Wortmannin potentiates integrase-mediated killing of lymphocytes and reduces the efficiency of stable transduction by retroviruses - PubMed (original) (raw)

Wortmannin potentiates integrase-mediated killing of lymphocytes and reduces the efficiency of stable transduction by retroviruses

R Daniel et al. Mol Cell Biol. 2001 Feb.

Erratum in

Mol Cell Biol 2001 Apr;21(7):2617

Abstract

Retroviral infection induces integrase-dependent apoptosis in DNA-PK-deficient murine scid lymphocytes. Furthermore, the efficiency of stable transduction of reporter genes is reduced in adherent cell lines that are deficient in cellular DNA-repair proteins known to mediate nonhomologous end joining (NHEJ), such as DNA-PK and XRCC4 (R. Daniel, R. A. Katz, and A. M. Skalka, Science 284:644-647, 1999). Here we report that wortmannin, an irreversible inhibitor of phosphatidylinositol 3-kinase (PI-3K)-related PKs, including the catalytic subunit of DNA-dependent protein kinase (DNA-PK(CS)) and ATM, sensitizes normal murine lymphocytes to retrovirus-mediated cell killing. We also show that the efficiency of stable transduction of reporter genes in human (HeLa) cells, mediated by either an avian sarcoma virus or a human immune deficiency virus type 1 vector, is reduced in the presence of wortmannin. The dose dependence of such reduction correlates with that for inhibition of PI-3K-related protein kinase activity in these cells. Results from wortmannin treatment of a panel of cell lines confirms that formation and/or survival of transductants is dependent on components of the NHEJ pathway. However, stable transduction is virtually abolished by wortmannin treatment of cells that lack ATM. These results suggest that ATM activity is required for the residual transduction observed in the NHEJ-deficient cells. Our studies support the hypothesis that DNA repair proteins of the NHEJ pathway and, in their absence, ATM are required to avoid integrase-mediated killing [corrected] and allow stable retroviral DNA transduction. The studies also suggest that cells can be sensitized to such killing and stable retroviral DNA integration blocked by drugs that inhibit cellular DNA repair pathways.

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Figures

FIG. 1

Effect of infection on the viability of wortmannin-treated, DNA repair-competent pre-B N2 cells. (A) Viability of N2 cells after infection with the IN+ ASV vector. Cells were infected at an MOI of 4 transducing units/cell in the absence of wortmannin (□), in the presence of 0.5 μM wortmannin (▿), or in the presence of 1 μM wortmannin ( formula image ). As a control, cells were mock infected in the absence of wortmannin (●), in the presence of 0.5 μM wortmannin (▴), or in the presence of 1 μM wortmannin (⧫). Cells were harvested at the indicated time points, and viability was measured by trypan blue dye exclusion. An average of two independent counts is shown. (B) Viability of wortmannin-treated N2 cells after infection with the integrase-defective (IN−) virus. Cells were infected with the IN− ASV vector at an MOI of 4 in the absence of wortmannin (●), in the presence of 0.5 μM wortmannin (▴), or in the presence of 1 μM wortmannin (⧫). As a positive control, cells were also infected with the IN+ virus (conditions and symbols as in Fig. 1A). Viability was again measured by trypan blue dye exclusion; two independent counts were made at each time point.

FIG. 2

Effect of infection on viability of wortmannin-treated scid (S33) cells. (A) Viability of scid cells after infection with the IN+ virus. Cells were infected at an MOI of 4 in the absence of wortmannin (□), in the presence of 1 μM wortmannin (▵), or in the presence of 2 μM wortmannin ( formula image ). As a control, cells were mock infected in the absence of wortmannin (●), in the presence of 1 μM wortmannin (▴), or in the presence of 2 μM wortmannin (⧫). Cells were harvested at the indicated times, and viability was measured by trypan blue dye exclusion. An average of two independent counts is shown. (B) Viability of wortmannin-treated S33 cells after infection with the integrase-defective, IN− virus. Cells were infected with the IN− virus at an MOI of 4 in the absence of wortmannin (●), in the presence of 1 μM wortmannin (▴), or in the presence of 2 μM wortmannin (⧫). In addition, cells were infected with the IN+ virus (controls and symbols as in Fig. 2A). Viability was again measured by trypan blue dye exclusion; two independent counts were made at each time point.

FIG. 3

Effect of antisense oligonucleotides on viability of control N2 and scid S33 cells infected with IN+ virus. Cells were mock infected or infected at an MOI of 4 and simultaneously treated with antisense oligonucleotides (final concentration, 10 μM) as indicated. Cells were then counted at 18 h postinfection. (A) Effect of antisense oligonucleotides on the viability of S33 cells. (B) Effect of antisense oligonucleotides on the viability of N2 cells. Open bars, mock-infected cells; shaded bars, IN+ virus-infected cells.

FIG. 4

(A) Retrovirus-mediated transduction in wortmannin-treated HeLa cells. HeLa cells were treated with 0 to 20 μM wortmannin (two dishes with 105 cells/dish for each point) and infected with a dilution of the IN+ virus to an MOI of ∼0.01. On the following day, wortmannin was removed and medium containing G418 at final concentration of 1 mg/ml was added. Resistant colonies were counted 2 weeks postinfection. The colony numbers were 329.5 ± 39 per dish in the absence of wortmannin, 110 ± 25 per dish at 5 μM wortmannin, 38 ± 6 per dish at 10 μM, and 6 ± 1 per dish at 20 μwortmannin. The results were plotted as a percentage of the number of colonies in the absence of wortmannin. (B) DNA-dependent protein kinase activity in wortmannin-treated HeLa cells. Cells were treated overnight with 0 to 10 μM wortmannin; nuclei were then isolated and lysed, and the DNA-dependent kinase activity was measured in the nuclear extracts as described (see Materials and Methods). The kinase activity was stimulated with sheared salmon sperm DNA, and the activity in the absence of salmon sperm DNA was subtracted from each datum point. The results were plotted as a percentage of the activity in the absence of wortmannin which was taken as 100%. (C) Transduction mediated by the HIV-1-based virus. HeLa cells (at 105 per dish) were treated with 0, 10, or 20 μM wortmannin and infected with the HIV-1-based virus vector. Cells were stained for β-galactosidase activity at 1 week postinfection, and stained (blue) colonies were counted on two dishes for each point.

FIG. 5

ATM expression in human and hamster (CHO) lines. Cells were lysed, and either whole-cell lysates (human cells) or immunoprecipitates obtained with ATM antibodies (CHO lines) were probed for ATM expression as described (see Materials and Methods). Cell line names are indicated.

FIG. 6

Cellular response to IN-mediated DNA damage. As noted in the text, possible IN-mediated damage signals include discontinuities in viral DNA or changes in cellular DNA or chromatin structure introduced during integration of the retroviral DNA. We propose that such damage is normally sensed (either directly or indirectly) by DNA-PK, together with other components of the NHEJ DNA repair pathway. The exact manner in which the NHEJ pathway mediates repair is still unknown. Activities might include signaling to other proteins and/or the recruitment of repair proteins. A direct interaction of NHEJ with IN can also not be excluded. Our results indicate that ATM can also respond to IN-mediated damage in the absence of DNA-PK. The manner in which ATM contributes to repair of the DNA damage is also unknown. It may also signal to other proteins and/or recruit repair proteins, or it may block the cell cycle until repair can be affected by another pathway.

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Wortmannin potentiates integrase-mediated killing of lymphocytes and reduces the efficiency of stable transduction by retroviruses - PubMed (original) (raw)