RHOAming Through the Nucleotide Excision Repair Pathway as a Mechanism of Cellular Response Against the Effects of UV Radiation

Abstract

Typical Rho GTPases include the enzymes RhoA, Rac1, and Cdc42 that act as molecular switches to regulate essential cellular processes in eukaryotic cells such as actomyosin dynamics, cell cycle, adhesion, death and differentiation. Recently, it has been shown that different conditions modulate the activity of these enzymes, but their functions still need to be better understood. Here we examine the interplay between RhoA and the NER (Nucleotide Excision Repair) pathway in human cells exposed to UVA, UVB or UVC radiation. The results show high levels and accumulation of UV-induced DNA lesions (strand breaks and cyclobutane pyrimidine dimers, CPDs) in different cells with RhoA loss of function (LoF), either by stable overexpression of negative dominant RhoA (RhoA-N19 mutant), by inhibition with C3 toxin or by transient silencing with siRNA. Cells under RhoA LoF showed reduced levels of γH2AX, p-Chk1 (Ser345) and p-p53 (Ser15) that reflected causally in their accumulation in G1/S phases, in low survival rates and in reduced cell proliferation, also in accordance with the energy of applied UV light. Even NER-deficient cells (XPA, XPC) or DNA translesion synthesis (TLS)-deficient cells (XPV) showed substantial hypersensitivity to UV effects when previously submitted to RhoA LoF. In contrast, analyses of apoptosis, necrosis, autophagy and senescence revealed that all cells displaying normal levels of active RhoA (RhoA-GTP) are more resistant to UV-promoted cell death. This work reaffirms the role of RhoA protein signaling in protecting cells from damage caused by UV radiation and demonstrates relevant communicating mechanisms between actin cytoskeleton and genomic stability.

RHOJ controls EMT-associated resistance to chemotherapy

“…We found, through genetic gain and loss of function studies in vitro and in vivo, the key role of RHOJ in regulating the resistance of EMT tumour cells to chemotherapy. Whereas small GTPases of the Rho family have been previously associated with DNA repair and response to therapy 42 , 43 , the role of RHOJ in regulating resistance to therapy in EMT tumour cells was unclear.…”

Downregulation of the Rho GTPase pathway abrogates resistance to ionizing radiation in wild-type p53 glioblastoma by suppressing DNA repair mechanisms

“…Cells were subjected to Rho inhibition by transient transfection of a vector containing the C3 toxin coding sequence, as previously described [ 15 ], or gene silencing by transfection of siRNAs specific for mDia1, PFN1, MYPT1 or p53 (MISSION® esiRNA, Invitrogen) using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol. Rho activity was also modulated by 48 h of starvation in serum-free medium prior to activation with medium containing 30% FBS.…”

“…Briefly, cellular protein extracts (DTT-free) were incubated with glutathione-Sepharose beads bound to the RBD-GST fusion protein. After the wash steps and recovery by centrifugation, the proportion of Rho-GTP bound to RBD-GST was quantified by immunoblotting as described [ 15 ] here in brief: specific amounts of protein extracts (quantified by the Bradford method) were denatured with the Laemmli protocol, separated by SDS‒PAGE and transferred to a nitrocellulose membrane (Millipore). After blocking with 5% low-fat milk/TBS-T for 30 min, the membranes were incubated with specific primary and secondary antibodies under the optimized incubation conditions described in the Supplementary data .…”

“…The high invasiveness of GBM is one of the major obstacles to clinical therapy, as these cells are intrinsically more resistant to current therapies [ 10 ]. All typical Rho GTPases have been shown to act in the early stages of DNA damage recognition, mainly through regulation of DDR pathways [ 11 – 15 ], and are also activated in response to genotoxic stressors [ 16 , 17 ]. Therefore, higher Rho GTPase activity seems to be necessary for DDR signaling and efficient repair of DSBs, among other types of DNA lesions [ 11 , 13 , 15 – 18 ].…”

“…All typical Rho GTPases have been shown to act in the early stages of DNA damage recognition, mainly through regulation of DDR pathways [ 11 – 15 ], and are also activated in response to genotoxic stressors [ 16 , 17 ]. Therefore, higher Rho GTPase activity seems to be necessary for DDR signaling and efficient repair of DSBs, among other types of DNA lesions [ 11 , 13 , 15 – 18 ]. The relationship between Rho and the p53 pathway has been pointed out in recent years and is purported to exert effects mainly via regulation of the cell cycle [ 19 – 22 ], gene expression [ 23 – 25 ] and cell migration [ 26 , 27 ]; however, the mechanism and interdependence of these pathways in DNA repair and tumor resistance have not yet been sufficiently explored and demonstrated.…”