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Daunorubicin-induced variations in gene transcription: commitment to proliferation arrest, senescence and apoptosis.

ABSTRACT: We used a human cDNA macroarray containing various oncogenes and tumour suppressor genes to assess gene expression profiles in early-passage Jurkat T lymphocytes treated with clinically relevant concentrations of the antitumour antibiotic daunorubicin. Several oncogenes and tumour suppressor genes were either up- or down-regulated depending on the daunorubicin concentration used. The expression levels of some of these genes were confirmed by semi-quantitative reverse transcriptase-PCR. We also compared the changes in cell-cycle distribution and the apoptotic morphological characteristics of the cells treated with daunorubicin, using flow cytometry and fluorescence microscopy. Exposure to 182 nM daunorubicin (its IC(75) in Jurkat T cells: where IC(75) is the drug concentration that inhibits growth by 75%) resulted in cell-cycle arrest in G(1) and almost immediate apoptosis. In contrast, decreasing the drug concentration to 91 nM (close to the IC(50)) caused G(2) arrest and cell senescence-like growth arrest, whereas features of apoptosis and necrosis appeared only after longer incubation times. Gene expression profiles, cell-cycle distribution, the presence of DNA damage and the time-dependent response of Jurkat T cells to cell death were correlated clearly. The general behaviour of the genes suggests that cell-cycle arrest and cell death follow distinct pathways depending on drug concentration.

SUBMITTER: Mansilla S

PROVIDER: S-EPMC1223450 | biostudies-literature | 2003 Jun

REPOSITORIES: biostudies-literature

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Daunorubicin-induced variations in gene transcription: commitment to proliferation arrest, senescence and apoptosis.

Mansilla Sylvia S Piña Benjamin B Portugal José J

The Biochemical journal 20030601 Pt 3

We used a human cDNA macroarray containing various oncogenes and tumour suppressor genes to assess gene expression profiles in early-passage Jurkat T lymphocytes treated with clinically relevant concentrations of the antitumour antibiotic daunorubicin. Several oncogenes and tumour suppressor genes were either up- or down-regulated depending on the daunorubicin concentration used. The expression levels of some of these genes were confirmed by semi-quantitative reverse transcriptase-PCR. We also c ...[more]

PMID: 12656675

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Project description:Vpr is an accessory protein of human immunodeficiency virus type 1 (HIV-1) with multiple functions. The induction of G2 arrest by Vpr plays a particularly important role in efficient viral replication because the transcriptional activity of the HIV-1 long terminal repeat is most active in G2 phase. The regulation of apoptosis by Vpr is also important for immune suppression and pathogenesis during HIV infection. However, it is not known whether Vpr-induced apoptosis depends on the ability of Vpr to induce G2 arrest, and the dynamics of Vpr-induced G2 arrest and apoptosis have not been visualized. We performed time-lapse imaging to examine the temporal relationship between Vpr-induced G2 arrest and apoptosis using HeLa cells containing the fluorescent ubiquitination-based cell cycle indicator2 (Fucci2). The dynamics of G2 arrest and subsequent long-term mitotic cell rounding in cells transfected with the Vpr-expression vector were visualized. These cells underwent nuclear mis-segregation after prolonged mitotic processes and then entered G1 phase. Some cells subsequently displayed evidence of apoptosis after prolonged mitotic processes and nuclear mis-segregation. Interestingly, Vpr-induced apoptosis was seldom observed in S or G2 phase. Likewise, visualization of synchronized HeLa/Fucci2 cells infected with an adenoviral vector expressing Vpr clearly showed that Vpr arrests the cell cycle at G2 phase, but does not induce apoptosis at S or G2 phase. Furthermore, time-lapse imaging of HeLa/Fucci2 cells expressing SCAT3.1, a caspase-3-sensitive fusion protein, clearly demonstrated that Vpr induces caspase-3-dependent apoptosis. Finally, to examine whether the effects of Vpr on G2 arrest and apoptosis were reversible, we performed live-cell imaging of a destabilizing domain fusion Vpr, which enabled rapid stabilization and destabilization by Shield1. The effects of Vpr on G2 arrest and subsequent apoptosis were reversible. This study is the first to characterize the dynamics of the morphological changes that occur during Vpr-induced G2 arrest and apoptosis.

Dataset Information

Daunorubicin-induced variations in gene transcription: commitment to proliferation arrest, senescence and apoptosis.

Publications

Daunorubicin-induced variations in gene transcription: commitment to proliferation arrest, senescence and apoptosis.

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