Differential Ras activation between caveolae/raft and non-raft microdomains.
ABSTRACT Although the consequences of Ras activation have been studied extensively in the context of oncogenesis, its regulation in physiological modes of signal transduction is not well understood. A fluorescent indicator, Raichu-Ras, was fused to the C-terminal hypervariable regions of H-Ras and K-Ras to create indicators for Ras activation within caveolae/rafts (Raichu-tH) and non-raft domains (Raichu-tK) of the plasma membrane, respectively. Raichu-tH was also found abundantly in endomembranes. To monitor Ras activation with high spatial resolution, it is imperative to observe sectioned images of the signals. We have developed a wide-field fluorescence microscope equipped with a digital micromirror device (DMD) to acquire optically sectioned images using fringe projection. This system provides reliable signals from fluorescence resonance energy transfer (FRET) between cyan and yellow mutants of green fluorescent protein. We have used this system to demonstrate that, upon stimulation with growth factors, the two indicators are activated in spatially and temporally unique patterns.
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ABSTRACT: Genome wide hypomethylation and regional hypermethylation of cancer cells and tissues remain a paradox, though it has received a convincing confirmation that epigenetic switching systems, including DNA-methylation represent a fundamental regulatory mechanism that has an impact on genome maintenance and gene transcription. Methylated cytosine residues of vertebrate DNA are transmitted by clonal inheritance through the strong preference of DNA methyltransferase, DNMT1, for hemimethylated-DNA. Maintenance of methylation patterns is necessary for normal development of mice, and aberrant methylation patterns are associated with many human tumours. DNMT1 interacts with many proteins during cell cycle progression, including PCNA, p53, EZH2 and HP1. Ras family of GTPases promotes cell proliferation by its oncogenic nature, which transmits signals by multiple pathways in both lipid raft dependent and independent fashion. DNA-methylation-mediated repression of DNA-repair protein O6-methylguanine DNA methyltransferase (MGMT) gene and increased rate of K-Ras mutation at codon for amino acids 12 and 13 have been correlated with a secondary role for Ras-effector homologues (RASSFs) in tumourigenesis. Lines of evidence suggest that DNA-methylation associated repression of tumour suppressors and apoptotic genes and ceaseless proliferation of tumour cells are regulated in part by Ras-signaling. Control of Ras GTPase signaling might reduce the aberrant methylation and accordingly may reduce the risk of cancer development.Experimental Cell Research 05/2008; 314(6):1193-201. · 3.58 Impact Factor
Article: Dissecting the different biological effects of oncogenic Ras isoforms in cancer cell lines: Could stimulation of oxidative stress be the one more weapon of H-Ras?: Regulation of oxidative stress and Ras biological effects.[show abstract] [hide abstract]
ABSTRACT: Ras proteins are small GTPase functioning as molecular switches that, in response to particular extracellular signalling, as growth factors, activate a diverse array of intracellular effector cascades regulating cell proliferation, differentiation and apoptosis. Human tumours frequently express Ras proteins (Ha-, Ki-, N-Ras) activated by point mutations which contribute to malignant phenotype, including invasiveness and angiogenesis. Despite the common signalling pathways leading to similar cellular responses, studies clearly demonstrate unique roles of the Ras family members in normal and pathological conditions and the lack of functional redundancy seems to be explainable, at least in part, by the ability of Ras isoforms to localize in different microdomains to plasma membrane and intracellular organelles. This different intracellular compartmentalization could help Ras isoforms to contact different downstream effectors finally leading to different biological outcomes. Interestingly, it has also been shown that Ha- and Ki-Ras exert an opposite role in regulating intracellular redox status. In this regard we suggest that H-Ras specific induction of ROS (reactive oxygen species) production could be one of the main determinants of the invasive phenotype which characterize cancer cells harbouring H-Ras mutations. In our hypothesis then, while K-Ras (not able to promote oxidative stress) could mainly contribute to cancer progression and invasiveness through activation of MAPK and PI3K, H-Ras-mediated oxidative stress could play a unique role in modulation of intercellular contacts leading to a loss of cell adhesion and eventually also to a metastatic spread.Medical Hypotheses 09/2012; · 1.39 Impact Factor