motif of human T-cell leukemia virus type 1 Tax oncoprotein augments the transforming activity in a rat fibroblast cell line. Virology. 2004, 318: 327-336. 10.1016/j.virol.2003.10.006.
Meertens L, Chevalier S, Weil R, Gessain A, Mahieux R: A 10-amino acid domain within human T-cell leukemia virus type 1 and type 2 tax protein sequences is responsible for their divergent subcellular distribution. J Biol Chem. 2004, 279: 43307-43320. 10.1074/jbc.M400497200.
Sieburg M, Tripp A, Ma JW, Feuer G: Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 tax oncoproteins modulate cell cycle progression and apoptosis. J Virol. 2004, 78: 10399-10409. 10.1128/JVI.78.19.10399-10409.2004.
Tsubata C, Higuchi M, Takahashi M, Oie M, Tanaka Y, Gejyo F, Fujii M: PDZ domain-binding motif of human T-cell leukemia virus type 1 Tax oncoprotein is essential for the interleukin 2 independent growth induction of a T-cell line. Retrovirology. 2005, 2: 46-10.1186/1742-4690-2-46.
Tripp A, Banerjee P, Sieburg M, Planelles V, Li F, Feuer G: Induction of cell cycle arrest by human T-cell lymphotropic virus type 1 Tax in hematopoietic progenitor (CD34+) cells: modulation of p21cip1/waf1 and p27kip1 expression. J Virol. 2005, 79: 14069-14078. 10.1128/JVI.79.22.14069-14078.2005.
Xie L, Yamamoto B, Haoudi A, Semmes OJ, Green PL: PDZ binding motif of HTLV-1 Tax promotes virus-mediated T-cell proliferation in vitro and persistence in vivo. Blood. 2006, 107: 1980-1988. 10.1182/blood-2005-03-1333.
Sheehy N, Lillis L, Watters K, Lewis M, Gautier V, Hall W: Functional analysis of human T lymphotropic virus type 2 Tax proteins. Retrovirology. 2006, 3: 20-10.1186/1742-4690-3-20.
Iwanaga Y, Tsukahara T, Ohashi T, Tanaka Y, Arai M, Nakamura M, Ohtani K, Koya Y, Kannagi M, Yamamoto N, Fujii M: Human T-cell leukemia virus type 1 tax protein abrogates interleukin-2 dependence in a mouse T-cell line. J Virol. 1999, 73: 1271-1277.
Niinuma A, Higuchi M, Takahashi M, Oie M, Tanaka Y, Gejyo F, Tanaka N, Sugamura K, Xie L, Green PL, Fujii M: Aberrant activation of the interleukin-2 autocrine loop through the nuclear factor of activated T cells by nonleukemogenic human T-cell leukemia virus type 2 but not by leukemogenic type 1 virus. J Virol. 2005, 79: 11925-11934. 10.1128/JVI.79.18.11925-11934.2005.
Tanaka Y, Yoshida A, Tozawa H, Shida H, Nyunoya H, Shimotohno K: Production of a recombinant human T-cell leukemia virus type-I trans-activator (tax1) antigen and its utilization for generation of monoclonal antibodies against various epitopes on the tax1 antigen. Int J Cancer. 1991, 48: 623-630.
Lewis MJ, Novoa P, Ishak R, Ishak M, Salemi M, Vandamme AM, Kaplan MH, Hall WW: Isolation, cloning, and complete nucleotide sequence of a phenotypically distinct Brazilian isolate of human T-lymphotropic virus type II (HTLV-II). Virology. 2000, 271: 142-154. 10.1006/viro.2000.0284.
We thank Dr. Hiroyuki Miyoshi at RIKEN Tsukuba Institute and Dr. William H. Hall for the CSII-EF-RfA plasmid and anti-Tax2 antibody, respectively. We also thank the Takeda Pharmaceutical Company for providing recombinant human IL-2. We would like to express our gratituded to Chika Yamamoto for her excellent technical assistance. This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas and for Scientific Research (C) of Japan.
The author(s) declare that they have no competing interests.
RK, MH, MT, LX, and YT carried out the establishing the cell lines and the functional analysis of the cell lines. MO, FG, and MF participated in the experimental design, data interpretation, and the writing of the manuscript.
Rie Kondo, Masaya Higuchi contributed equally to this work.