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Aysegul Tura, Matthias Lueke, Mihaela Reinsberg, Mahdy Ranjbar, Anna Kraus, Salvatore Grisanti, Julia Lueke; Elevated RhoC expression and Rho-kinase activity in the uveal melanoma cells with Monosomy-3. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5903. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Rho-GTPase family is an important regulator of actin cytoskeleton organization, proliferation, migration, adhesion, matrix remodelling, and gene expression. The best characterized member of the Rho-GTPases is RhoA, which is encoded by a gene on Chromosome-3. The RhoC isoform, which can be upregulated in the absence of RhoA, was recently detected in elevated levels in more invasive forms of various tumors. In this study, we analyzed the levels of RhoA and RhoC as well as the proliferation rate and Rho-kinase activity in uveal melanoma (UM) cells with Monosomy-3 compared to the cells with Disomy-3.
UM cells were isolated from the primary tumor of a patient (female, 48 years) operated in our clinic, who developed liver metastases within 2 years after enucleation. Cultured cells (Passages 1-3) were dissociated by dispase, seeded on to 8-well chamber slides, and incubated in culture medium (RPMI-1640 supplemented with 1% fetal bovine serum, 2 mM L-Glutamine and penicillin/streptomycin) with or without insulin-like growth factor-1 (IGF-1, 50 ng/ml) and the Rho-kinase inhibitor H-1152 (5 µM) for 3 days. An Immuno-FISH assay was developed for the simultaneous detection of the proteins of interest and the copy number of chromosome-3.
Monosomy-3 was detected in a minimum of 55% of the cultured cells. RhoA protein was expressed at approximately 30% lower levels whereas RhoC was expressed at 40% higher levels in the cells with Monosomy-3 compared to the cells with Disomy-3 (P<0.05). IGF-1 led to a significant increase in the upregulation of the proliferation marker Ki-67 and the levels of myosin phosphatase-1 phosphorylation in the cells with Monosomy-3, whereas the Rho-kinase inhibitor suppressed these events.
Our preliminary findings demonstrate the upregulation of the RhoC protein in UM cells with Monosomy-3, possibly as a compensation for RhoA. This might lead to an elevated Rho-kinase activity and proliferation rate in response to IGF-1, which might underlie the higher metastatic potential of UM cells with Monosomy-3.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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