Nucleolar localization of Small G protein RhoA is associated with active RNA synthesis in human carcinoma HEp-2 cells

Abstract

Previous studies have demonstrated that the nuclear localization of ras homolog family member A (RhoA), with prominent concentration in the nucleolus, is a common feature in human cancer tissues and cancer cell lines. Although a previous study has demonstrated that the nuclear translocation of RhoA occurs via active transport, a process that occurs through importin α in a nuclear factor-κB-dependent manner, the mechanism, biological function and pathological meaning of the nucleolar residency of RhoA remain to be elucidated. As the cell nucleolus is the site of ribosome biosynthesis, the aim of the present study was to investigate the association between RNA synthesis and the nucleolar localization of RhoA, as well as the molecular mechanisms underlying the residency of RhoA in the nucleolus of HEp-2 (human larynx epithelial carcinoma) cells. Indirect immunofluorescence microscopy was used to evaluate the subcellular distribution of nuclear RhoA, and immunoblotting analysis was used to determine the total cellular protein level of RhoA. Consistent with the results of previous studies, untreated HEp-2 cells exhibited bright nucleolar staining, indicating an increased concentration of RhoA in the nucleoli. Treatment with actinomycin D for the inhibition of RNA synthesis caused a redistribution of RhoA from the nucleoli to the nucleoplasm with a speckled staining pattern. Immunoblotting revealed that neither the total cellular amount of RhoA nor the integrity of RhoA was affected by treatment with actinomycin D. In cells that were treated at a decreased concentration (0.05 mg/l) of actinomycin D, the redistribution of RhoA was reversible following the removal of the drug from the culture medium. However, this reversal was not observed at an increased drug concentration (1 mg/l). Overall, to the best of our knowledge, the results of the present study provide the first in situ evidence that the inhibition of RNA synthesis induces a redistribution of nucleolar RhoA to the nucleoplasm, and additionally suggest that the nucleolar residency of RhoA in HEp-2 cells may be associated with active RNA synthesis.

VPRBP Functions Downstream of the Androgen Receptor and OGT to Restrict p53 Activation in Prostate Cancer

“…4G ). Fibrillarin showed exclusive nucleolar localization with a redistribution to small nucleoplasmic entities in the VPRBP knockdown cells, similar to previously described with actinomycin D treatment ( 39 ). To quantify nucleolar fragmentation, we measured the individual nucleolar area based on the fibrillarin staining as previously described ( 40 ), which showed that VPRBP knockdown cells have a much smaller nucleolar size compared (∼1.7 µm 2 ) with scrambled si-transfected cells (9.8 µm 2 ; Supplementary Fig S6A).…”

VPRBP functions downstream of the androgen receptor and OGT to restrict p53 activation in prostate cancer

“…4G). Fibrillarin showed exclusive nucleolar localization with a redistribution to small nucleoplasmic entities in the VPRBP knockdown cells, similar to previously described with actinomycin D treatment 34 . A significant (~25%) reduction in fibrillarin and 40S ribosomal protein S8 (RPS8) expression, was observed with VPRBP siRNA2 (Fig.…”

CX-5461 causes nucleolar compaction, alteration of peri- and intranucleolar chromatin arrangement, an increase in both heterochromatin and DNA damage response

“…Intranucleolar chromatin appears frequently at the interface between FCs and DFCs and the perinucleolar chromatin extends forming a bright DAPI-positive ring around nucleoli, containing mainly condensed chromatin partly including NORs and some stretches of decondensed chromatin. Right: treatment with high doses of AMD resolves FC/DFC architecture causing spatial segregation of the three nucleolar components; rDNA retracts into NORs as does most of pol I while the majority of fibrillarin redistributes into the nucleoplasm; NORs and perinucleolar chromatin appear in patches forming nucleolar caps (scheme compiled with data from 10 , 59 , 60 ). …”

“…Intranucleolar chromatin appears frequently at the interface between FCs and DFCs and the perinucleolar chromatin extends forming a bright DAPI-positive ring around nucleoli, containing mainly condensed chromatin partly including NORs and some stretches of decondensed chromatin. Right: treatment with high doses of AMD resolves FC/DFC architecture causing spatial segregation of the three nucleolar components; rDNA retracts into NORs as does most of pol I while the majority of fibrillarin redistributes into the nucleoplasm; NORs and perinucleolar chromatin appear in patches forming nucleolar caps (scheme compiled with data from 10,59,60 www.nature.com/scientificreports/ Summing up, we showed that CX-5461 treatment in cultured cells has a profound impact on nucleolar morphology and function of cultured cells. Nucleoli diminish in size and acquire a compact shape surrounded by an increased shell of perinucleolar heterochromatin enriched in repressive chromatin markers.…”