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Cell Cycle Arrest and Apoptosis

a cell cycle and cell cycle technology, applied in the field of biotechnology, can solve the problems of inability to extrapolate the findings relating to the cell cycle, inability to understand the mechanism by which vpr induces apoptosis, and inability to activate the proapoptotic signaling cascad

Inactive Publication Date: 2012-05-17
PLANELLES VICENTE +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is not possible to extrapolate the findings relating to cell cycle arrest to apoptosis, as the pathways do not completely overlap or follow one from the other.
However, the mechanism by which Vpr induces apoptosis was not understood.
However, the initial event induced by Vpr towards activation of the proapoptotic signaling cascade was not elucidated.

Method used

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  • Cell Cycle Arrest and Apoptosis
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  • Cell Cycle Arrest and Apoptosis

Examples

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example i

[0085]Rad17 is necessary for Vpr-mediated G2 arrest. To examine the role of Rad17 in Vpr-mediated G2 arrest, RNA interference was used to reduce endogenous Rad17 levels. Transfected siRNA duplex oligonucleotides targeted at Rad17 mRNA (54) were used to knock down Rad17, in parallel, siRNA with a nonspecific target sequence and mock transfection was used as controls. In these experiments, endogenous Rad17 protein levels were reduced by approximately 85%, relative to those of mock-transfected or nonspecific siRNA-transfected cells (FIG. 1A). Following transfection, cells were transduced with lentivirus vectors expressing either Vpr and GFP cDNAs separated by an internal ribosome entry site (pHR-Vpr) or GFP alone (pHRGFP) (40) (79). At 48 h after transduction, cells were analyzed by flow cytometry for infection efficiency and DNA content, as reported by GFP expression and propidium iodide staining, respectively. Cell cycle distributions of the various experimental cell populations were...

example ii

[0086]Hus1 is necessary for Vpr-mediated G2 arrest. Finding that Rad17 is necessary for Vpr-induced G2 arrest, another constituent of the ATR signaling pathway, Hus1, was examined. To examine the role of Hus1 in Vpr-induced G2 arrest, Hus1− / − p21− / − mouse embryonic fibroblasts (49) were used. Both Hus1− / − p21− / − cells and Hus1+ / + p21− / − cells exhibited normal cell cycle distributions when mock infected or infected with pHR-GFP (FIG. 2). Hus1− / − p21− / − cells, how however, failed to arrest in G2 after infection with pHR-Vpr, whereas their Hus1+ / + counterparts exhibited robust G2 arrest (FIG. 2). These experiments illustrate a requirement for Hus1 in Vpr-induced G2 arrest.

[0087]Taken together, the observations indicate that HIV-1 Vpr activates the G2 checkpoint in a manner that is mechanistically similar to that of certain genotoxic agents (specifically, HU) that cause replication inhibition. Because recognition of DNA damage via ATR can lead to dramatic cellular changes, other than ch...

example iii

[0088]Vpr expression induces γ-H2AX and BRCA1 focus formation. It was possible that Vpr, through ATR activation, would induce γ-H2AX (9) and. BRCA1 (45) focus formation. To test this hypothesis, HeLa cells were infected with pHR-Vpr or pHR-GFP and, 48 h later, immunostained with γ-H2AX- or BRCA1-specific antibodies. As a positive control, nontransduced cells were treated for 1 h with 10 mM HU 10 min prior to immunostaining (46). Samples then were visualized by fluorescence scanning confocal microscopy (FIGS. 3A and B). Cells with multiple (10 or more), intense nuclear foci were manually counted. These quantitations are presented in FIG. 3C. Approximately 93 or 69% of Vpr-expressing cells exhibited significant γ-H2AX or BRCA1 focal staining, respectively, whereas only 9 or 16% of pHR-GFPinfected cells exhibited any γ-H2AX or BRCA1 foci, respectively. Less than 8% of mock-infected cells exhibited γ-H2AX or BRCA1 foci. Approximately 94 or 65% of HU-treated cells exhibited γ-H2AX or BRC...

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Abstract

The HIV-1 accessory gene vpr encodes a conserved 96-amino acid protein that is necessary and sufficient for the HIV-1-induced block of cellular proliferation and induction of apoptosis. Expression of vpr in CD4.sup.+ lymphocytes results in G2 arrest, followed by apoptosis. ATR, as a cellular factor that mediates Vpr-induced cell cycle arrest, is required for activation of the Breast Cancer-Associated Protein-1 (BRCA1). In addition, the Growth Arrest and DNA Damage protein (GADD45) is upregulated by Vpr in an ATR-dependent manner. Posttranscriptional silencing of either ATR or GADD45 leads to nearly complete suppression of the pro-apoptotic and / or cell cycle arrest effect of Vpr.

Description

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT[0001]Work described herein was supported in part by National Institute of Health Grant Nos. AI49057 and AI054188. The United States Government may have certain rights in the invention.TECHNICAL FIELD[0002]This invention relates generally to biotechnology, more particularly, to compounds, compositions, and / or methods useful in the regulation of apoptosis and / or G2 cell cycle arrest.BACKGROUND[0003]Human immunodeficiency virus type 1 (HIV-1) has four genes, vif, vpr, vpu, and nef, termed “accessory genes,” that are dispensable for viral replication in vitro (14). Many important functions related to HIV-1 pathogenesis have been ascribed to these accessory genes. Specifically, Vpr has been implicated in long terminal repeat transactivation, nuclear import of the preintegration complex, induction of G2 cell cycle arrest, and apoptosis. Recent studies identifying single amino acid changes in Vpr in a cohort of HIV-1-infected ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/16A61P43/00C40B30/04A61P3/04C07K19/00C12N5/071C12N15/113
CPCA61K35/13A61K38/10G01N2510/00C12N2310/14G01N33/5008C12N15/113A61P3/04A61P31/00A61P43/00
Inventor PLANELLES, VICENTEANDERSEN, JOSHUA L.ZIMMERMAN, ERIK
Owner PLANELLES VICENTE
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