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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

Increased caspase 8 and 9 activation in N-ras-/-and H-ras-/-/N-ras-/- fibroblasts. Caspase 8 and 9 activities were measured as described in Materials and methods in WT, N-ras-/- and H-ras-/-/N-ras-/- cell lines that had been subjected to different culture conditions, including, as indicated, serum starvation for 24 hours or subsequent stimulation with serum for 1 hour or 8 hours. Three separate determinations were performed where all individual assays were carried out in triplicate. All values were normalized against their respective N-ras WT controls. Error bars indicate standard deviation (*P < 0.05; **P < 0.01).
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Figure 8: Increased caspase 8 and 9 activation in N-ras-/-and H-ras-/-/N-ras-/- fibroblasts. Caspase 8 and 9 activities were measured as described in Materials and methods in WT, N-ras-/- and H-ras-/-/N-ras-/- cell lines that had been subjected to different culture conditions, including, as indicated, serum starvation for 24 hours or subsequent stimulation with serum for 1 hour or 8 hours. Three separate determinations were performed where all individual assays were carried out in triplicate. All values were normalized against their respective N-ras WT controls. Error bars indicate standard deviation (*P < 0.05; **P < 0.01).

Mentions: Two major pathways regulate apoptosis induction in mammalian cells. In the extrinsic pathway, apoptosis is induced through specialized surface receptors such as FAS or tumor necrosis factor-α [72,73], whereas in the intrinsic pathway, this process is mainly induced through release of mitochondrial pro-apoptotic factors [72,74]. Our proteomic data showed increased expression of proteins involved in both the intrinsic (Bax, p53) and extrinsic (Casp8, FAS) pathways, together with some effector caspases and Bid, which connect both pathways. We confirmed these data and checked the functionality of both apoptotic pathways by measuring Casp8 (extrinsic pathway) and Casp9 (intrinsic pathway) activity in N-ras-/- and H-ras-/-/N-ras-/- fibroblasts (Figure 8). These assays showed increased activity of both caspases in the knockout cell lines compared to the WT controls and did not show predominance of either pathway in our ras knockout cell lines. All together, these results support our genomic and proteomic data and demonstrate an increase in the apoptotic response associated with the absence of N-Ras in N-ras-/- and H-ras-/-/N-ras-/- fibroblasts.


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)

Increased caspase 8 and 9 activation in N-ras-/-and H-ras-/-/N-ras-/- fibroblasts. Caspase 8 and 9 activities were measured as described in Materials and methods in WT, N-ras-/- and H-ras-/-/N-ras-/- cell lines that had been subjected to different culture conditions, including, as indicated, serum starvation for 24 hours or subsequent stimulation with serum for 1 hour or 8 hours. Three separate determinations were performed where all individual assays were carried out in triplicate. All values were normalized against their respective N-ras WT controls. Error bars indicate standard deviation (*P < 0.05; **P < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Increased caspase 8 and 9 activation in N-ras-/-and H-ras-/-/N-ras-/- fibroblasts. Caspase 8 and 9 activities were measured as described in Materials and methods in WT, N-ras-/- and H-ras-/-/N-ras-/- cell lines that had been subjected to different culture conditions, including, as indicated, serum starvation for 24 hours or subsequent stimulation with serum for 1 hour or 8 hours. Three separate determinations were performed where all individual assays were carried out in triplicate. All values were normalized against their respective N-ras WT controls. Error bars indicate standard deviation (*P < 0.05; **P < 0.01).
Mentions: Two major pathways regulate apoptosis induction in mammalian cells. In the extrinsic pathway, apoptosis is induced through specialized surface receptors such as FAS or tumor necrosis factor-α [72,73], whereas in the intrinsic pathway, this process is mainly induced through release of mitochondrial pro-apoptotic factors [72,74]. Our proteomic data showed increased expression of proteins involved in both the intrinsic (Bax, p53) and extrinsic (Casp8, FAS) pathways, together with some effector caspases and Bid, which connect both pathways. We confirmed these data and checked the functionality of both apoptotic pathways by measuring Casp8 (extrinsic pathway) and Casp9 (intrinsic pathway) activity in N-ras-/- and H-ras-/-/N-ras-/- fibroblasts (Figure 8). These assays showed increased activity of both caspases in the knockout cell lines compared to the WT controls and did not show predominance of either pathway in our ras knockout cell lines. All together, these results support our genomic and proteomic data and demonstrate an increase in the apoptotic response associated with the absence of N-Ras in N-ras-/- and H-ras-/-/N-ras-/- fibroblasts.

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