Research ArticleRETROVIROLOGY

Abacavir, an anti–HIV-1 drug, targets TDP1-deficient adult T cell leukemia

See allHide authors and affiliations

Science Advances  24 Apr 2015:
Vol. 1, no. 3, e1400203
DOI: 10.1126/sciadv.1400203
  • Fig. 1 ABC selectively kills HTLV-1–infected and ATL cell lines.

    (A) Viability of the indicated cells after treatment (2 and 4 days) with 100 μM of the indicated NRTI. MTS values of treated cells relative to untreated cells are shown. Results are expressed as means ± SD of three independent experiments. (B) Viability of the indicated cells after treatment (2 days) with the indicated ABC dose. MTS values of treated cells relative to untreated cells are shown. (C) MT-2 and ED-40515(−) cells were treated with ABC or AZT together with IFN-α (0, 100, or 1000 IU/ml) for 2 days and analyzed with an MTS assay. MTS values of treated cells relative to untreated cells are shown.

  • Fig. 2 ABC induced S/G2-phase arrest and apoptosis.

    (A) Flow cytometry was used to analyze cell cycle status after propidium iodide (PI) staining. ED-40515(−) and Jurkat cells were treated with and without 100 μM ABC for 2 days and analyzed using flow cytometry. Results are expressed as means ± SD of three independent experiments. (B) Representation of flow cytometric analysis of cell cycle after PI staining. (C) Flow cytometric analysis of apoptosis after annexin V and PI double staining. ED-40515(−), MT-2, Hut-102, ATL-43T, and Jurkat cells were treated with the indicated dose of ABC or Adriamycin (ADR; 1 μg/ml) for 48 hours. Dark and light bars represent early and late apoptosis, respectively. Results are expressed as means ± SD of three independent experiments.

  • Fig. 3 ABC-induced DSB accumulation in ATL cells as a result of the impairment of DNA repair.

    (A) Chromosome breaks induced by ABC. ED-40515(−) cells treated with 100 μM ABC for 2 days were evaluated for chromosome breaks (yellow arrow). (B) ED-40515(−) and Jurkat cells were treated with the indicated doses of ABC and AZT for 2 days. Percentages of chromosome break–positive cells are shown. Results are expressed as means ± SD of three independent experiments. n.s., not significant. (C) Expression kinetics of γH2AX foci after acute exposure to ABC. ED-40515(−) and Jurkat cells were treated with 500 μM ABC for 12 hours. After treatment, the cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) and anti-γH2AX at the indicated times. (D) Percentages of γH2AX focus–positive cells at the indicated times. Focus-positive cells are those containing more than five γH2AX foci. (E) Cell proliferation of ED-40515(−) and Jurkat cells based on an MTS assay. MTS values relative to time zero are shown. (F) Expression kinetics of γH2AX foci at the indicated times after 2-Gy γ-irradiation. Irradiated ED-40515(−) and Jurkat cells were stained with DAPI and γH2AX. (G) Percentages of γH2AX focus–positive cells at the indicated times. (H) Cell proliferation of ED-40515(−) and Jurkat cells evaluated by an MTS assay. Data shown are as in (E).

  • Fig. 4 ABC-induced prolonged activation of homologous recombination–dependent repair in ATL cells.

    (A) Immunocytochemical staining with DAPI, anti-γH2AX, and anti-RAD51 recombinase. ED-40515(−) and Jurkat cells were treated with or without 100 μM ABC for 2 days. The percentages of γH2AX and RAD51 focus–positive cells, defined as cells containing more than five foci per cell, are shown. (B) Immunocytochemical staining with DAPI, anti-γH2AX, and anti-RAD51. ED-40515(−) and Jurkat cells were treated with or without 100 μM ABC for 2 days. The merged images display frequent colocalization of γH2AX and RAD51.

  • Fig. 5 Impaired TDP1 responsible for ATL cell sensitivity to ABC.

    (A) Venn diagram of DNA repair genes defective in mutant chicken DT40 cell lines sensitive to ABC and down-regulated DNA repair genes in ATL cells. (B) Relative sensitivities of Tdp1-deficient DT40 cells treated with six NRTIs. Results are expressed as means ± SD of three independent experiments. (C) Analysis of mRNA expression levels based on microarray data from the GEO database (available at www.ncbi.nlm.nih.gov/geo/; accession number GSE33615). The relative expression levels of TDP1 in peripheral blood cells obtained from ATL patients and normal healthy donor controls are shown. The box plot uses the median, the approximate quartiles, and the lowest and highest data points to convey the level, spread, and symmetry of the distribution of data values. Statistical analysis was performed using Student’s t test to compare the mRNA expression levels between the ATL cases (n = 52) and the normal controls (n = 21). (D) Western blot analysis of TDP1 expression in HTLV-1–infected cell lines and in Jurkat cells. (E) Quantitative reverse transcription–polymerase chain reaction (RT-PCR) for TDP1 in primary ATL cells from 10 patients and in primary CD4+ T cells from 5 healthy donors. Data shown as in (C). (F) Western blot analysis of TDP1 in primary ATL cells from two patients and primary CD4+ T cells from two healthy donors.

  • Fig. 6 TDP1 removes ABC from DNA ends in vitro.

    (A) Schematic diagram of in vitro biochemical assays for TDP1 activity. Both substrates contained the same sequence and conjugated CBV or tyrosine as a 3′-blocking lesion via a phosphodiester linkage. The substrates (S) were radiolabeled at the 5′ end with 32P. The products that are removed from the 3′-blocking lesion from the substrates by TDP1 are labeled “P.” Y: Tyr. (B) A representative gel demonstrating the processing of the indicated substrates by increasing amount of total cell lysates from Tdp1−/− DT40 cells or Tdp1−/− DT40 cells stably transfected with hTDP1 transgene. The substrates were incubated with serially diluted total cell lysates ranging from 0.03 to 7.5 μg. Reaction proceeded for 15 min at 25°C before being quenched and analyzed on 16% denaturing gels. (C) The percentage of product yield is plotted against increasing lysate concentration. Results are expressed as means ± SD of three independent experiments.

  • Fig. 7 TDP1 catalytic activity requirement for cellular tolerance to ABC.

    (A) Control siRNA or siTDP1 mixture was transfected into Jurkat cells. Depletion of TDP1 expression was confirmed by Western blot analysis 48 hours after transfection (right panel). Jurkat cells transfected with control siRNA or siTDP1 were treated with or without 300 μM ABC for 48 hours. MTS values of treated relative to untreated cells are shown (left panel). Results are expressed as means ± SD of three independent experiments. (B) MT-2 cells stably transfected with either wild-type (WT) TDP1, H263A TDP1, or H493R TDP1 transgenes. Stably transfected clones and control cells (mock) were treated with the indicated dose of ABC for 48 hours. Western blot analysis was conducted for expression of transgenes (right panel). Data shown as in (A, left panel). (C) Cell cycle analysis is shown as in Fig. 2A. MT-2/TDP1WT, MT-2/TDP1H263A, and MT-2/TDP1H493R cells were treated with or without 100 μM ABC for 24 hours and subjected to flow cytometry. (D) The extent of apoptosis is shown as the percentage of annexin V–positive cells (y axis). Cells were treated as in (C). (E) The indicated cells were treated as in Fig. 3C. Data shown as in Fig. 3C. (F) Statistical analysis of data in (E). MT-2/TDP1WT cells, but not MT-2/TDP1H263A or MT-2/TDP1H493R cells, are resistant to ABC. (G) Proliferation of the indicated cells is shown as in Fig. 3E.

  • Fig. 8 ABC inhibits the growth of ATL cell xenografts in NOD/SCID mice.

    (A) Tumor sizes of xenografted NOD/SCID mice treated with or without ABC. The mean sizes of individual tumors were plotted (control group, n = 7; ABC group, n = 6). P values were determined using Student’s t test (*P < 0.05). Error bars represent the SEM. (B) Kaplan-Meier survival curves for the ABC-treated and control mice. P values were determined using the Wilcoxon test.

  • Fig. 9 Specific lethality of ABC on ATL cells due to a defect in TDP1.

    ABC is phosphorylated in a unique stepwise anabolism and is converted to the triphosphate of CBV. During DNA synthesis, triphosphorylated ABC was incorporated into host chromosomal DNA by replicative DNA polymerases, leading to premature termination of DNA replication. In normal cells, TDP1 removes ABC quickly and DNA synthesis continues. However, in HTLV-1(+) cells, the collapse of the replication fork is induced because of the deficiency of TDP1, leading to DSB formation and apoptosis.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/1/3/e1400203/DC1

    Fig. S1. ABC selectively kills HTLV-1–infected and ATL cell lines.

    Fig. S2. Cell counting assays showing ABC cytotoxicity to MT-2 cells.

    Fig. S3. ABC-induced apoptosis.

    Fig. S4. Relative sensitivities to six NRTIs of DT40 cells deficient in the indicated gene.

    Fig. S5. The relative sensitivity of candidate gene-deficient DT40 cells to the six individual NRTIs and the level of the mRNA expression in ATL cells.

    Fig. S6. Ectopic expression of TDP1WT, TDP1H263A, and TDP1H493R in MT-2 cells.

    Fig. S7. ABC and AZT cytotoxicity in MT-2/TDP1WT cells.

    Fig. S8. CPT11 and veliparib enhance the lethality of ABC on ATL cells.

    Table S1. Inhibitory concentration (IC50) values of ABC for each cell line.

    Table S2. Isogenic mutant chicken DT40 cell lines used in this study.

    Table S3. Characteristics of two ATL patients.

    Table S4. List of HTLV-1–infected and ATL cell lines.

    References (4884)

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. ABC selectively kills HTLV-1–infected and ATL cell lines.
    • Fig. S2. Cell counting assays showing ABC cytotoxicity to MT-2 cells.
    • Fig. S3. ABC-induced apoptosis.
    • Fig. S4. Relative sensitivities to six NRTIs of DT40 cells deficient in the indicated gene.
    • Fig. S5. The relative sensitivity of candidate gene-deficient DT40 cells to the six individual NRTIs and the level of the mRNA expression in ATL cells.
    • Fig. S6. Ectopic expression of TDP1WT, TDP1H263A, and TDP1H493R in MT-2 cells. Fig. S7. ABC and AZT cytotoxicity in MT-2/TDP1WT cells.
    • Fig. S8. CPT11 and veliparib enhance the lethality of ABC on ATL cells.
    • Table S1. Inhibitory concentration (IC50) values of ABC for each cell line.
    • Table S2. Isogenic mutant chicken DT40 cell lines used in this study.
    • Table S3. Characteristics of two ATL patients.
    • Table S4. List of HTLV-1–infected and ATL cell lines.
    • References (48–84)

    Download PDF

    Files in this Data Supplement:

Stay Connected to Science Advances

Navigate This Article