Alteration of Golgi structure in senescent cells and its regulation by a G protein γ subunit
Introduction
Replicatively senescent cells or cells that have undergone genotoxic stress exhibit increased secretion of a number of factors including cytokines, growth factors, metalloproteinases and extracellular matrix proteins [1]. The enhanced secretion of these factors is known to induce inflammation and has been demonstrated to facilitate epithelial mesenchymal transition which promotes tumorigenesis [1]. Since cellular secretion is mediated by the Golgi complex, we examined the status of the Golgi in senescent cells resulting from stress or replicative exhaustion. Based on previous reports, 5-bromo 2-deoxyuridine (BrdU) exposure to cells was used as a stress induced model for senescence [2], [3], [4] which mimics the properties of replicative senescence. It has been shown that BrdU treatment induces cellular senescence likely by inducing the DNA-damage response [5]. DNA damage has been shown to trigger senescence [6]. It has been shown that it induces senescence in stem cells and inhibits proliferation of cancer cells [15], [16]. We also confirmed a previous finding that a heterotrimeric G protein subunit, γ11 (GNG11) is upregulated in senescent cells [7]. The γ11 subunit is capable of translocation from the plasma membrane to the Golgi on receptor activation as a βγ complex [8], [9] and regulates the structure of the Golgi [10]. We therefore examined the possibility that the G protein γ11 subunit plays a role in the regulation of the Golgi structure in senescence.
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Constructs, cell lines and chemicals
The tagged and untagged G protein constructs, various Golgi markers and PH-mCh used in this study have been previously described [9], [10], [11], [12]. Mammalian expression vector containing cDNA encoding γ11 shRNA and control scrambled shRNAs were from the TRC library of Broad Institute (Sigma) and CFP-tubulin was from E. Bertrand (CNRS, Montpellier, France). The HeLa cell line was from ATCC; the WI38 and IMR90 cell lines were from NIA Aging Cell Repository at Coriell Institute for Medical
BrdU (5-bromo deoxyuridine) induces cellular senescence
We used BrdU treated HeLa cells as a rapidly assayable model for cellular senescence. It has been observed that BrdU induces cellular senescence by activating the DNA damage response [5], [14]. Consistent with previous reports [2], [4], [5], [14], [15], [17] we found that BrdU induces cellular senescence. HeLa cells treated with 200 μM BrdU for 24–48 h showed several characteristics similar to replicatively senescent IMR90 cells. BrdU treated cells slowed down in growth, changed shape (Fig. 1A
Discussion
The structure of the Golgi complex in senescent cells has not been examined before to our knowledge. Results here show that the Golgi complex is compact in non-senescent or pre-senescent cells but is dispersed in senescent cells. The consistently altered appearance of the Golgi in both stress induced senescent cells and replicatively senescent cells suggests that the Golgi structure could serve as a useful marker for cellular senescence. Senescent cells show significantly increased secretion of
Acknowledgements
This work was supported by NIH grants (GM69027 and GM080558) to N.G. and an AHA postdoctoral fellowship (D.K.S.).
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These authors contributed equally to this work.