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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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Microarray analysis of differential gene expression in wild-type and knockout fibroblasts (H-ras-/-, N-ras-/- and H-ras-/-/N-ras-/-) subjected to serum starvation or stimulation.  Graphical representation of numbers of probesets showing differential gene expression in pair-wise SAM comparisons between the microarray hybridization data of WT fibroblasts that were serum-starved for 24 hours (Control) and corresponding microarray hybridization data of fibroblasts of the indicated WT and ras knockout genotypes obtained before (0 h) or after short-term (1 h) or mid-term (8 h) post-starvation incubation of the cultures in the presence of 20% fetal bovine serum (FBS). Four independent microarray hybridizations were performed for all conditions involving WT samples, and at least three independent hybridizations were performed with RNA of each of the different knockout genotypes analyzed. Numbers shown indicate the amount of induced (red) or repressed (green), differentially expressed probesets that were identified in each case using a stringent false discovery rate cut-off parameter value of 0.09.
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Figure 1: Microarray analysis of differential gene expression in wild-type and knockout fibroblasts (H-ras-/-, N-ras-/- and H-ras-/-/N-ras-/-) subjected to serum starvation or stimulation. Graphical representation of numbers of probesets showing differential gene expression in pair-wise SAM comparisons between the microarray hybridization data of WT fibroblasts that were serum-starved for 24 hours (Control) and corresponding microarray hybridization data of fibroblasts of the indicated WT and ras knockout genotypes obtained before (0 h) or after short-term (1 h) or mid-term (8 h) post-starvation incubation of the cultures in the presence of 20% fetal bovine serum (FBS). Four independent microarray hybridizations were performed for all conditions involving WT samples, and at least three independent hybridizations were performed with RNA of each of the different knockout genotypes analyzed. Numbers shown indicate the amount of induced (red) or repressed (green), differentially expressed probesets that were identified in each case using a stringent false discovery rate cut-off parameter value of 0.09.

Mentions: To ascertain whether or not the different members of the Ras family control the expression of specific gene sets in response to the absence or presence of serum in cell cultures, we used commercial oligonucleotide microarrays to compare the genomic expression profile of serum-starved or serum-treated, WT, immortalized fibroblasts with those of similarly treated fibroblasts derived from knockout mice harboring single- or double- mutations for the H-ras and N-ras loci (H-ras-/-, N-ras-/-, H-ras-/-/N-ras-/-). For this purpose, we analyzed representative RNA samples extracted from cell cultures of the mentioned WT and ras knockout genotypes that had been subjected to 24 hours of serum deprivation (Figure 1, 0 h), or to incubation in the presence of serum for 1 hour or 8 hours after the previous 24-hour starvation period (Figure 1, 1 h or 8 h). The results from microarray hybridizations corresponding to cell cultures subjected to serum starvation for 24 hours were instrumental to characterize the transcriptional profile of non-proliferating, off-cycle fibroblasts arrested in G0 because of the absence of growth factors caused by serum withdrawal from the cultures. Addition of serum to the starved (G0) cell cultures causes re-entry of the growth-arrested cells into the cell cycle, thus starting progression through G1 in a process involving an absolute requirement for the participation of Ras proteins [37,39,42]. In this regard, the transcriptional profiles corresponding to cell cultures incubated in the presence of serum for a short period (1 hour) are expected to include loci belonging to the population of immediate early (IE) genes known to be expressed immediately after exposure of serum-depleted fibroblasts to growth factors or serum [43-47]. On the other hand, the transcriptional profiles corresponding to cell cultures incubated in the presence of serum for 8 hours represent the transcriptomic pattern associated with the early stages of G1 progression known to lead to entry into S phase after Rb phosphorylation and subsequent E2F-dependent transcriptional activation [48].


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)

Microarray analysis of differential gene expression in wild-type and knockout fibroblasts (H-ras-/-, N-ras-/- and H-ras-/-/N-ras-/-) subjected to serum starvation or stimulation.  Graphical representation of numbers of probesets showing differential gene expression in pair-wise SAM comparisons between the microarray hybridization data of WT fibroblasts that were serum-starved for 24 hours (Control) and corresponding microarray hybridization data of fibroblasts of the indicated WT and ras knockout genotypes obtained before (0 h) or after short-term (1 h) or mid-term (8 h) post-starvation incubation of the cultures in the presence of 20% fetal bovine serum (FBS). Four independent microarray hybridizations were performed for all conditions involving WT samples, and at least three independent hybridizations were performed with RNA of each of the different knockout genotypes analyzed. Numbers shown indicate the amount of induced (red) or repressed (green), differentially expressed probesets that were identified in each case using a stringent false discovery rate cut-off parameter value of 0.09.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Microarray analysis of differential gene expression in wild-type and knockout fibroblasts (H-ras-/-, N-ras-/- and H-ras-/-/N-ras-/-) subjected to serum starvation or stimulation. Graphical representation of numbers of probesets showing differential gene expression in pair-wise SAM comparisons between the microarray hybridization data of WT fibroblasts that were serum-starved for 24 hours (Control) and corresponding microarray hybridization data of fibroblasts of the indicated WT and ras knockout genotypes obtained before (0 h) or after short-term (1 h) or mid-term (8 h) post-starvation incubation of the cultures in the presence of 20% fetal bovine serum (FBS). Four independent microarray hybridizations were performed for all conditions involving WT samples, and at least three independent hybridizations were performed with RNA of each of the different knockout genotypes analyzed. Numbers shown indicate the amount of induced (red) or repressed (green), differentially expressed probesets that were identified in each case using a stringent false discovery rate cut-off parameter value of 0.09.
Mentions: To ascertain whether or not the different members of the Ras family control the expression of specific gene sets in response to the absence or presence of serum in cell cultures, we used commercial oligonucleotide microarrays to compare the genomic expression profile of serum-starved or serum-treated, WT, immortalized fibroblasts with those of similarly treated fibroblasts derived from knockout mice harboring single- or double- mutations for the H-ras and N-ras loci (H-ras-/-, N-ras-/-, H-ras-/-/N-ras-/-). For this purpose, we analyzed representative RNA samples extracted from cell cultures of the mentioned WT and ras knockout genotypes that had been subjected to 24 hours of serum deprivation (Figure 1, 0 h), or to incubation in the presence of serum for 1 hour or 8 hours after the previous 24-hour starvation period (Figure 1, 1 h or 8 h). The results from microarray hybridizations corresponding to cell cultures subjected to serum starvation for 24 hours were instrumental to characterize the transcriptional profile of non-proliferating, off-cycle fibroblasts arrested in G0 because of the absence of growth factors caused by serum withdrawal from the cultures. Addition of serum to the starved (G0) cell cultures causes re-entry of the growth-arrested cells into the cell cycle, thus starting progression through G1 in a process involving an absolute requirement for the participation of Ras proteins [37,39,42]. In this regard, the transcriptional profiles corresponding to cell cultures incubated in the presence of serum for a short period (1 hour) are expected to include loci belonging to the population of immediate early (IE) genes known to be expressed immediately after exposure of serum-depleted fibroblasts to growth factors or serum [43-47]. On the other hand, the transcriptional profiles corresponding to cell cultures incubated in the presence of serum for 8 hours represent the transcriptomic pattern associated with the early stages of G1 progression known to lead to entry into S phase after Rb phosphorylation and subsequent E2F-dependent transcriptional activation [48].

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