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Abstract
A Ha-ras transformant '7-4', derived from mouse NIH/3T3 fibroblasts, was used to study the relationship between overexpression of activated Ha-ras and cell apoptosis. This cell line contains an inducible Ha-rasVal12 oncogene, which was under the regulation of the Escherichia coli (E. coli) lac operator/repressor system. We demonstrate that overexpression of activated Ha-ras oncogene by exogenous isopropyl-beta-D-thiogalactoside (IPTG) under serum-depleted conditions can stimulate cell apoptosis. Cell cycle analysis showed that most of the 7-4 cells with Ha-ras overexpression accumulated at S-phase and that the expression level of p34cdc2 kinase was decreased, suggesting that p34cdc2 may be involved in 7-4 cell apoptosis. Overexpression of bcl-2 transgene in these cells blocked Ha-ras-induced apoptosis, and this blockage was confirmed downstream of Ha-ras gene expression. Cycloheximide blocked the apoptosis of 7-4 cells in a dose-dependent manner, indicating that specific protein regulating apoptosis may be synthesized through Ha-ras overexpression. Ha-ras overexpression-triggered apoptosis was also prevented in the 7-4 derivatives that express either dominant-negative rasAsn17 or dominant-negative raf-1C4B to suppress Ha-ras signal transduction at different stages, indicating that overexpression of activated Ha-ras can induce cell apoptosis and that raf-1 pathway activity is required for this process.
Highlights
Ha-ras transgene was overexpressed in the transformants in the presence of IPTG under serum-deprived conditions
The activated Ha-ras oncogene in these two cell lines can be overexpressed by IPTG induction under normal conditions (Liu et al, 1992)
To ensure that the Ha-ras oncogene could be overexpressed in the cells by IPTG induction under serum-deprived
Summary
Mouse fibroblast NIH/3T3 cells and their transgenic derivatives (7-4, 7-4-2 and dominant-negative ras and raf-l cells) were maintained in a-modified Eagle medium (a-MEM; Gibco-BRL, USA) containing 10% calf serum (Gibco) and incubated at 37°C in a carbon dioxide incubator. IPTG (Gold biotechnology, USA), a non-metabolizable lactose analogue, was added to induce expression of Ha-ras transgene in 7-4 and 7-4-2 cells. The cells were maintained in 0.2% calf serum-containing medium in the absence or presence of 2.5 mM. The probes used were an [a-32P]dCTP-labelled 4-kb BamHl fragment of Ha-ras DNA from plasmid pSVlacOras and a 2-kb BamHl fragment of f-actin DNA from plasmid pHFBA-1 (Liu et al, 1992). The probes used were an [a-32P]dCTP-labelled 4-kb BamHl fragment of Ha-ras DNA from plasmid pSVlacOras and a 2-kb BamHl fragment of f-actin DNA from plasmid pHFBA-1 (Liu et al, 1992). f-Actin is used as an internal control
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