Ras-induced Changes Research Articles

Ras-Induced Changes in H3K27me3 Occur after Those in Transcriptional Activity

Oncogenic signaling pathways regulate gene expression in part through epigenetic modification of chromatin including DNA methylation and histone modification. Trimethylation of histone H3 at lysine-27 (H3K27), which correlates with transcriptional repression, is regulated by an oncogenic form of the small GTPase Ras. Although accumulation of trimethylated H3K27 (H3K27me3) has been implicated in transcriptional regulation, it remains unclear whether Ras-induced changes in H3K27me3 are a trigger for or a consequence of changes in transcriptional activity. We have now examined the relation between H3K27 trimethylation and transcriptional regulation by Ras. Genome-wide analysis of H3K27me3 distribution and transcription at various times after expression of oncogenic Ras in mouse NIH 3T3 cells identified 115 genes for which H3K27me3 level at the gene body and transcription were both regulated by Ras. Similarly, 196 genes showed Ras-induced changes in transcription and H3K27me3 level in the region around the transcription start site. The Ras-induced changes in transcription occurred before those in H3K27me3 at the genome-wide level, a finding that was validated by analysis of individual genes. Depletion of H3K27me3 either before or after activation of Ras signaling did not affect the transcriptional regulation of these genes. Furthermore, given that H3K27me3 enrichment was dependent on Ras signaling, neither it nor transcriptional repression was maintained after inactivation of such signaling. Unexpectedly, we detected unannotated transcripts derived from intergenic regions at which the H3K27me3 level is regulated by Ras, with the changes in transcript abundance again preceding those in H3K27me3. Our results thus indicate that changes in H3K27me3 level in the gene body or in the region around the transcription start site are not a trigger for, but rather a consequence of, changes in transcriptional activity.

ROS up-regulation mediates Ras-induced changes of cell morphology and motility

Expression of activated Ras causes an increase in intracellular content of reactive oxygen species (ROS). To determine the role of ROS up-regulation in mediation of Ras-induced morphological transformation and increased cell motility, we studied the effects of hydrogen peroxide and antioxidant NAC on morphology of REF52 rat fibroblasts and their ability to migrate into the wound in vitro. Treatment with low dosages of hydrogen peroxide leading to 1.5- to 2-fold increase in intracellular ROS levels induced changes of cell shape, actin cytoskeleton organization, cell adhesions and migration resembling those in Ras-transformed cells. On the other hand, treatment with NAC attenuating ROS up-regulation in cells with conditional or constitutive expression of activated Ras led to partial reversion of morphological transformation and decreased cell motility. The effect of ROS on cell morphology and motility probably results from modulation of activity of Rac1, Rho, and cofilin proteins playing a key role in regulation of actin dynamics. The obtained data are consistent with the idea that ROS up-regulation mediates two key events in Ras-induced morphological transformation and cell motility: it is responsible for Rac1 activation and is necessary (though insufficient) for realization of Ras-induced cofilin dephosphorylation.

Ras-inducible immortalized fibroblasts: focus formation without cell cycle deregulation.

The Ras oncogene transforms cultured murine fibroblasts into malignant, focus-forming cells, whose lack of contact inhibition is evidenced by high saturation densities. In order to investigate the reversibility of Ras transformation, as well as the kinetics of Ras-induced changes, cell lines that conditionally express oncogenic Ras were constructed. Both focus formation and increased saturation density were inducible and fully reversible. In exponentially growing cells, oncogenic Ras-expression had no effect on proliferation rates, Erk phosphorylation, or the level of cyclin D1, and Ras-induction did not confer serum-independent growth. As expected, growth to high density in uninduced cells led to quiescence with a low level of cyclin D1 and no active Erk; in this setting, Ras induction prevented full downregulation of cyclin D1 and inactivation of Erk. Our results show that Ras expression to a level sufficient for transformation leads to relatively subtle effects on known downstream targets, and that the focus formation and increased saturation density growth induced by Ras is not a result of growth factor independence.

Keratin expression and steroidogenesis in rat granulosa cells, transformed with the Kirsten- ras and SV40 oncogenes singly and in combination

The keratins are a component of the cytoskeleton that is present in fetal and neonatal rat granulosa cells (ROG), but disappears as the cells undergo postnatal steroidogenic differentiation. Steroidogenesis is initiated in the fetus as a low level constitutive function which is cAMP responsive, but becomes responsive to gonadotrophic hormones only after birth. ROG from PMSG-primed immature rats, like mature ROG, are keratin negative, highly steroidogenic and gonadotrophin-responsive, but rapidly lose their steroidogenic capacity in culture. In such cultured cells, transformation with the Kirsten- ras oncogene (v-Ki- ras) maintains low levels of constitutive steroidogenesis and responsiveness to cAMP, and induces the expression of keratin. To determine whether similar changes would occur in cells expressing both the SV40 and v-Ki- ras, cultured ROG were transformed with SV40 early genes, with Kirsten murine sarcoma virus (KiMSV), or with both agents concurrently. Keratin was demonstrated by fluorescence microscopy and Western blots, and progesterone production by RIA. ROG transformed with SV40 alone became immortalized but secreted little steroid and lacked keratin. In contrast, three cell lines, co-transformed with SV40 plus KiMSV, acquired keratin as well as the capacity to secrete progesterone in response to cAMP, closely resembling cells transformed with Ki- ras alone. KiMSV-transformed muscle fascia fibroblasts lacked both steroidogenic potential and keratin. The results show that the complex, v-Ki- ras-induced changes in steroidogenesis and keratin expression are reproducible and tissue specific. The phenotypic resemblance between singly and doubly transformed ROG indicates that the v-Ki- ras oncogene does not act by overcoming SV40-mediated inhibition of differentiation. The enhancement of keratin expression concurrently with gonadotrophin-independent steroidogenesis in SV40 v -Ki -ras- transformed cells resembles the phenotype of mutipotential embryonic ROG precursors.

Mitotic and post mitotic consequences of genomic instability induced by oncogenic Ha-Ras

Induced expression of a mutant human Ha-ras oncogene in NIH3T3 cells leads to the rapid production of multicentric chromosomes, acentric chromosome fragments, double minute chromosomes, increased heteroploidy, and increased capacity to undergo gene amplification. In this study we have used fluorescent-in-situ hybridization (FISH) to demonstrate that induction of the Ha-ras oncogene also leads to disruption of the mitotic machinery, resulting in aberrant mitoses and abnormal daughter cells. Cells induced to express an oncogenic Ha-ras transgene accumulate chromosomes that lag outside of the rest of the chromosomal architecture, chromosomes that form bridges between daughter nuclei at anaphase, and that form micronuclei. Many of these mitotic aberrations contain structurally abnormal chromosomes. These ras-induced changes were suppressed by the introduction of a gene encoding the dominant negative effector of ras, raf 301. Expression of raf301 in cells induced to express Ha-ras reduced the level of growth in soft agar, chromosome aberrations, mitotic aberrations, and frequency of gene amplification. These data provide evidence for an association between Ha-ras induced transformation, chromosome aberrations and gene amplification. Furthermore they offer insight into how the cell responds to the formation of aberrant chromosomes, and how disrupting chromosomal architecture could lead to further imbalances in the distribution of genetic material between daughter cells.