HTLV-I Tax-dependent and -independent events associated with immortalization of human primary T lymphocytes

Abstract

Human T-cell leukemia virus type I (HTLV-I IntroductionHuman T-cell leukemia virus type I (HTLV-I) transforms human CD4 ϩ T cells in vitro and in vivo causing adult T-cell leukemia/ lymphoma (ATL). 1 ATL has an extremely poor prognosis for survival, characterized by an aggressive proliferation of leukemic cells. 2 The molecular basis for HTLV-I-mediated transformation of T cells is unclear. Tax has been shown to inactivate several tumor suppressors, disrupt cell cycle and DNA repair checkpoints, and stimulate cell growth, while protecting against apoptosis. [3][4][5][6][7] Importantly, the oncogenic activities of Tax have been studied mainly in transgenic models and in rodent fibroblasts. [8][9][10] Although these models provided useful information, they have limitations, because the transformation of human cells in vitro requires more oncogenic events than the transformation of rodent cells. 11 The role of Tax in transformation is based largely on forced overexpression of Tax, which results in cell-cycle abnormalities, faulty mitosis, aneuploidy, and the formation of multinucleated cells. A major caveat to these studies is that they were performed in established cell-culture lines, which are already dysfunctional in key regulatory pathways (tumor suppressor, cell cycle, and DNA repair checkpoints), and therefore are much more susceptible than normal primary cells to Tax-induced alterations. Studying Tax in human primary T cells is preferable, yet, to date, immortalized Tax-expressing human T-cell lines able to proliferate indefinitely in culture have not been established. In one study, the pX region of HTLV-I (which expresses the Tax gene among other viral genes) was found to transform human T cells in the context of a Herpesvirus saimiri vector, 12 although there is no evidence to refute that the herpes genes from the vector synergized or were activated by Tax to promote transformation or that other viral genes were involved. This prompted us to investigate the ability of Tax to immortalize human primary T cells. In the following study, we demonstrate that Tax-induced immortalization of peripheral blood mononuclear cells (PBMCs) is a very rare event. Tax alone enhanced proliferation, but appears insufficient to sustain permanent proliferation. Although most attempts were unsuccessful, we finally obtained an immortalized Tax-expressing CD4 human T-cell line, WT4, which was maintained in continuous culture for more than 4 years. Our results suggest that Tax alone is a poor oncogene that increases the life span and rate of T-cell proliferation in the presence of interleukin-2 (IL-2), thereby increasing the probability for accumulation of genetic defects leading to immortalization. Methods Plasmids and cell linesThe HTLV-I tax gene was cloned into the HRCMV or HRЈ lentiviral vectors ( Figure 1A). Pseudotype virus particles were produced as previously reported. 13 For cell-cycle analysis, tax was cloned into an UBC-IRES-GFP lentiviral vector. The p53 gene of WT4 was sequenced and cloned as previously described. 14 ...

Polycomb-dependent epigenetic landscape in adult T-cell leukemia

“…The Venus-positive transduced cells rapidly expanded and were then immortalized by WT Tax expression ( Figure 6B and supplemental Figure 9B). 51 The functional importance of NF-kB and cAMP response element-binding protein activities in this process was supported by results from Tax mutants. 52,53 EZH2 inhibition completely prevented cell growth, indicating that the enzymatic activity of EZH2 is necessary for Tax-dependent cell growth and subsequent immortalization, which is a relevant surrogate for studying the epigenome of preleukemic cells.…”

Permissive Sense and Antisense Transcription from the 5′ and 3′ Long Terminal Repeats of Human T-Cell Leukemia Virus Type 1

“…In support of this hypothesis, recent studies have shown that HBZ counteracts Tax-induced senescence of HeLa cells, which also involves activation NF-B signaling and the accumulation of reactive oxygen species (25). Considering that Tax alone is able to immortalize primary T cells, albeit inefficiently (50), it would be interesting to examine whether coexpression of HBZ augments this process or possibly leads to T-cell transformation.…”

HTLV-2 Tax Immortalizes Human CD4+ Memory T Lymphocytes by Oncogenic Activation and Dysregulation of Autophagy

“…It is conceivable that the oncogenic potential of the Tax proteins is best determined in human primary T cells, the natural host cells for HTLV-1 or -2 infection, rather than in rodent cells that are highly susceptible to viral or cellular oncogene-mediated transformation. Indeed, devoid of other viral components, Tax1 appeared to be a weak oncoprotein as Tax1-mediated immortalization of primary human T cells was a rare incidence (21). The ability of Tax2 in immortalizing human T cells has not been previously reported.…”

“…Unlike ATL leukemia cells in which Tax expression is typically lost, the growth of Tax-established primary T cells requires constitutive production of the Tax protein in order to promote T cell survival and proliferation. The activity of a heterologous viral promoter, such as cytomegalovirus promoter (CMV), which was previously utilized to establish Tax1-immortalized primary T cells (21), may be deleterious during prolonged cell growth due to a possible promoter silencing (34). In the Tax1-immortalized T cell line WT4, Tax1 was expressed at a much lower level than that in MT-2 cells in p40tax form (21).…”

“…The activity of a heterologous viral promoter, such as cytomegalovirus promoter (CMV), which was previously utilized to establish Tax1-immortalized primary T cells (21), may be deleterious during prolonged cell growth due to a possible promoter silencing (34). In the Tax1-immortalized T cell line WT4, Tax1 was expressed at a much lower level than that in MT-2 cells in p40tax form (21). MT-2 cells express abundant amount of p68 Env-Tax fusion protein but produce extremely low level of p40Tax (35).…”