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Comparative Study
. 1999 Oct 12;96(21):12039-43.
doi: 10.1073/pnas.96.21.12039.

Lentiviral delivery of HIV-1 Vpr protein induces apoptosis in transformed cells

S A Stewart  1 , B Poon, J B Jowett, Y Xie, I S Chen
Affiliations
Comparative Study

Lentiviral delivery of HIV-1 Vpr protein induces apoptosis in transformed cells

S A Stewart et al. Proc Natl Acad Sci U S A. .

Abstract

Most current anticancer therapies act by inducing tumor cell stasis followed by apoptosis. HIV-1 Vpr effectively induces apoptosis of T cells after arrest of cells at a G(2)/M checkpoint. Here, we investigated whether this property of Vpr could be exploited for use as a potential anticancer agent. As a potentially safer alternative to transfer of genes encoding Vpr, we developed a method to efficiently introduce Vpr protein directly into cells. Vpr packaged into HIV-1 virions lacking a genome induced efficient cell cycle arrest and apoptosis. Introduction of Vpr into tumor cell lines of various tissue origin, including those bearing predisposing mutations in p53, XPA, and hMLH1, induced cell cycle arrest and apoptosis with high efficiency. Significantly, apoptosis mediated by virion-associated Vpr was more effective on rapidly dividing cells compared with slow-growing cells, thus, in concept, providing a potential differential effect between some types of tumor cells and surrounding normal cells. This model system provides a rationale and proof of concept for the development of potential cancer therapeutic agents based on the growth-arresting and apoptotic properties of Vpr.

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Figures

Figure 1
Figure 1
Virion-associated Vpr is sufficient to induce apoptosis. (a) HeLa cells (5 × 104) were infected and analyzed by annexin V staining at 24, 48, and 72 h postinfection. Concurrent Thy 1.2 analysis revealed that 66.2% and 81.7% of cells were infected with HR’Thy(Vpr−) and HR’Thy(Vpr+), respectively. The y-axis represents the percent of cells that are annexin V-positive and 7AAD-negative and thus undergoing apoptosis. Results are for mock (⧫)-, HR’Thy(Vpr−) (■)-, and HR’Thy(Vpr+) (▴)-infected cells. The data shown are representative of eight independent experiments. (b) The percent of dead cells in the cultures was determined at the same time as the cells were analyzed for annexin V staining. The y-axis represents the cumulative number of dead cells within the culture. Symbols are as described for a.
Figure 2
Figure 2
Vpr is packaged into virions. (a) Concentrated virus preparations were analyzed for the presence of Gag. Blots were probed with a human hyperimmune sera. The Gag species, p24 and p17, are indicated by arrows. (b) Concentrated virus preparations were subject to Western blot analysis and probed with an anti-Vpr antibody. pHR’Vpr produces wild-type Vpr and Vpr containing an N-terminal influenza hemagglutinin tag caused by the presence of a second initiation codon before Vpr. The protein doublet in the HR’Vpr lane is caused by the detection of both forms by the anti-Vpr sera. The arrow indicates wild-type Vpr.
Figure 3
Figure 3
Vpr packaged into an empty virion is sufficient to induce apoptosis. (a) 5 × 103 infected HeLa cells were analyzed by annexin V staining at 24, 48, and 72 h postinfection. DNA analysis at 48 h postinfection revealed that 27.7%, 31.0%, 80.6%, 95.9%, 27.7%, and 85.9% of mock-, HR’Thy(Vpr−)-, HR’Thy(Vpr+)-, HR’Vpr-, RNA-(Vpr−)- and RNA-(Vpr+)-infected cells, respectively, were in the G2 phase of the cell cycle, indicating the presence of 1.6 × 104 to 4.8 × 104 infectious units per infection. Results are for mock (⧫)-, HR’Thy(Vpr−) (■)-, HR’Thy(Vpr+) (▴)-, HR’Vpr (×)-, RNA-(Vpr−) (□)-, and RNA-(Vpr+) (●)-infected cells. The data shown are representative of four independent experiments. (b) The percent of dead cells in the cultures was determined at the same time as the cells were analyzed for annexin V staining. Symbols are as described for a.
Figure 4
Figure 4
Virion-associated Vpr results in more cell death than γ-radiation. (a) HeLa cells (5 × 104) were infected with 1.7 × 104 infectious units or irradiated with 4,000 rads of γ-irradiation, and analyzed for DNA content at 24 h postinfection. Relative cell numbers are indicated on the y-axis, and DNA content is indicated on the x-axis. Percent of cells in G2 is indicated at the upper right of each panel. (b) 5 × 103 HeLa cells were analyzed for annexin V staining at 24, 48, and 72 h postinfection. Results are for mock (⧫)-, HR’Thy(Vpr−) (■)-, HR’Thy(Vpr+) (▴)-infected cells, and γ-irradiated (×) cells. (c) The percent of dead cells in the cultures was determined at the same time as the cells were analyzed for annexin V staining. Symbols are as described for b.
Figure 5
Figure 5
Vpr preferentially kills rapidly dividing cells. (a) Human fibroblasts were grown in Iscove’s plus 1%-depleted FCS or 10% FCS. Cells (2 × 104) were plated on day 0 and counted on days 1, 3, 5, 7, and 9. (b) Normal human fibroblasts were grown in 10% FCS and mock-infected (⧫) or infected with HIV-1NL4–3Thyenv(-) (■), HR’Thy(Vpr−) (▴), or HR’Thy(Vpr+) (×). At 144 h postinfection, infection with HIV-1NL4–3Thyenv(-), HR’Thy(Vpr−), and HR’Thy(Vpr+) resulted in 63%, 66%, and 71% Thy 1.2-positive cells, respectively. At 72, 144, and 216 h postinfection, Thy 1.2-positive cells were analyzed for annexin V staining. (c) Normal human fibroblasts were grown in 1% depleted FCS and mock-infected (⧫) or infected with HIV-1NL4–3Thyenv(-) (■), HR’Thy (Vpr−) (▴), or HR’Thy(Vpr+) (×). At 144 h postinfection, infection with HIV-1NL4–3Thyenv(-), HR’Thy(Vpr−), and HR’Thy(Vpr+) resulted in 48%, 65%, and 82% Thy 1.2-positive cells, respectively. At 72, 144, and 216 h postinfection, Thy 1.2-positive cells were analyzed for annexin V staining.
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