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The human immunodeficiency virus type 1 Vpr protein and its carboxy-terminally truncated form induce apoptosis in tumor cells.

Nonaka M, Hashimoto Y, Takeshima SN, Aida Y - Cancer Cell Int. (2009)

Bottom Line: We have reported previously that C81, a carboxy-terminally truncated form of Vpr, interferes with cell proliferation and results in apoptosis without G2 arrest.In contrast, Vpr resulted in G2 arrest and apoptosis in HeLa and 293 T cells.Overall, Vpr and C81 have potential as novel therapeutic agents for treatment of cancer.

View Article: PubMed Central - HTML - PubMed

Affiliation: Viral Infectious Diseases Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. mizuho-624.1128@s7.dion.ne.jp

ABSTRACT
The human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr induces apoptosis after cell cycle arrest at the G2 phase in primate cells. We have reported previously that C81, a carboxy-terminally truncated form of Vpr, interferes with cell proliferation and results in apoptosis without G2 arrest. Here, we investigated whether this property of Vpr and C81 could be exploited for use as a potential anticancer agent. First, we demonstrated that C81 induced G1 arrest and apoptosis in all tumor cells tested. In contrast, Vpr resulted in G2 arrest and apoptosis in HeLa and 293 T cells. Vpr also suppressed the damaged-DNA-specific binding protein 1 (DDB1) in HepG2 cells, thereby inducing apoptosis without G2 arrest. G2 arrest was restored when DDB1 was overexpressed in cells that also expressed Vpr. Surprisingly, C81 induced G2 arrest when DDB1 was overexpressed in HepG2 cells, but not in HeLa or 293 T cells. Thus, the induction of Vpr- and C81-mediated cell cycle arrest appears to depend on the cell type, whereas apoptosis was observed in all tumor cells tested. Overall, Vpr and C81 have potential as novel therapeutic agents for treatment of cancer.

No MeSH data available.


Related in: MedlinePlus

Vpr and C81 induce apoptosis in tumor cells. (A) HeLa, HepG2, and 293 T cells were transfected with pME18Neo encoding Flag-tagged wild-type Vpr or C81, or the control pME18Neo-Flag. At 48 h post-transfection, cells were fixed and stained with anti-Flag, followed by Alexa-488-conjugated anti-mouse IgG. Finally, cells were stained with Hoechst 33258 to monitor the nuclear morphology. Apoptotic bodies (arrowheads) were identified using confocal laser scanning microscopy. (B) HeLa, HepG2, and 293 T cells were transfected with the pME18Neo plasmid encoding Flag-tagged wild-type Vpr or C81, or the control pME18Neo-Flag, together with pSV-β-galactosidase. Cells were treated with (filled columns) or without (open columns) inhibitors of caspase-3. At 30 h (293 T), 36 h (HeLa), or 48 h (HepG2) post-transfection, caspase-3 activity was measured and normalized to the β-galactosidase activity. Each column and error bar represents the mean ± SD of measurements from three samples. (C) HeLa, HepG2, and 293 T cells were transfected with pME18Neo encoding Flag-tagged wild-type Vpr or C81, or the control pME18Neo-Flag with or without the GFP expression vector pEGFP-N1. GFP was used as a reporter to discriminate between transfected and untransfected cells. At 47 h (293 T), 38 h (HeLa), or 58 h (HepG2) post-transfection, cells were stained with PE-Annexin V and 7-AAD to identify apoptotic cells, or anti-mouse IgG2b-PE (Nippon BD Company Ltd) and 7-AAD as a negative control. The percentages of Annexin V-positive and 7-AAD negative cells relative to GFP-positive cells indicate the level of Vpr or C81 associated apoptosis.
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Figure 2: Vpr and C81 induce apoptosis in tumor cells. (A) HeLa, HepG2, and 293 T cells were transfected with pME18Neo encoding Flag-tagged wild-type Vpr or C81, or the control pME18Neo-Flag. At 48 h post-transfection, cells were fixed and stained with anti-Flag, followed by Alexa-488-conjugated anti-mouse IgG. Finally, cells were stained with Hoechst 33258 to monitor the nuclear morphology. Apoptotic bodies (arrowheads) were identified using confocal laser scanning microscopy. (B) HeLa, HepG2, and 293 T cells were transfected with the pME18Neo plasmid encoding Flag-tagged wild-type Vpr or C81, or the control pME18Neo-Flag, together with pSV-β-galactosidase. Cells were treated with (filled columns) or without (open columns) inhibitors of caspase-3. At 30 h (293 T), 36 h (HeLa), or 48 h (HepG2) post-transfection, caspase-3 activity was measured and normalized to the β-galactosidase activity. Each column and error bar represents the mean ± SD of measurements from three samples. (C) HeLa, HepG2, and 293 T cells were transfected with pME18Neo encoding Flag-tagged wild-type Vpr or C81, or the control pME18Neo-Flag with or without the GFP expression vector pEGFP-N1. GFP was used as a reporter to discriminate between transfected and untransfected cells. At 47 h (293 T), 38 h (HeLa), or 58 h (HepG2) post-transfection, cells were stained with PE-Annexin V and 7-AAD to identify apoptotic cells, or anti-mouse IgG2b-PE (Nippon BD Company Ltd) and 7-AAD as a negative control. The percentages of Annexin V-positive and 7-AAD negative cells relative to GFP-positive cells indicate the level of Vpr or C81 associated apoptosis.

Mentions: Next, we detected apoptotic cells by monitoring fluorescence after staining the cells with Hoechst 33258. HeLa, HepG2, and 293 T cells were transfected with the pME18Neo empty vector, or the expression plasmids for Vpr or C81. At 48 h post-transfection, cells were fixed and stained with anti-Flag followed by Alexa-488-conjugated anti-mouse IgG. To monitor nuclear morphology, cells were stained with Hoechst 33258. The cells that expressed either Vpr or C81 had condensed chromatin, a hallmark of cells undergoing apoptosis (Fig. 2A). Furthermore, we assessed apoptosis in Vpr- and C81-expressing cells by measuring the activity of caspase-3, which plays an essential role in the induction of apoptosis (Fig. 2B). Caspase-3 activity was significantly higher in the Vpr- and C81-expressing cells compared to the control vector transfected cells. Caspase-3 activity was approximately two-five fold higher than in the cells transfected with the control pME18Neo empty vector. Interestingly, caspase-3 activity was significantly higher in cells that had been transfected with the C81 expression vector, compared to cells transfected with the Vpr expression vector. Conversely, treatment with the caspase-3 specific inhibitor, Z-DEVD-FMK, suppressed the activity of caspase-3 in all transfected cells, including cells transfected with the control pME18Neo empty vector. These results strongly suggested that Vpr and C81 can induce apoptosis in the HepG2 and HeLa cell lines.


The human immunodeficiency virus type 1 Vpr protein and its carboxy-terminally truncated form induce apoptosis in tumor cells.

Nonaka M, Hashimoto Y, Takeshima SN, Aida Y - Cancer Cell Int. (2009)

Vpr and C81 induce apoptosis in tumor cells. (A) HeLa, HepG2, and 293 T cells were transfected with pME18Neo encoding Flag-tagged wild-type Vpr or C81, or the control pME18Neo-Flag. At 48 h post-transfection, cells were fixed and stained with anti-Flag, followed by Alexa-488-conjugated anti-mouse IgG. Finally, cells were stained with Hoechst 33258 to monitor the nuclear morphology. Apoptotic bodies (arrowheads) were identified using confocal laser scanning microscopy. (B) HeLa, HepG2, and 293 T cells were transfected with the pME18Neo plasmid encoding Flag-tagged wild-type Vpr or C81, or the control pME18Neo-Flag, together with pSV-β-galactosidase. Cells were treated with (filled columns) or without (open columns) inhibitors of caspase-3. At 30 h (293 T), 36 h (HeLa), or 48 h (HepG2) post-transfection, caspase-3 activity was measured and normalized to the β-galactosidase activity. Each column and error bar represents the mean ± SD of measurements from three samples. (C) HeLa, HepG2, and 293 T cells were transfected with pME18Neo encoding Flag-tagged wild-type Vpr or C81, or the control pME18Neo-Flag with or without the GFP expression vector pEGFP-N1. GFP was used as a reporter to discriminate between transfected and untransfected cells. At 47 h (293 T), 38 h (HeLa), or 58 h (HepG2) post-transfection, cells were stained with PE-Annexin V and 7-AAD to identify apoptotic cells, or anti-mouse IgG2b-PE (Nippon BD Company Ltd) and 7-AAD as a negative control. The percentages of Annexin V-positive and 7-AAD negative cells relative to GFP-positive cells indicate the level of Vpr or C81 associated apoptosis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Vpr and C81 induce apoptosis in tumor cells. (A) HeLa, HepG2, and 293 T cells were transfected with pME18Neo encoding Flag-tagged wild-type Vpr or C81, or the control pME18Neo-Flag. At 48 h post-transfection, cells were fixed and stained with anti-Flag, followed by Alexa-488-conjugated anti-mouse IgG. Finally, cells were stained with Hoechst 33258 to monitor the nuclear morphology. Apoptotic bodies (arrowheads) were identified using confocal laser scanning microscopy. (B) HeLa, HepG2, and 293 T cells were transfected with the pME18Neo plasmid encoding Flag-tagged wild-type Vpr or C81, or the control pME18Neo-Flag, together with pSV-β-galactosidase. Cells were treated with (filled columns) or without (open columns) inhibitors of caspase-3. At 30 h (293 T), 36 h (HeLa), or 48 h (HepG2) post-transfection, caspase-3 activity was measured and normalized to the β-galactosidase activity. Each column and error bar represents the mean ± SD of measurements from three samples. (C) HeLa, HepG2, and 293 T cells were transfected with pME18Neo encoding Flag-tagged wild-type Vpr or C81, or the control pME18Neo-Flag with or without the GFP expression vector pEGFP-N1. GFP was used as a reporter to discriminate between transfected and untransfected cells. At 47 h (293 T), 38 h (HeLa), or 58 h (HepG2) post-transfection, cells were stained with PE-Annexin V and 7-AAD to identify apoptotic cells, or anti-mouse IgG2b-PE (Nippon BD Company Ltd) and 7-AAD as a negative control. The percentages of Annexin V-positive and 7-AAD negative cells relative to GFP-positive cells indicate the level of Vpr or C81 associated apoptosis.
Mentions: Next, we detected apoptotic cells by monitoring fluorescence after staining the cells with Hoechst 33258. HeLa, HepG2, and 293 T cells were transfected with the pME18Neo empty vector, or the expression plasmids for Vpr or C81. At 48 h post-transfection, cells were fixed and stained with anti-Flag followed by Alexa-488-conjugated anti-mouse IgG. To monitor nuclear morphology, cells were stained with Hoechst 33258. The cells that expressed either Vpr or C81 had condensed chromatin, a hallmark of cells undergoing apoptosis (Fig. 2A). Furthermore, we assessed apoptosis in Vpr- and C81-expressing cells by measuring the activity of caspase-3, which plays an essential role in the induction of apoptosis (Fig. 2B). Caspase-3 activity was significantly higher in the Vpr- and C81-expressing cells compared to the control vector transfected cells. Caspase-3 activity was approximately two-five fold higher than in the cells transfected with the control pME18Neo empty vector. Interestingly, caspase-3 activity was significantly higher in cells that had been transfected with the C81 expression vector, compared to cells transfected with the Vpr expression vector. Conversely, treatment with the caspase-3 specific inhibitor, Z-DEVD-FMK, suppressed the activity of caspase-3 in all transfected cells, including cells transfected with the control pME18Neo empty vector. These results strongly suggested that Vpr and C81 can induce apoptosis in the HepG2 and HeLa cell lines.

Bottom Line: We have reported previously that C81, a carboxy-terminally truncated form of Vpr, interferes with cell proliferation and results in apoptosis without G2 arrest.In contrast, Vpr resulted in G2 arrest and apoptosis in HeLa and 293 T cells.Overall, Vpr and C81 have potential as novel therapeutic agents for treatment of cancer.

View Article: PubMed Central - HTML - PubMed

Affiliation: Viral Infectious Diseases Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. mizuho-624.1128@s7.dion.ne.jp

ABSTRACT
The human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr induces apoptosis after cell cycle arrest at the G2 phase in primate cells. We have reported previously that C81, a carboxy-terminally truncated form of Vpr, interferes with cell proliferation and results in apoptosis without G2 arrest. Here, we investigated whether this property of Vpr and C81 could be exploited for use as a potential anticancer agent. First, we demonstrated that C81 induced G1 arrest and apoptosis in all tumor cells tested. In contrast, Vpr resulted in G2 arrest and apoptosis in HeLa and 293 T cells. Vpr also suppressed the damaged-DNA-specific binding protein 1 (DDB1) in HepG2 cells, thereby inducing apoptosis without G2 arrest. G2 arrest was restored when DDB1 was overexpressed in cells that also expressed Vpr. Surprisingly, C81 induced G2 arrest when DDB1 was overexpressed in HepG2 cells, but not in HeLa or 293 T cells. Thus, the induction of Vpr- and C81-mediated cell cycle arrest appears to depend on the cell type, whereas apoptosis was observed in all tumor cells tested. Overall, Vpr and C81 have potential as novel therapeutic agents for treatment of cancer.

No MeSH data available.


Related in: MedlinePlus