Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of ouse fibroblasts lacking H-Ras and/or NRas with those of matching, WT controls - reveals serum-dependent transcriptional networks identify distinct functional roles for H-ras and N-ras

ABSTRACT: Serum-dependent transcriptional networks identify distinct functional roles for H-ras and N-ras during initial stages of the cell cycle; Using oligonucleotide microarrays, we compared gene expression transcriptional profiles corresponding to the initial cell cycle stages of mouse fibroblasts lacking H-Ras and/or NRas with those of matching, WT controls. The similarity of transcription profiles among serum-starved fibroblasts of all different WT and ras knockout genotypes tested, indicated that H-Ras and N-Ras do not play significant roles in control of transcriptional responses to serum deprivation stress. In contrast, genomic disruption of H-ras or N-ras, individually or in combination, determined highly specific, differential gene expression profiles in response to post-starvation stimulation with serum for 1 hour (G0/G1 transition) or for 8 hours (mid-G1 progression). The absence of N-Ras caused significantly higher changes than the absence of H-Ras on the wave of transcriptional activation linked to G0/G1 transition. In contrast, the absence of H-Ras affected more potently the profile of the transcriptional wave detected during mid-G1 progression. Functional analysis demonstrated a predominant functional association of H-Ras with growth and proliferation, whereas N-Ras exhibited a closer functional link to development or cell cycle regulation as well as immunomodulation and apoptosis. Mechanistic analysis indicated that ERK-dependent activation of 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 signaling. Our observations support previous reports of an absolute requirement for different peaks of Ras activity during the initial stages of the cell cycle and confirm the notion of functional specificity for the H-Ras and N-Ras isoforms. Experiment Overall Design: Mus musculus cell lines from the appropriate ras genotype were harvested on Dulbeccoâs modified Eagleâs medium (DMEM) supplemented with fetal bovine serum (10% FBS), glutamine (2mM), penicillin (100 U/ml) and streptomycin (100 mg/ml). Cultures were grown in a humidified CO2 (5%) atmosphere at 37Â°C and when subconfluent cells were starved for 24 hours. After starvation cells were either used for RNA isolation, or induced for 1 hour or 8 hours with 20% fetal bovine serum and then RNA extraction and isolation was carried out for hybridization on Affymetrix microarrays.

ORGANISM(S): Mus musculus

SUBMITTER: Javier De Las Rivas

PROVIDER: E-GEOD-14829 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Transcription profiling of ouse fibroblasts lacking H-Ras and/or NRas with those of matching, WT controls - reveals serum-dependent transcriptional networks identify distinct functional roles for H-ras and N-ras

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