Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 tax oncoproteins modulate cell cycle progression and apoptosis

Abstract

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia and lymphoma, an aggressive clonal malignancy of human CD4-bearing T lymphocytes. HTLV-2, although highly related to HTLV-1 at the molecular level, has not been conclusively linked to development of lymphoproliferative disorders. Differences between the biological activities of the respective tax gene products (Tax1 and Tax2) may be one factor which accounts for the differential pathogenicities associated with infection. To develop an in vitro model to investigate and compare the effects of constitutive expression of Tax1 and Tax2, Jurkat T-cell lines were infected with lentivirus vectors encoding Tax1 and Tax2 in conjunction with green fluorescent protein, and stably transduced clonal cell lines were generated by serial dilution in the absence of drug selection. Jurkat cells that constitutively express Tax1 and Tax2 (Tax1/Jurkat and Tax2/Jurkat, respectively) showed notably reduced kinetics of cellular replication, and Tax1 inhibited cellular replication to a higher degree in comparison to Tax2. Tax1 markedly activated transcription from the cdk inhibitor p21(cip1/waf1) promoter in comparison to Tax2, suggesting that upregulation of p21(cip1/waf1) may account for the differential inhibition of cellular replication kinetics displayed by Tax1/Jurkat and Tax2/Jurkat cells. The presence of binucleated and multinucleated cells, reminiscent of large lymphocytes with cleaved or cerebriform nuclei often seen in HTLV-1- and -2-seropositive patients, was noted in cultures expressing Tax1 and Tax2. Although Tax1 and Tax2 expression mediated elevated resistance to apoptosis in Jurkat cells after serum deprivation, Tax1 was unique in protection from apoptosis after exposure to camptothecin and etoposide, inhibitors of topoisomerase I and II, respectively. Characterization of the unique phenotypes displayed by Tax1 and Tax2 in vitro will provide information as to the relative roles of these oncoproteins and their contribution to HTLV-1 and -2 pathogenesis in vivo.

FIG. 1.

Characterization of Jurkat cell lines constitutively expressing Tax1 and Tax2. (A) Flow cytometric analysis of GFP expression. Jurkat cells were infected with LVs (MOI = 3), as previously described (62). Cells were serially diluted and clonally plated in 96-well plates at 48 h postinfection. After expansion, clones were randomly selected and analyzed for GFP expression by flow cytometry. GFP expression was assayed at 1, 6, and 12 weeks after clonal expansion, and GFP expression measured at 12 weeks is shown. The data were analyzed by using WinMDI 2.8 software. The clonal cell lines and LVs used for infection are as follows: Tax1/Jurkat, HR′CMV-Tax1/GFP; Tax1(−)/Jurkat, HR′CMV-Tax1(−)/GFP; and Tax2B/Jurkat and Tax2C/Jurkat, HR′CMV-Tax2/GFP. (B) Luciferase activity resulting from transfection of Jurkat clones with HTLV-1-LTR-Luc. Jurkat cell lines (10⁷) were transfected with HTLV-1-LTR-Luc (24 μg; a gift from Kuan-Teh Jeang, National Institutes of Health, Bethesda Md.) by using Lipofectamine 2000 (Invitrogen). Cells were lysed in cell culture lysis reagent (Promega), and the luciferase activity was normalized for the amount of protein in each extract, as determined by a Bradford assay. SLB-1 is an HTLV-1-transformed T-cell line and contains multiple proviral integrations of HTLV-1. The experiment was performed three times, and the luciferase assays were carried out in triplicate. Error bars represent one standard deviation, and statistical analysis was performed by using the Student t test (P < 0.05), with Tax1(−)/Jurkat cells as a control. The transfection efficiencies ranged from 80 to 95% in SLB-1 cells as determined by GFP expression after cotransfection of a GFP reporter gene construct and HTLV-1-LTR-Luc.

FIG. 2.

Effects of Tax1 and Tax2 on Jurkat cell proliferation. Unsynchronized Jurkat cell lines (10⁵) were plated in 3 ml of complete IMDM medium in six-well plates. Cells were stained with 20 μl of trypan blue (Gibco-BRL), and viable cells were quantified by microscopy every 24 h. The absolute number of cells in the culture on days 1 through 6 is represented on the left y axis. In one experiment, cell replication kinetics were quantified out to 14 days after plating, and these data are represented on the right y axis. These experiments were repeated three times. Error bars represent one standard deviation. Single-tailed analysis of variance (ANOVA) and Student t tests analysis was performed. ✽, P < 0.05, with Tax1(−)/Jurkat cells as a control.

FIG.3.

Multinucleation in Tax1/Jurkat and Tax2/Jurkat cell cultures. LV transduced Jurkat cells (10⁵) were plated onto poly-l-lysine-coated chamber slides, incubated in 1:1 methanol-acetone, and stained with hematoxylin for 2 min and eosin for 1 min (H&E). Cell lines were also stained separately with DAPI, a DNA-specific stain, for 10 min at room temperature. Photographs were taken with a SPOT digital microscope camera (Diagnostic Instruments, Inc., Sterling Heights, Mich.) on a Nikon Eclipse microscope. (A) The presence of multinucleated cells was detected by H&E staining (magnification, ×40). The blue circle represents a cell undergoing mitosis for reference. The red circles represent large, multilobulated cells representing ∼1% of total cells in the Tax1/Jurkat, Tax2B/Jurkat, and Tax2C/Jurkat cell cultures. Each enlarged field is photographed at ×80. (B) Hematoxylin-and-eosin (H&E)- and DAPI-stained Jurkat cell lines. Aliquots of cells (10⁶) were stained with H&E (left panels) or DAPI (middle panels) and observed by microscopy (magnification, ×20). DAPI-stained cells were also analyzed by flow cytometry (Becton Dickinson). The shaded overlay represents LV-transduced Jurkat cells stained with DAPI compared to the parental Jurkat cell line stained with DAPI (right panels).

FIG. 4.

Tax1 and Tax2 transactivation of the p21^cip1/waf1 promoter. LV constructs encoding GFP, Tax1, Tax2, or the HTLV-1 tax gene in the antisense orientation [Tax1(−)] were cotransfected into 293T cells with plasmids encoding the p21^cip1/waf1 promoter initiating expression of luciferase (a gift from Fatah Kashanchi, George Washington University, Washington, D.C.). Transfection efficiencies ranged from between 90 to 95%, as determined by fluorescence microscopic analysis of GFP expression. Luciferase activity was measured at 48 h posttransfection. (A) Luciferase activity resulting from cotransfection of LVs encoding GFP, Tax1, Tax1(−), or Tax2 and 2-Luc, a construct encoding 1,481 nucleotides of the p21 promoter (14). The number above each column represents the average luciferase activity. “Mock DNA” is a cotransfection of the p21 promoter construct (2-Luc) with pUC19 carrier DNA and represents the basal level of luciferase activity. (B) Tax1 and Tax2 transactivation of p21^cip1/waf1 promoter deletion constructs. Luciferase activity resulting from cotransfection of LVs and p21 promoter constructs which retain either 173 nucleotides (8-Luc) or 49 nucleotides (11-Luc) of the 5′ p21 promoter sequences. A full-length cloned p21 promoter (0-Luc) and the locations of the p53 binding sites within the promoter (S1 and S2) are provided as a reference. Transfections were repeated three times, and luciferase reactions were carried out in triplicate. Error bars represent one standard deviation. Statistical analysis was performed by using the Student t test. ✽, P < 0.05, with Tax1(−)/Jurkat cells as a control.

FIG. 5.

Cell cycle and apoptosis analysis of Jurkat clones after serum starvation. Clonal Jurkat cell lines (10⁶) were serum starved by plating them in 3 ml of IMDM medium supplemented with pen/strep and glutamate in the absence of serum. After 48 h, cells were replated in 3 ml of IMDM supplemented with 10% FBS. An aliquot of cells (5 × 10⁵) was incubated in 70% ethanol, washed with PBS, and stained with propidium iodide and analyzed by flow cytometry immediately after 48 h of serum starvation (Baseline) and 48 h after refeeding of cells in the presence serum (48 Hours). Sub-G₀ (apoptotic cells) and cycling cells (S+G₂/M) were quantified for each Jurkat cell line by flow cytometry, and percentages of cells in each fraction are indicated. The percentage of apoptotic cells (Sub-G₀) and actively cycling cells (S+G₂/M) after serum withdrawal and at 48 h after refeeding cells in the presence of serum are indicated. WinMDI 2.8 was used to quantify the cell subpopulations and analyze the data. Experiments were performed three times; the error bars represent one standard deviation. Statistical analysis was performed by using single-tailed ANOVA and Student t tests ✽, P < 0.05, with Tax1(−)/Jurkat cells as a control.

FIG. 6.

Flow cytometric analysis of Jurkat cell lines after incubation with CPT, TXL, and ETP. Jurkat cell lines were cultured in the presence of either 1.0 μM CPT, an inhibitor of Top1 (□); 1.0 μM ETP, an inhibitor of Top2 (▥); or 5 ng of TXL/ml (▪). An aliquot of the cell culture (10⁶ cells) was analyzed by flow cytometry at 24 and 36 h after drug application, and apoptosis was assayed by staining for Annexin V (A and C) and 7-AAD (B and D). The data were compiled from three independent experiments, and statistical analysis was performed by using single-tailed ANOVA and Student t tests. ✽, P < 0.05, with Tax1(−)/Jurkat cells as a control. The error bars represent one standard deviation.