Adeno-associated virus type 2 Rep78 induces apoptosis through caspase activation independently of p53

Abstract

Adeno-associated virus (AAV) type 2 Rep78 is a multifunctional protein required for AAV DNA replication, integration, and gene regulation. The biochemical activities of Rep78 have been described, but the effects of Rep proteins on the cell have not been characterized. We have analyzed Rep-mediated cytotoxicity. We demonstrated that Rep78 expression is sufficient to induce cell death and disruption of the cell cycle. Cell death was found to be mediated by apoptosis. Rep78 expression resulted in the activation of caspase-3, a terminal caspase directly involved in the execution of cell death. A peptidic inhibitor of caspase-3, Z-Asp-Glu-Val-Asp-fluoromethylketone (Z-DEVD-FMK), abrogated Rep78-induced apoptosis, indicating that Rep78-mediated apoptosis is caspase-3 dependent. Rep78 induced apoptosis in wild-type p53-containing human embryonal carcinoma NT-2 cells and in p53-null promyelocytic human HL-60 cells, indicating that at least one pathway of Rep78-induced apoptosis is p53 independent. Apoptosis was shown to occur during the G(1) and early S phases of the cell cycle. By analyzing the effects of Rep78 mutations on cell viability, the cause of cell death was attributed in part to two biochemical activities of Rep78, DNA binding and ATPase/helicase activity. The endonuclease activity of Rep78 did not contribute to apoptosis induction.

FIG. 1

Rep78 expression is cytotoxic. The viability of pNLS-GFP- or pRep78-GFP-transfected HL-60 cells was determined by PI staining (50 μg/ml) and flow cytometry. Percent viability represents the fraction of cells that excluded PI times 100. The abscissa is the time after electroporation. ■, pNLS-GFP; ●, pRep78-GFP.

FIG. 2

Rep78 induces apoptosis. Shown are representative diagrams of Annexin V staining of pNLS-GFP-transfected (A) or pRep78-GFP-transfected (B) HL-60 cells at 24 h posttransfection. The x axis represent the green fluorescence intensity (NLS-GFP expression in panel A and Rep78-GFP expression in panel B). The fluorescence intensity of Annexin V-PE is represented on the y axis. The populations in the upper quadrants are apoptotic cells, while cells in the lower quadrants are nonapoptotic. Left quadrants are GFP-negative cells, while cells in the right quadrants are those expressing NLS-GFP (A) or Rep78-GFP (B). Cells displayed were gated for the exclusion of ViaProbe (i.e., dead cells). The percentage of Rep78-mediated apoptosis was calculated by division of the percentage of Annexin V-positive, Rep78-GFP-expressing cells by the percentage of the total Rep78-GFP subpopulation [9.3%/(9.3% + 23.6%) = 28.2%]. (C) Time course of induction of apoptosis by Rep78. Data are means ± standard errors of values from three experiments. Percent apoptosis was determined as in described for panel B.

FIG. 2

Rep78 induces apoptosis. Shown are representative diagrams of Annexin V staining of pNLS-GFP-transfected (A) or pRep78-GFP-transfected (B) HL-60 cells at 24 h posttransfection. The x axis represent the green fluorescence intensity (NLS-GFP expression in panel A and Rep78-GFP expression in panel B). The fluorescence intensity of Annexin V-PE is represented on the y axis. The populations in the upper quadrants are apoptotic cells, while cells in the lower quadrants are nonapoptotic. Left quadrants are GFP-negative cells, while cells in the right quadrants are those expressing NLS-GFP (A) or Rep78-GFP (B). Cells displayed were gated for the exclusion of ViaProbe (i.e., dead cells). The percentage of Rep78-mediated apoptosis was calculated by division of the percentage of Annexin V-positive, Rep78-GFP-expressing cells by the percentage of the total Rep78-GFP subpopulation [9.3%/(9.3% + 23.6%) = 28.2%]. (C) Time course of induction of apoptosis by Rep78. Data are means ± standard errors of values from three experiments. Percent apoptosis was determined as in described for panel B.

FIG. 3

Rep78 expression results in activation of caspase-3. pNLS-GFP- or pRep78-GFP transfected HL-60 cells were stained with PI and sorted by flow cytometry for the expression of GFP or Rep78-GFP and exclusion of PI (i.e., living cells) at 12 h posttransfection. Extracts of sorted cells were assayed for caspase-3 activity toward the substrate DEVD-pNA. Caspase-3 activity is proportional to the optical density (OD) at 405 nm. Data are means ± standard errors of values from three experiments.

FIG. 4

The synthetic peptide inhibitor Z-DEVD-FMK reduced levels of Rep78-induced apoptosis. HL-60 cells were transfected with pNLS-GFP or pRep78-GFP. The caspase-3 inhibitor Z-DEVD-FMK (100 μM) was added 1 h after transfection and was present throughout the experiment. Cells were analyzed for apoptosis by Annexin V staining and flow cytometry. Data are means ± standard errors of values from three experiments. ■, pNLS-GFP; □, pNLS-GFP plus Z-DEVD-FMK; ●, pRep78-GFP; ○, pRep78-GFP plus Z-DEVD-FMK.

FIG. 5

Rep78 expression results in an accumulation of cells in the G₁ phase of the cell cycle. Twelve hours after transfection of HL-60 cells with pNLS-GFP (A) or pRep78-GFP (B), the cell cycle distribution was analyzed by staining for DNA content with the dye Hoechst 33342. Diagrams on the right show cells [GFP(+)] which have been gated for GFP expression (i.e., NLS-GFP in panel A and Rep78-GFP in panel B, whereas those on the left [GFP(−)] show data for GFP-negative cells. Dead cells detected by ViaProbe staining were excluded from this analysis.

FIG. 6

Rep78-induced apoptosis is G₁/early-S-phase specific. HL-60 cells were transfected with pRep78-GFP. Twelve hours after transfection, cells were analyzed for DNA content by staining with the dye Hoechst 33342 (x axis) and for apoptosis by Annexin V staining (y axis). Cells analyzed were gated for expression of Rep78-GFP [GFP(+); right panel) or nonexpression [GFP(−); left panel). Cells in the left quadrants of the panels are in G₁ or early S phase, and cells in the right quadrants are in late S or G₂/M phase of the cell cycle. The upper quadrants of each panel represent apoptotic cells, while the lower quadrants show nonapoptotic cells. Dead cells were excluded from this analysis by costaining with ViaProbe. Data shown are for cells which had been gated for size, granularity, and exclusion of ViaProbe.

FIG. 7

Mutation of the ATP binding site of Rep78 reduces cytotoxicity. The viability of pNLS-GFP-, pRep78-GFP-, pRep78(Y156F)-GFP-, or pRep78(K340H)-GFP-transfected HL-60 cells was assayed by PI staining (50 μg/ml) and flow cytometry. Data are means ± standard errors of values from three experiments. ■, pNLS-GFP; ●, pRep78-GFP; ○, pRep78(Y156F)-GFP; ⧫, pRep78(K340H)-GFP; ▴, pRep52-GFP.

FIG. 8

Rep78 induces apoptosis in NT2 cells. NT2 cells were transfected with pNLS-GFP or pRep78-GFP and analyzed for apoptosis by Annexin V staining. (A) Representative data for cells at 24 h posttransfection. The x axis represents the green fluorescence intensity (i.e., NLS-GFP or Rep78-GFP). The fluorescence intensity of Annexin V-PE is represented on the y axis. The populations in the upper quadrants show apoptotic cells, while cells in the lower quadrants are nonapoptotic. Cells were gated for the exclusion of ViaProbe (i.e., dead cells). (B) Time course of the experiment shown in panel A.

FIG. 8

Rep78 induces apoptosis in NT2 cells. NT2 cells were transfected with pNLS-GFP or pRep78-GFP and analyzed for apoptosis by Annexin V staining. (A) Representative data for cells at 24 h posttransfection. The x axis represents the green fluorescence intensity (i.e., NLS-GFP or Rep78-GFP). The fluorescence intensity of Annexin V-PE is represented on the y axis. The populations in the upper quadrants show apoptotic cells, while cells in the lower quadrants are nonapoptotic. Cells were gated for the exclusion of ViaProbe (i.e., dead cells). (B) Time course of the experiment shown in panel A.