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. 2013;8(2):e55989.
doi: 10.1371/journal.pone.0055989. Epub 2013 Feb 6.

Activation of WIP1 phosphatase by HTLV-1 Tax mitigates the cellular response to DNA damage

Tajhal Dayaram  1 , Francene J Lemoine, Lawrence A Donehower, Susan J Marriott
Affiliations

Activation of WIP1 phosphatase by HTLV-1 Tax mitigates the cellular response to DNA damage

Tajhal Dayaram et al. PLoS One. 2013.

Abstract

Genomic instability stemming from dysregulation of cell cycle checkpoints and DNA damage response (DDR) is a common feature of many cancers. The cancer adult T cell leukemia (ATL) can occur in individuals infected with human T cell leukemia virus type 1 (HTLV-1), and ATL cells contain extensive chromosomal abnormalities, suggesting that they have defects in the recognition or repair of DNA damage. Since Tax is the transforming protein encoded by HTLV-1, we asked whether Tax can affect cell cycle checkpoints and the DDR. Using a combination of flow cytometry and DNA repair assays we showed that Tax-expressing cells exit G(1) phase and initiate DNA replication prematurely following damage. Reduced phosphorylation of H2AX (γH2AX) and RPA2, phosphoproteins that are essential to properly initiate the DDR, was also observed in Tax-expressing cells. To determine the cause of decreased DDR protein phosphorylation in Tax-expressing cells, we examined the cellular phosphatase, WIP1, which is known to dephosphorylate γH2AX. We found that Tax can interact with Wip1 in vivo and in vitro, and that Tax-expressing cells display elevated levels of Wip1 mRNA. In vitro phosphatase assays showed that Tax can enhance Wip1 activity on a γH2AX peptide target by 2-fold. Thus, loss of γH2AX in vivo could be due, in part, to increased expression and activity of WIP1 in the presence of Tax. siRNA knockdown of WIP1 in Tax-expressing cells rescued γH2AX in response to damage, confirming the role of WIP1 in the DDR. These studies demonstrate that Tax can disengage the G(1)/S checkpoint by enhancing WIP1 activity, resulting in reduced DDR. Premature G(1) exit of Tax-expressing cells in the presence of DNA lesions creates an environment that tolerates incorporation of random mutations into the host genome.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Tax expression alters the G1 phase arrest following DNA damage.
Asynchronous CREF-neo and CREF-Tax cells were exposed to 30 J/m2 of UV irradiation. The percent of cells in G1 phase (A) and S phase (B) are displayed at the indicated times post-irradiation. Results shown are the average of three independent experiments (error bars represent standard error of the mean; * p-value≤0.1, ** p-value≤0.05).
Figure 2
Figure 2. Tax expression accelerates S-phase entry following DNA damage.
Synchronized CREF-neo and CREF-Tax cells were exposed to 30 J/m2 of UV irradiation 12 hours after release from G0, which is shown as “0” h post irradiation. The percent of cells in G1 phase (A) and S phase (B) are displayed at the indicated times post-irradiation. Results shown are the average of three independent experiments. (error bars represent standard error of the mean; * p-value≤0.1, ** p-value≤0.05).
Figure 3
Figure 3. Tax represses repair of UV irradiation-induced DNA damage.
Asynchronously growing CREF-neo and CREF-Tax cells were exposed to 15 J/m2 UV irradiation. Thymine dimers in genomic DNA were detected at indicated times following UV or mock-treatment.
Figure 4
Figure 4. Tax expressing cells fail to form UV-induced γH2AX and p-RPA foci.
(A) UV irradiated CREF-neo and CREF-Tax cells were stained with either anti- p-RPA2(S33), anti- p-RPA2(S4/8), or anti-γH2AX (all stained in red). Cells were counterstained with Dapi (blue) to visualize nuclei. (B) CREF-Neo and CREF-Tax cells were either mock-irradiated or damaged with 30 J/m2 UV. Cells were collected at the indicated timepoints and stained for γH2AX (red) and DAPI (blue). (C) Quantitation of γH2AX immunofluorescence intensity of 12 fields of at least 20 cells. Error bars represent standard error and ‡ represents a p-value≤0.001.
Figure 5
Figure 5. Tax expression inhits γH2AX in a dose-dependent manner.
(A) CREF-neo and CREF-Tax cells were exposed to 30 J/m2 UV and harvested at the indicated timepoints. Whole cell extracts were analyzed by western blot for Actin, Tax and γH2AX. (B) 293 cells were untransfected (No Tax) or transfected with the indicated amounts of a Tax expression vector and exposed to 30 J/m2 UV. Cells were harvested at 10 minutes post-UV and whole cell extracts were analyzed by western blot for Tax, Actin and γH2AX.
Figure 6
Figure 6. Tax-expressing cells upregulate WIP1 mRNA following UV-damage.
(A) Jpx9 cells were induced for Tax expression with 20 uM CdCl2 and harvested at the indicated timepoints to assay for Tax expression by western blot. Uninduced or Jpx9 cells induced for 48 hours when Tax expression was strong were undamaged or exposed to 50 J/m2 UV and harvested at the indicated times for quantitative RT-PCR analysis. The y-axis represents WIP1 mRNA levels normalized to GAPDH. Relative WIP1 mRNA is shown in comparison to undamaged, uninduced Jpx9 cells. The average of three independent experiments is shown. Error bars represent standard error and asterisks indicate significant differences between Tax-expressing and uninduced cells at each timepoint (* = p≤0.1, ** = p≤0.05 and ***≤0.01). (B) Jurkat and Jpx9 cells were left untreated or treated with 20 µM CdCl2 for 48 hours. Cells were then harvested and resulting RNA subjected to quantitative RT-PCR for WIP1 and GAPDH. Relative WIP1 mRNA of treated cells is shows in comparison to untreated cells.
Figure 7
Figure 7. Tax interacts with WIP1.
(A) 293 cells were transfected with either a Flag-WIP1 construct alone or Flag-WIP1 along with a Tax expression construct. Lysates were immunoprecipitated using either anti-WIP1 or anti-Tax antibodies followed by western blotting with anti-Flag or anti-Tax antibodies. (B) Coomassie stain of purified His-WIP1 and GST-Tax used in panel C. (C) His-WIP1 was incubated with either Glutathione sepharose beads or GST-Tax immobilized on Glutathione sepharose. Reactions were analyzed by western blot with anti-Tax and anti-His antibodies.
Figure 8
Figure 8. Tax enhances WIP1 phosphatase activity.
In vitro phosphatase assays were performed using purified His-WIP1, GST or GST-Tax, and H2AX (pS139), p38 (pT180) and UNG2 (pT31) phosphopeptides. Free phosphate levels for each reaction are shown. The average of three independent experiments is shown. Error bars represent standard error and asterisks indicate significance (* = p≤0.1 and ** = p≤0.05).
Figure 9
Figure 9. Inhibition of WIP1 in Tax-expressing cells restores γH2AX levels following DNA damage.
(A) CREF-Tax cells were transfected with a control siRNA or siRNA targeted to WIP1. 48 hours post-transfection cells were treated with 30 J/m2 UV, allowed to recover for 4 hours, and analyzed by western blot for γH2AX and actin. (B) UV-damaged control siRNA transfected and WIP1 siRNA transfected CREF-Tax cells were analyzed by quantitative RT-PCR for WIP1 expression. (C) Uninfected (CEM) and HTLV-1 infected (MT4) cells (untreated or treated with the UV-mimetic drug 4-NQO) were harvested at the indicated times and analyzed by western blot. (D) CEM and MT4 cells were left untreated (−), treated with 4-NQO (+), or treated with the WIP1 inhibitor Compound M for 1 hour followed by 4-NQO (+M) and harvested after a 4 hour recovery followed by analysis by western blot for γH2AX.
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