Figure 1.
HTLV-I Tax stimulates the formation of DDSB in the genome.
A) Accumulation of DDSB in HTLV-I-transformed cells MT-4 and Tax-only immortalized human T cells WT4, WT4I and WT4B was analyzed by comet assays. Normal PBMCs were used as a negative control. B) Western blots analyses showing physiological levels of Tax expression in human T-cell lines immortalized by Tax only. C) Western blot analyses for γ-H2AX and Tax expression in HTLV-I-transformed MT-4 cells, Tax-immortalized WT-4 T-cells or Jurkat control cells. H2AX was used as a loading control for each sample. D) γ-H2AX foci were readily detected by immunofluorescence in HTLV-I transformed cells and Tax-expressing T-cells but not in normal PBMCs. E) Quantification of the percentage of cells harboring 10 or more γ-H2AX foci per cell in PBMC, WT-4, MT-4, MT-2 and C8166. Results were derived from counting 100 nuclei for PBMC and each cell line. F) Tax-inducible Jurkat JPX9 cells and Jurkat control cells were treated with CdCl to induce Tax expression in the former and analyzed by immunofluorescence for presence of DDSB as shown by γ-H2AX foci. G) Western blot analyses using γ-H2AX and Tax-specific antibodies confirmed an increased γ-H2AX expression in Tax-expressing cells only. H) Representative image of DDSB detected by comet assays in JPX9 cells only after induction of Tax and not Jurkat cells treated under the same conditions. I) Jurkat cells were transduced with VSV-pseudotype viruses expressing Tax or GFP as described in materials and methods. After 48 hours proteins were extracted and analyzed by western blot for γ-H2AX expression. Actin was used as a loading control.
Figure 2.
Tax-associated DDSB occur in S phase during DNA replication.
A) HTLV-I-transformed MT-2 cells were pulse-labeled with BrDU to identify cells in S phase of the cell cycle. Immunofluorescence experiments indicated that γ-H2AX foci are detected in BrDU positive cells only. Cells were counter stained with DAPI. B) HTLV-I-transformed MT-2 cells were dual stained with Cyclin A, a marker of S phase, and γ-H2AX, a marker of DDSB foci. Cells were counter-stained with DAPI. C) HTLV-I-transformed MT-2 cells were dual stained with PCNA, which appear as a punctuated pattern in S phase, and γ-H2AX, to reveal DDSB foci. Cells were counter-stained with DAPI. D) Accumulation of DDSB foci in S phase was confirmed in Tax-only expressing cells using Tax-inducible JPX9 cells and Jurkat control, both treated with CdCl and dual-stained with Cyclin A and γ-H2AX antibodies. E) JPX9 cells were synchronized in G0/G1 by exposure to hydroxyurea (HU) overnight. G0/G1 JPX9 cells and non-synchronized JPX9 cells were exposed to CdCl to induce Tax expression and analyzed for presence of γ-H2AX DDSB foci. F) Efficacy of HU treatment was demonstrated by cell cycle analyses by FACS after PI staining. From top to bottom, untreated cells, HU G1-arrested cells, HU G1-arrested cells released by washing out HU and culturing 20 hours. G) Western blot analyses confirmed that HU treatment did not affect Tax expression.
Figure 3.
HTLV-I-transformed cells and normal PBMC are equally sensitive to DNA damaging agents and have an overall similar rate of DNA repair.
HTLV-I-transformed cells (MT-2, MT-4, C8166, 1185) and Tax-immortalized cells (WT4, WT4B, WT4I) and PBMC were exposed to 10 nM of Doxorubicin, washed and rate of repair was evaluated by immunostaining of γ-H2AX -revealed DDSB foci and quantified by microscopy at 0 h, 2.5 h, 5 h and 10 h after treatment. Because of various numbers of breaks in the absence of treatment, DNA breaks were normalized at 0 for time T = 0. Data were generated from counting 100 cells. Rate of repair is represented by the decreasing number of γ-H2AX foci at 2.5, 5 and 10 hours after doxorubicin treatment. Data were generated from counting 100 cells.
Figure 4.
Tax inhibits the DDSB HR DNA repair through activation of NF-kB.
A) Representation of in vivo HR assays using the DR-GFP HR reporter and the pI-SceI vectors. B) Jurkat T cells were cotransfected with DR-GFP vector either with the control vector or with the pI-SceI, and along with Tax or Tax mutants M47 or M22 using Amaxa electroporation. Forty-eight hours later the expression of GFP was assessed by FACS analysis. Relative percentage of GFP-expressing cells was represented by histograms corresponding to the average of 3 independent experiments. C) Activation of NF-kB reporter luciferase vector by HTLV-I Tax and Tax mutants M47 and M22. D and E) In vivo HR assay was performed in the presence of Tax and along with coexpression of a phosphorylation-defective dominant negative IkBα mutant that efficiently blocks NF-kB activation by Tax. F) Inhibition of HR by Tax was investigated using HTLV-I molecular clones expressing Tax or Tax mutants M47 and M22 at physiological levels.
Figure 5.
Tax-associated DDSB are not repaired by SSA or MMEJ.
A and B) Tax-inducible cell line JPX9 and Jurkat control cells were induced with CdCl, γ-irradiated with a dose of 2Gy and treated with or without DNA-PK inhibitor NU7026. γH2AX foci were detected by microscopy at time t = 0, 2 h, 4 h, 6 h and 24 h. Image results provided are representative of the experiment performed in duplicate. Average number of γ-H2AX foci per cell was quantified by microscopy. The error bars in the figures represent the standard error of the mean (SEM) for 30 to 50 cells per sample. The images were obtained using epifluorescence Nikon Ti-s and have been treated with the deconvolution feature provided with the Nikon's NIS-Element software. C) HTLV-I Tax-expressing cells MT-4 were untreated (left) or incubated with DNA-PK inhibitor for 2 days (middle panel) or 6 days (inhibitor was replenished every 2 days). Cell cycle was analyzed by FACS after propidium iodide staining. Arrows indicate cell cycle arrest in G2/M and aneuploidy.
Figure 6.
Tax expression switch DDSB repair from HR to error-prone NHEJ.
A) Colocalization of Ku80 (specific to NHEJ DNA repair) and γ-H2AX in MT-2 and WT4 cells 30 min after gamma-irradiation. B) Colocalization of γH2AX foci and Ku80 in Tax-inducible JPX9 after induction of Tax expression by CdCl. Non-induced JPX9 and CdCl treated Jurkat control cells were used to demonstrate specificity to Tax expression. The images were obtained using epifluorescence Nikon Ti-s and have been treated with the deconvolution feature provided with Nikon's NIS-Elements software. C) Colocalization of γ-H2AX foci and Rad51 (specific to HR DNA repair) after Tax induction in JPX9 and in Jurkat cells that were used as control. The images were obtained using epifluorescence Nikon Ti-s and have been treated with the deconvolution feature provided with Nikon's NIS-Elements software.