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Serum-dependent transcriptional networks identify distinct functional roles for H-Ras and N-Ras during initial stages of the cell cycle.

Castellano E, Guerrero C, Núñez A, De Las Rivas J, Santos E - Genome Biol. (2009)

Bottom Line: The absence of N-Ras caused significantly higher changes than the absence of H-Ras in the wave of transcriptional activation linked to G0/G1 transition.Mechanistic analysis indicated that extracellular signal-regulated kinase (ERK)-dependent activation of signal transducer and activator of transcription 1 (Stat1) mediates the regulatory effect of N-Ras on defense and immunity, whereas the pro-apoptotic effects of N-Ras are mediated through ERK and p38 mitogen-activated protein kinase signaling.Our observations confirm the notion of an absolute requirement for different peaks of Ras activity during the initial stages of the cell cycle and document the functional specificity of H-Ras and N-Ras during those processes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centro de Investigación del Cáncer, IBMCC (CSIC-USAL), University of Salamanca, Campus Unamuno, 37007 Salamanca, Spain. Esther.Castellano@cancer.org.uk

ABSTRACT

Background: Using oligonucleotide microarrays, we compared transcriptional profiles corresponding to the initial cell cycle stages of mouse fibroblasts lacking the small GTPases H-Ras and/or N-Ras with those of matching, wild-type controls.

Results: Serum-starved wild-type and knockout ras fibroblasts had very similar transcriptional profiles, indicating that H-Ras and N-Ras do not significantly control transcriptional responses to serum deprivation stress. In contrast, genomic disruption of H-ras or N-ras, individually or in combination, determined specific differential gene expression profiles in response to post-starvation stimulation with serum for 1 hour (G0/G1 transition) or 8 hours (mid-G1 progression). The absence of N-Ras caused significantly higher changes than the absence of H-Ras in the wave of transcriptional activation linked to G0/G1 transition. In contrast, the absence of H-Ras affected the profile of the transcriptional wave detected during G1 progression more strongly than did the absence of N-Ras. H-Ras was predominantly functionally associated with growth and proliferation, whereas N-Ras had a closer link to the regulation of development, the cell cycle, immunomodulation and apoptosis. Mechanistic analysis indicated that extracellular signal-regulated kinase (ERK)-dependent activation of signal transducer and activator of transcription 1 (Stat1) mediates the regulatory effect of N-Ras on defense and immunity, whereas the pro-apoptotic effects of N-Ras are mediated through ERK and p38 mitogen-activated protein kinase signaling.

Conclusions: Our observations confirm the notion of an absolute requirement for different peaks of Ras activity during the initial stages of the cell cycle and document the functional specificity of H-Ras and N-Ras during those processes.

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Related in: MedlinePlus

Overlapping of differential gene expression patterns from wild-type and ras knockout fibroblasts after serum stimulation for 1 hour or 8 hours.  (a) Venn diagrams showing number of probesets contained in the intersections among the different lists of differentially expressed genes occurring simultaneously in WT, H-ras-/-, N-ras-/- or H-ras-/-/N-ras-/-fibroblasts after incubation of serum-starved cells in the presence of serum for 1 hour or 8 hours. (b) Venn diagrams showing overlapping among the lists of differentially expressed genes of H-ras-/-, N-ras-/- or H-ras-/-/N-ras-/- fibroblasts generated after excluding from them those loci showing similar values of differential expression (ratio of the R-fold values within the range 0.6 to 1.5) in the corresponding 1-hour or 8-hour WT controls.
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Figure 2: Overlapping of differential gene expression patterns from wild-type and ras knockout fibroblasts after serum stimulation for 1 hour or 8 hours. (a) Venn diagrams showing number of probesets contained in the intersections among the different lists of differentially expressed genes occurring simultaneously in WT, H-ras-/-, N-ras-/- or H-ras-/-/N-ras-/-fibroblasts after incubation of serum-starved cells in the presence of serum for 1 hour or 8 hours. (b) Venn diagrams showing overlapping among the lists of differentially expressed genes of H-ras-/-, N-ras-/- or H-ras-/-/N-ras-/- fibroblasts generated after excluding from them those loci showing similar values of differential expression (ratio of the R-fold values within the range 0.6 to 1.5) in the corresponding 1-hour or 8-hour WT controls.

Mentions: Our initial analysis of the microarray hybridization data generated in this study focused on identifying the loci sharing differential expression among the different genotypes and experimental conditions tested (Figure 2). Figure 2a identifies and quantifies the overlapping of differentially expressed probesets occurring among all the WT, H-ras-/-, N-ras-/- or H-ras-/-/N-ras-/- genotypes analyzed, after 1 hour or 8 hours of serum treatment. On the other hand, in order to better identify the genes whose differential expression is exclusively due to the presence/absence of Ras proteins in the fibroblasts, Figure 2b shows the intersections occurring among the lists of differentially expressed genes for the H-ras-/-, N-ras-/- or H-ras-/-/N-ras-/- genotypes that were generated after excluding from them all the loci showing similar values of differential expression in their corresponding (1 hour or 8 hours) WT controls. Thus, Tables S4, S5 and S6 in Additional data file 1 list, respectively, the individual gene probeset composing the wave of differential expression occurring after 1 hour of serum stimulation in only the H-ras-/-, N-ras-/- or H-ras-/-/N-ras-/- fibroblasts but not in the WT control cells. Similarly, Tables S7, S8 and S9 in Additional data file 1 describe the wave of differentially expressed genes occurring only in H-ras-/-, N-ras-/- or H-ras-/-/N-ras-/- fibroblasts, respectively, but not in WT fibroblasts, after 8 h of serum incubation. To facilitate the detailed analysis of our microarray expression data, all these tables present gene lists categorized according to their degree of overexpression/repression and functional category.


Serum-dependent transcriptional networks identify distinct functional roles for H-Ras and N-Ras during initial stages of the cell cycle.

Castellano E, Guerrero C, Núñez A, De Las Rivas J, Santos E - Genome Biol. (2009)

Overlapping of differential gene expression patterns from wild-type and ras knockout fibroblasts after serum stimulation for 1 hour or 8 hours.  (a) Venn diagrams showing number of probesets contained in the intersections among the different lists of differentially expressed genes occurring simultaneously in WT, H-ras-/-, N-ras-/- or H-ras-/-/N-ras-/-fibroblasts after incubation of serum-starved cells in the presence of serum for 1 hour or 8 hours. (b) Venn diagrams showing overlapping among the lists of differentially expressed genes of H-ras-/-, N-ras-/- or H-ras-/-/N-ras-/- fibroblasts generated after excluding from them those loci showing similar values of differential expression (ratio of the R-fold values within the range 0.6 to 1.5) in the corresponding 1-hour or 8-hour WT controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3091317&req=5

Figure 2: Overlapping of differential gene expression patterns from wild-type and ras knockout fibroblasts after serum stimulation for 1 hour or 8 hours. (a) Venn diagrams showing number of probesets contained in the intersections among the different lists of differentially expressed genes occurring simultaneously in WT, H-ras-/-, N-ras-/- or H-ras-/-/N-ras-/-fibroblasts after incubation of serum-starved cells in the presence of serum for 1 hour or 8 hours. (b) Venn diagrams showing overlapping among the lists of differentially expressed genes of H-ras-/-, N-ras-/- or H-ras-/-/N-ras-/- fibroblasts generated after excluding from them those loci showing similar values of differential expression (ratio of the R-fold values within the range 0.6 to 1.5) in the corresponding 1-hour or 8-hour WT controls.
Mentions: Our initial analysis of the microarray hybridization data generated in this study focused on identifying the loci sharing differential expression among the different genotypes and experimental conditions tested (Figure 2). Figure 2a identifies and quantifies the overlapping of differentially expressed probesets occurring among all the WT, H-ras-/-, N-ras-/- or H-ras-/-/N-ras-/- genotypes analyzed, after 1 hour or 8 hours of serum treatment. On the other hand, in order to better identify the genes whose differential expression is exclusively due to the presence/absence of Ras proteins in the fibroblasts, Figure 2b shows the intersections occurring among the lists of differentially expressed genes for the H-ras-/-, N-ras-/- or H-ras-/-/N-ras-/- genotypes that were generated after excluding from them all the loci showing similar values of differential expression in their corresponding (1 hour or 8 hours) WT controls. Thus, Tables S4, S5 and S6 in Additional data file 1 list, respectively, the individual gene probeset composing the wave of differential expression occurring after 1 hour of serum stimulation in only the H-ras-/-, N-ras-/- or H-ras-/-/N-ras-/- fibroblasts but not in the WT control cells. Similarly, Tables S7, S8 and S9 in Additional data file 1 describe the wave of differentially expressed genes occurring only in H-ras-/-, N-ras-/- or H-ras-/-/N-ras-/- fibroblasts, respectively, but not in WT fibroblasts, after 8 h of serum incubation. To facilitate the detailed analysis of our microarray expression data, all these tables present gene lists categorized according to their degree of overexpression/repression and functional category.

Bottom Line: The absence of N-Ras caused significantly higher changes than the absence of H-Ras in the wave of transcriptional activation linked to G0/G1 transition.Mechanistic analysis indicated that extracellular signal-regulated kinase (ERK)-dependent activation of signal transducer and activator of transcription 1 (Stat1) mediates the regulatory effect of N-Ras on defense and immunity, whereas the pro-apoptotic effects of N-Ras are mediated through ERK and p38 mitogen-activated protein kinase signaling.Our observations confirm the notion of an absolute requirement for different peaks of Ras activity during the initial stages of the cell cycle and document the functional specificity of H-Ras and N-Ras during those processes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centro de Investigación del Cáncer, IBMCC (CSIC-USAL), University of Salamanca, Campus Unamuno, 37007 Salamanca, Spain. Esther.Castellano@cancer.org.uk

ABSTRACT

Background: Using oligonucleotide microarrays, we compared transcriptional profiles corresponding to the initial cell cycle stages of mouse fibroblasts lacking the small GTPases H-Ras and/or N-Ras with those of matching, wild-type controls.

Results: Serum-starved wild-type and knockout ras fibroblasts had very similar transcriptional profiles, indicating that H-Ras and N-Ras do not significantly control transcriptional responses to serum deprivation stress. In contrast, genomic disruption of H-ras or N-ras, individually or in combination, determined specific differential gene expression profiles in response to post-starvation stimulation with serum for 1 hour (G0/G1 transition) or 8 hours (mid-G1 progression). The absence of N-Ras caused significantly higher changes than the absence of H-Ras in the wave of transcriptional activation linked to G0/G1 transition. In contrast, the absence of H-Ras affected the profile of the transcriptional wave detected during G1 progression more strongly than did the absence of N-Ras. H-Ras was predominantly functionally associated with growth and proliferation, whereas N-Ras had a closer link to the regulation of development, the cell cycle, immunomodulation and apoptosis. Mechanistic analysis indicated that extracellular signal-regulated kinase (ERK)-dependent activation of signal transducer and activator of transcription 1 (Stat1) mediates the regulatory effect of N-Ras on defense and immunity, whereas the pro-apoptotic effects of N-Ras are mediated through ERK and p38 mitogen-activated protein kinase signaling.

Conclusions: Our observations confirm the notion of an absolute requirement for different peaks of Ras activity during the initial stages of the cell cycle and document the functional specificity of H-Ras and N-Ras during those processes.

Show MeSH
Related in: MedlinePlus