Endogenous HIV-1 Vpr-mediated apoptosis and proteome alteration of human T-cell leukemia virus-1 transformed C8166 cells

Abstract

HIV-1 viral protein R (Vpr) can induce cell cycle arrest and cell death, and may be beneficial in cancer therapy to suppress malignantly proliferative cell types, such as adult T-cell leukemia (ATL) cells. In this study, we examined the feasibility of employing the HIV-vpr gene, via targeted gene transfer, as a potential new therapy to kill ATL cells. We infected C8166 cells with a recombinant adenovirus carrying both vpr and GFP genes (rAd-vpr), as well as the vector control virus (rAd-vector). G(2)/M phase cell cycle arrest was observed in the rAd-vpr infected cells. Typical characteristics of apoptosis were detected in rAd-vpr infected cells, including sub-diploid peak exhibition in DNA content assay, the Hoechst 33342 accumulation, apoptotic body formation, mitochondrial membrane potential and plasma membrane integrity loss. The proteomic assay revealed apoptosis related protein changes, exhibiting the regulation of caspase-3 activity indicator proteins (vimentin and Rho GDP-dissociation inhibitor 2), mitochondrial protein (prohibitin) and other regulatory proteins. In addition, the up-regulation of anti-inflammatory redox protein, thioredoxin, was identified in the rAd-vpr infected group. Further supporting these findings, the increase of caspase 3&7 activity in the rAd-vpr infected group was observed. In conclusion, endogenous Vpr is able to kill HTLV-1 transformed C8166 cells, and may avoid the risks of inducing severe inflammatory responses through apoptosis-inducing and anti-inflammatory activities.

Fig. 1

Functional Vpr expressed endogenously in C8166 cells. a GFP expression in mock-, rAd-vector and rAd-vpr infected groups. Cells with either mock or viral infections were cultured for 24 h. GFP was detected at FL1 channel in flowcytometry and data shown are representative of 3 independent experiments. M1 region shows GFP positive cells. b Representative images of GFP expression in different groups (scale bar = 10 µm), followed by western blotting confirmation of HIV-1 Vpr in C8166 cells. c Cell cycle and cell death of C8166 cells. Cells were stained with propidium iodide and analyzed for DNA content to determine cell populations at 48 and 72 h after mock, rAd-vector and rAd-vpr infection. In the order of increasing DNA content, dead cells are shown in the sub-diploid peaks, G1 phase populations in diploid peaks, S phase cells in the plateau area and G₂/M cells in the polyploidy peaks.

Fig. 2

Mitochondrial membrane potential loss was induced by endogenous Vpr expression in C8166 cells. Cells with mock- and viral infections as well as CPT (0.5 µmol/l) treatment were cultured for 24 h prior to JC-1 staining. a JC-1 aggregate and monomer observation. Images shown are representative observations from 3 independent experiments. Red images indicate the JC-1 aggregate fluorescence from healthy mitochondria, while green images exhibit cytosolic JC-1 monomers. Merged images indicated the co-localization of JC-1 aggregates and monomers. Scale bar = 10 µm. b MFI quantification of JC-1 aggregate in mock-, rAd-vector and rAd-vpr infected groups. c MFI quantification of JC-1 monomer in different groups. d MMP loss assay by FACS. Data are shown as mean ± SEM. One-way-ANOVA analysis, n = 3. ** P<0.01.

Fig. 3

Apoptosis and plasma membrane integrity changes induced by endogenously expressed Vpr in C8166 cells. Mock-, rAd-vector and rAd-vpr infected as well as CPT (0.5 µmol/l) treated cells were cultured for 24 h and subjected to Hoechst 33342 and PI double staining. a Observations of apoptotic bodies and plasma membrane integrity changes. Images shown are representative observations from 3 independent experiments. Green images indicate GFP positive cells, blue images show Hoechst 33342 fluorescence, and red images exhibit PI staining of nucleic acids. Arrow heads indicate condensed DNA and apoptotic bodies. Scale bar = 20 µm. b MFI quantification of Hoechst 33342 staining in mock-, rAd-vector and rAd-vpr infected groups. c FACS analysis of both Hoechst 33342 and PI staining. Data are shown as mean ± SEM. One-way-ANOVA analysis, n = 3. ** P<0.01.

Fig. 4

Two-dimensional electrophoresis analysis of endogenous Vpr induced C8166 cell death. Cells were cultured 48 h after mock or viral infection before lysing. Protein separation of cell lysates obtained by 2DE gels and images were acquired after post-staining with colloidal Coomassie Blue. After analysis and normalization by PDQuest software, images are shown for mock- (a), rAd-vector (b), and rAd-vpr (c) infected C8166 cells. Circled spots with identical spot numbers across gels indicate proteins at the same gel positions. The numbering is also consistent with Table 1, which provides protein identifications generated by mass spectrometry. RT-PCR was performed on the 14-3-3 (d) and GSTP1 (e) proteins, and sample loading was controlled by GAPDH mRNA quantification, respectively.

Fig. 5

Endogenously expressed Vpr induces up-regulation of caspase 3&7 activity in C8166 cells. Mock-, rAd-vector and rAd-vpr infected as well as CPT (0.5 µmol/l) treated cells were cultured for 24 h before being labeled by red FLICA. Images in the upper row show the active caspases 3&7 in the C8166 cells of different groups, while those in the lower row indicate the merged images of GFP, active caspases 3&7 and white field. Scale bar = 20µm.