Artigo Acesso aberto Revisado por pares

Early nucleolar responses differentiate mechanisms of cell death induced by oxaliplatin and cisplatin

2021; Elsevier BV; Volume: 296; Linguagem: Inglês

10.1016/j.jbc.2021.100633

ISSN

1083-351X

Autores

Emily C. Sutton, Victoria J. DeRose,

Tópico(s)

Metal complexes synthesis and properties

Resumo

Recent reports provide evidence that the platinum chemotherapeutic oxaliplatin causes cell death via ribosome biogenesis stress, while cisplatin causes cell death via the DNA damage response (DDR). Underlying differences in mechanisms that might initiate disparate routes to cell death by these two broadly used platinum compounds have not yet been carefully explored. Additionally, prior studies had demonstrated that cisplatin can also inhibit ribosome biogenesis. Therefore, we sought to directly compare the initial influences of oxaliplatin and cisplatin on nucleolar processes and on the DDR. Using pulse-chase experiments, we found that at equivalent doses, oxaliplatin but not cisplatin significantly inhibited ribosomal RNA (rRNA) synthesis by Pol I, but neither compound affected rRNA processing. Inhibition of rRNA synthesis occurred as early as 90 min after oxaliplatin treatment in A549 cells, concurrent with the initial redistribution of the nucleolar protein nucleophosmin (NPM1). We observed that the nucleolar protein fibrillarin began to redistribute by 6 h after oxaliplatin treatment and formed canonical nucleolar caps by 24 h. In cisplatin-treated cells, DNA damage, as measured by γH2AX immunofluorescence, was more extensive, whereas nucleolar organization was unaffected. Taken together, our results demonstrate that oxaliplatin causes early nucleolar disruption via inhibition of rRNA synthesis accompanied by NPM1 relocalization and subsequently causes extensive nucleolar reorganization, while cisplatin causes early DNA damage without significant nucleolar disruption. These data support a model in which, at clinically relevant doses, cisplatin kills cells via the canonical DDR, and oxaliplatin kills cells via ribosome biogenesis stress, specifically via rapid inhibition of rRNA synthesis. Recent reports provide evidence that the platinum chemotherapeutic oxaliplatin causes cell death via ribosome biogenesis stress, while cisplatin causes cell death via the DNA damage response (DDR). Underlying differences in mechanisms that might initiate disparate routes to cell death by these two broadly used platinum compounds have not yet been carefully explored. Additionally, prior studies had demonstrated that cisplatin can also inhibit ribosome biogenesis. Therefore, we sought to directly compare the initial influences of oxaliplatin and cisplatin on nucleolar processes and on the DDR. Using pulse-chase experiments, we found that at equivalent doses, oxaliplatin but not cisplatin significantly inhibited ribosomal RNA (rRNA) synthesis by Pol I, but neither compound affected rRNA processing. Inhibition of rRNA synthesis occurred as early as 90 min after oxaliplatin treatment in A549 cells, concurrent with the initial redistribution of the nucleolar protein nucleophosmin (NPM1). We observed that the nucleolar protein fibrillarin began to redistribute by 6 h after oxaliplatin treatment and formed canonical nucleolar caps by 24 h. In cisplatin-treated cells, DNA damage, as measured by γH2AX immunofluorescence, was more extensive, whereas nucleolar organization was unaffected. Taken together, our results demonstrate that oxaliplatin causes early nucleolar disruption via inhibition of rRNA synthesis accompanied by NPM1 relocalization and subsequently causes extensive nucleolar reorganization, while cisplatin causes early DNA damage without significant nucleolar disruption. These data support a model in which, at clinically relevant doses, cisplatin kills cells via the canonical DDR, and oxaliplatin kills cells via ribosome biogenesis stress, specifically via rapid inhibition of rRNA synthesis. The nucleolus is the site of ribosome biogenesis, a process that includes transcription of ribosomal RNA (rRNA) by RNA Polymerase I (Pol I), processing of rRNA, and assembly of ribosomal subunits. Nucleolar morphology can be used as a prognostic factor for tumor severity, and nucleolar size is correlated to node and receptor status in breast cancer as well as length of disease-free survival period (1Derenzini M. Montanaro L. Treré D. What the nucleolus says to a tumour pathologist.Histopathology. 2009; 54: 753-762Crossref PubMed Scopus (141) Google Scholar). Functionally, ribosome biogenesis is closely associated with cellular processes such as the cell cycle, further linking the dysregulation of this process to cancer and other diseases (2Tsai R.Y.L. Pederson T. Connecting the nucleolus to the cell cycle and human disease.FASEB J. 2014; 28: 3290-3296Crossref PubMed Scopus (59) Google Scholar). While upregulated ribosome biogenesis is associated with cancer, inhibition of ribosome biogenesis can lead to activation of the tumor suppressor protein p53 via the nucleolar stress response. This has made ribosome biogenesis and the nucleolus desirable targets for potential chemotherapeutic agents (3Pickard A.J. Bierbach U. The cell's nucleolus: An emerging target for chemotherapeutic intervention.ChemMedChem. 2013; 8: 1441-1449Crossref PubMed Scopus (41) Google Scholar, 4Woods S.J. Hannan K.M. Pearson R.B. Hannan R.D. The nucleolus as a fundamental regulator of the p53 response and a new target for cancer therapy.Biochim. Biophys. Acta. 2015; 1849: 821-829Crossref PubMed Scopus (85) Google Scholar, 5Bywater M.J. Poortinga G. Sanij E. Hein N. Peck A. Cullinane C. Wall M. Cluse L. Drygin D. Anderes K. Huser N. Proffitt C. Bliesath J. Haddach M. Schwaebe M.K. et al.Inhibition of RNA polymerase I as a therapeutic strategy to promote cancer-specific activation of p53.Cancer Cell. 2012; 22: 51-65Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar). A few small molecules induce a cytotoxic nucleolar stress response by selectively inhibiting rRNA synthesis by Pol I. Actinomycin D (ActD) is known to selectively inhibit Pol I at low doses, an effect attributed to its propensity to target GC-rich regions of DNA, including ribosomal DNA (rDNA) (6Peltonen K. Colis L. Liu H. Trivedi R. Moubarek M.S. Moore H.M. Bai B. Rudek M.A. Bieberich C.J. Laiho M. A targeting modality for destruction of RNA polymerase I that possesses anticancer activity.Cancer Cell. 2014; 25: 77-90Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). It is used effectively to treat specific types of tumors, but its clinical efficacy has been limited in scope (2Tsai R.Y.L. Pederson T. Connecting the nucleolus to the cell cycle and human disease.FASEB J. 2014; 28: 3290-3296Crossref PubMed Scopus (59) Google Scholar). BMH-21 was identified in a screen for compounds that induce a p53 response (7Peltonen K. Colis L. Liu H. Jäämaa S. Moore H.M. Enbäck J. Laakkonen P. Vaahtokari A. Jones R.J. af Hällström T.M. Laiho M. Identification of novel p53 pathway activating small-molecule compounds reveals unexpected similarities with known therapeutic agents.PLoS One. 2010; 5e12996Crossref PubMed Scopus (45) Google Scholar) and later found to inhibit Pol I (6Peltonen K. Colis L. Liu H. Trivedi R. Moubarek M.S. Moore H.M. Bai B. Rudek M.A. Bieberich C.J. Laiho M. A targeting modality for destruction of RNA polymerase I that possesses anticancer activity.Cancer Cell. 2014; 25: 77-90Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). CX-5461 was identified in a screen for selective Pol I inhibitors (8Drygin D. Lin A. Bliesath J. Ho C.B. O'Brien S.E. Proffitt C. Omori M. Haddach M. Schwaebe M.K. Siddiqui-Jain A. Streiner N. Quin J.E. Sanij E. Bywater M.J. Hannan R.D. et al.Targeting RNA polymerase I with an oral small molecule CX-5461 inhibits ribosomal RNA synthesis and solid tumor growth.Cancer Res. 2011; 71: 1418-1430Crossref PubMed Scopus (307) Google Scholar). The ability of BMH-21 to inhibit rRNA transcription is currently believed to be due to stalling of Pol I transcription followed by degradation of the Pol I subunit RPA194 (6Peltonen K. Colis L. Liu H. Trivedi R. Moubarek M.S. Moore H.M. Bai B. Rudek M.A. Bieberich C.J. Laiho M. A targeting modality for destruction of RNA polymerase I that possesses anticancer activity.Cancer Cell. 2014; 25: 77-90Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 9Wei T. Najmi S.M. Liu H. Peltonen K. Kucerova A. Schneider D.A. Laiho M. Small-molecule targeting of RNA polymerase I activates a conserved transcription elongation checkpoint.Cell Rep. 2018; 23: 404-414Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The mechanism of CX-5461 remains more elusive, with recent evidence suggesting that it may primarily exert its cytotoxic effects by way of topoisomerase II inhibition rather than Pol I inhibition as previously believed (10Bruno P.M. Lu M. Dennis K.A. Inam H. Moore C.J. Sheehe J. Elledge S.J. Hemann M.T. Pritchard J.R. The primary mechanism of cytotoxicity of the chemotherapeutic agent CX-5461 is topoisomerase II poisoning.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 4053-4060Crossref PubMed Scopus (34) Google Scholar, 11Haddach M. Schwaebe M.K. Michaux J. Nagasawa J. O'Brien S.E. Whitten J.P. Pierre F. Kerdoncuff P. Darjania L. Stansfield R. Drygin D. Anderes K. Proffitt C. Bliesath J. Siddiqui-Jain A. et al.Discovery of CX-5461, the first direct and selective inhibitor of RNA polymerase I, for cancer therapeutics.ACS Med. Chem. Lett. 2012; 3: 602-606Crossref PubMed Scopus (62) Google Scholar). Platinum anticancer compounds have also been shown to disrupt nucleolar function. Until relatively recently, it was believed that all platinum-based chemotherapeutic agents, including cisplatin and oxaliplatin (Fig. 1A), exerted their cytotoxic effects by triggering the DNA damage response (DDR) (12Kelland L. The resurgence of platinum-based cancer chemotherapy.Nat. Rev. Cancer. 2007; 7: 573-584Crossref PubMed Scopus (3259) Google Scholar, 13Siddik Z.H. Cisplatin: Mode of cytotoxic action and molecular basis of resistance.Oncogene. 2003; 22: 7265-7279Crossref PubMed Scopus (2398) Google Scholar). In 2017, Bruno et al. (14Bruno P.M. Liu Y. Park G.Y. Murai J. Koch C.E. Eisen T.J. Pritchard J.R. Pommier Y. Lippard S.J. Hemann M.T. A subset of platinum-containing chemotherapeutic agents kills cells by inducing ribosome biogenesis stress.Nat. Med. 2017; 23: 461-471Crossref PubMed Scopus (199) Google Scholar) used an RNAi screening approach to identify ribosome biogenesis inhibition as a primary mechanism of oxaliplatin cytotoxicity in cancer cells, whereas cisplatin acts via the canonical DDR. This distinction has been supported by later studies demonstrating more extensive redistribution of the protein nucleophosmin (NPM1) from the nucleolus to the nucleoplasm—a hallmark of the nucleolar stress response—upon treatment with oxaliplatin and derivates (15Sutton E.C. McDevitt C.E. Prochnau J.Y. Yglesias M.V. Mroz A.M. Yang M.C. Cunningham R.M. Hendon C.H. DeRose V.J. Nucleolar stress induction by oxaliplatin and derivatives.J. Am. Chem. Soc. 2019; 141: 18411-18415Crossref PubMed Scopus (10) Google Scholar). Because prior studies have suggested a more complex relationship between nucleolar stress processes and these platinum drugs, further investigation into the differences between cisplatin and oxaliplatin with regard to their ability to disrupt nucleolar processes is warranted. While no studies to date have fully characterized how oxaliplatin might induce ribosome biogenesis stress, its ability to do so is documented in existing literature. Robust oxaliplatin-induced redistribution of NPM1 has been demonstrated (14Bruno P.M. Liu Y. Park G.Y. Murai J. Koch C.E. Eisen T.J. Pritchard J.R. Pommier Y. Lippard S.J. Hemann M.T. A subset of platinum-containing chemotherapeutic agents kills cells by inducing ribosome biogenesis stress.Nat. Med. 2017; 23: 461-471Crossref PubMed Scopus (199) Google Scholar, 15Sutton E.C. McDevitt C.E. Prochnau J.Y. Yglesias M.V. Mroz A.M. Yang M.C. Cunningham R.M. Hendon C.H. DeRose V.J. Nucleolar stress induction by oxaliplatin and derivatives.J. Am. Chem. Soc. 2019; 141: 18411-18415Crossref PubMed Scopus (10) Google Scholar, 16McDevitt C.E. Yglesias M.V. Mroz A.M. Sutton E.C. Yang M.C. Hendon C.H. DeRose V.J. Monofunctional platinum(II) compounds and nucleolar stress: Is phenanthriplatin unique?.J. Biol. Inorg. Chem. 2019; 24: 899-908Crossref PubMed Scopus (6) Google Scholar, 17Burger K. Mühl B. Harasim T. Rohrmoser M. Malamoussi A. Orban M. Kellner M. Gruber-Eber A. Kremmer E. Hölzel M. Eick D. Chemotherapeutic drugs inhibit ribosome biogenesis at various levels.J. Biol. Chem. 2010; 285: 12416-12425Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar), and it has been shown to inhibit rRNA transcription and induce rearrangement of other nucleolar proteins at low doses (6.25 μM) in human fibrosarcoma cells (17Burger K. Mühl B. Harasim T. Rohrmoser M. Malamoussi A. Orban M. Kellner M. Gruber-Eber A. Kremmer E. Hölzel M. Eick D. Chemotherapeutic drugs inhibit ribosome biogenesis at various levels.J. Biol. Chem. 2010; 285: 12416-12425Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Proteomics studies have shown changes in levels of proteins related to ribosome biogenesis upon oxaliplatin treatment (18Ozdian T. Holub D. Maceckova Z. Varanasi L. Rylova G. Rehulka J. Vaclavkova J. Slavik H. Moudry P. Znojek P. Stankova J. de Sanctis J.B. Hajduch M. Dzubak P. Proteomic profiling reveals DNA damage, nucleolar and ribosomal stress are the main responses to oxaliplatin treatment in cancer cells.J. Proteomics. 2017; 162: 73-85Crossref PubMed Scopus (13) Google Scholar). However, in that study it was concluded that nucleolar stress was a consequence of DNA damage. The relationship between nucleolar stress induction and cisplatin is more complex. While there is evidence for cisplatin's ability to affect ribosome biogenesis and other nucleolar processes, recent data and an evaluation of existing literature suggest that these observed mechanisms may not be clinically relevant. Some examinations of cisplatin treatment in HeLa cells have demonstrated a reduction in rRNA synthesis by transcription run-on assay, along with redistribution of Pol I and Upstream Binding Factor (UBF), a transcription factor for Pol I (19Jordan P. Carmo-Fonseca M. Cisplatin inhibits synthesis of ribosomal RNA in vivo.Nucleic Acids Res. 1998; 26: 2831-2836Crossref PubMed Scopus (105) Google Scholar). Morphological changes in the nucleolus have been observed at early stages of cell death induced by high doses (40 μM) of cisplatin (20Horky M. Wurzer G. Kotala V. Anton M. Vojtĕsek B. Vácha J. Wesierska-Gadek J. Segregation of nucleolar components coincides with caspase-3 activation in cisplatin-treated HeLa cells.J. Cell Sci. 2001; 114: 663-670Crossref PubMed Google Scholar). Reduction of transcription follows colocalization of Pol I and coilin 6 h after cisplatin treatment in HeLa cells, and this effect on transcription can be reversed by siRNA silencing of coilin (21Gilder A.S. Do P.M. Carrero Z.I. Cosman A.M. Broome H.J. Velma V. Martinez L. a Hebert M.D. Coilin participates in the suppression of RNA polymerase I in response to cisplatin-induced DNA damage.Mol. Biol. Cell. 2011; 22: 1070-1079Crossref PubMed Scopus (36) Google Scholar). Hamdane et al. (22Hamdane N. Herdman C. Mars J.-C. Stefanovsky V. Tremblay M.G. Moss T. Depletion of the cisplatin targeted HMGB-box factor UBF selectively induces p53-independent apoptotic death in transformed cells.Oncotarget. 2015; 6: 27519-27536Crossref PubMed Scopus (16) Google Scholar) have shown that 30 μM of cisplatin treatment leads to displacement of UBF from rDNA and inhibition of rRNA transcription in mouse embryonic fibroblasts, and high doses (50–100 μM) have been shown to inhibit rRNA transcription and induce nucleolar disruption in human fibrosarcoma cells (17Burger K. Mühl B. Harasim T. Rohrmoser M. Malamoussi A. Orban M. Kellner M. Gruber-Eber A. Kremmer E. Hölzel M. Eick D. Chemotherapeutic drugs inhibit ribosome biogenesis at various levels.J. Biol. Chem. 2010; 285: 12416-12425Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Cisplatin does not induce the same robust level of NPM1 translocation as oxaliplatin does (14Bruno P.M. Liu Y. Park G.Y. Murai J. Koch C.E. Eisen T.J. Pritchard J.R. Pommier Y. Lippard S.J. Hemann M.T. A subset of platinum-containing chemotherapeutic agents kills cells by inducing ribosome biogenesis stress.Nat. Med. 2017; 23: 461-471Crossref PubMed Scopus (199) Google Scholar, 15Sutton E.C. McDevitt C.E. Prochnau J.Y. Yglesias M.V. Mroz A.M. Yang M.C. Cunningham R.M. Hendon C.H. DeRose V.J. Nucleolar stress induction by oxaliplatin and derivatives.J. Am. Chem. Soc. 2019; 141: 18411-18415Crossref PubMed Scopus (10) Google Scholar). In sum, there is a clear difference between the ability of these highly similar platinum compounds to affect nucleolar processes. An overarching goal of research into these compounds is to better understand the mechanisms behind their differential efficacy and resistance profiles, and recent evidence suggests that it is decreasingly likely that any effects of cisplatin on nucleolar processes bear relevance to its clinical applications, with the inverse being true of oxaliplatin. Further exploration of the mechanisms of action of these drugs and an examination of their ability to induce nucleolar stress are warranted. Previously, our lab has demonstrated some structural constraints for Pt(II) drugs to cause stress. The diaminocyclohexane (DACH) ligand of oxaliplatin proved critical, and while some changes to its size and aromaticity could be tolerated, compounds with different ring orientations did not induce stress (15Sutton E.C. McDevitt C.E. Prochnau J.Y. Yglesias M.V. Mroz A.M. Yang M.C. Cunningham R.M. Hendon C.H. DeRose V.J. Nucleolar stress induction by oxaliplatin and derivatives.J. Am. Chem. Soc. 2019; 141: 18411-18415Crossref PubMed Scopus (10) Google Scholar). This proved valuable in understanding what types of Pt(II) molecules can lead to this response, but the biological mechanism by which oxaliplatin-like compounds induce nucleolar stress and cause NPM1 redistribution is still not well understood. For example, Pt(II) compounds are known to cause DNA damage, which can cause nucleolar stress indirectly by inhibiting Pol I (23Korsholm L.M. Lund C.C. Larsen D.H. Recent advances in the nucleolar responses to DNA double-strand breaks.Nucleic Acids Res. 2020; 48: 9449-9461Crossref PubMed Scopus (6) Google Scholar). This relationship between DNA damage and nucleolar stress has yet to be examined in the specific context of Pt(II)-induced nucleolar stress. Some previous work has closely examined the ability of oxaliplatin and cisplatin, among other chemotherapeutic compounds, to inhibit rRNA synthesis and processing as well as rearrangement of specific nucleolar proteins (17Burger K. Mühl B. Harasim T. Rohrmoser M. Malamoussi A. Orban M. Kellner M. Gruber-Eber A. Kremmer E. Hölzel M. Eick D. Chemotherapeutic drugs inhibit ribosome biogenesis at various levels.J. Biol. Chem. 2010; 285: 12416-12425Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). However, these studies examined cisplatin's nucleolar effects at doses likely above clinical relevance and did not directly link this Pt(II)-induced inhibition to p53 stabilization after treatment with relevant Pt(II) doses. We sought to build on this work by utilizing similar techniques at time points and doses relevant to these critical processes. This paper marks a step forward in understanding which nucleolar processes do and do not cause Pt(II)-induced nucleolar stress by examining rRNA synthesis and processing alongside NPM1 redistribution at relevant time points, assessing DNA damage, and further characterizing protein redistribution behavior in Pt(II) stress conditions. We find that rRNA transcription is inhibited at early time points by oxaliplatin, but not cisplatin, and that this inhibition correlates with onset of NPM1 redistribution and precedes the formation of canonical fibrillarin-containing nucleolar caps. We also conclude that rRNA transcription inhibition is not preceded by a DDR during oxaliplatin treatment and that previous observations of cisplatin-induced nucleolar stress are likely reflecting events downstream of DDR. The protein NPM1 normally resides mainly in the periphery of the nucleolus and its relocalization to the nucleoplasm is a hallmark of the nucleolar stress response (Fig. 1B). It has previously been shown that both cisplatin and oxaliplatin can cause redistribution of NPM1 but with much higher doses of cisplatin (100 μM) than oxaliplatin (6.25 μM) in the same cell lines (17Burger K. Mühl B. Harasim T. Rohrmoser M. Malamoussi A. Orban M. Kellner M. Gruber-Eber A. Kremmer E. Hölzel M. Eick D. Chemotherapeutic drugs inhibit ribosome biogenesis at various levels.J. Biol. Chem. 2010; 285: 12416-12425Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Additionally, there has been little exploration of the molecular events preceding NPM1 redistribution upon drug treatment. We have previously measured NPM1 redistribution after 24 h of treatment with Pt(II) compounds (15Sutton E.C. McDevitt C.E. Prochnau J.Y. Yglesias M.V. Mroz A.M. Yang M.C. Cunningham R.M. Hendon C.H. DeRose V.J. Nucleolar stress induction by oxaliplatin and derivatives.J. Am. Chem. Soc. 2019; 141: 18411-18415Crossref PubMed Scopus (10) Google Scholar). In order to directly compare cisplatin and oxaliplatin and to initially bracket a time course for onset of NPM1 distribution, A549 cells were treated for 4, 8, and 12 h with 10 μM cisplatin or oxaliplatin. A treatment concentration of 10 μM was chosen because it is a sufficient concentration to induce nucleolar stress without killing a large portion of the cellular population based on our previous findings (15Sutton E.C. McDevitt C.E. Prochnau J.Y. Yglesias M.V. Mroz A.M. Yang M.C. Cunningham R.M. Hendon C.H. DeRose V.J. Nucleolar stress induction by oxaliplatin and derivatives.J. Am. Chem. Soc. 2019; 141: 18411-18415Crossref PubMed Scopus (10) Google Scholar). Cells were then fixed, permeabilized, and immunostained for NPM1 (Fig. 1, C and D). The extent of NPM1 redistribution was calculated as previously described (15Sutton E.C. McDevitt C.E. Prochnau J.Y. Yglesias M.V. Mroz A.M. Yang M.C. Cunningham R.M. Hendon C.H. DeRose V.J. Nucleolar stress induction by oxaliplatin and derivatives.J. Am. Chem. Soc. 2019; 141: 18411-18415Crossref PubMed Scopus (10) Google Scholar, 16McDevitt C.E. Yglesias M.V. Mroz A.M. Sutton E.C. Yang M.C. Hendon C.H. DeRose V.J. Monofunctional platinum(II) compounds and nucleolar stress: Is phenanthriplatin unique?.J. Biol. Inorg. Chem. 2019; 24: 899-908Crossref PubMed Scopus (6) Google Scholar) by determining the coefficient of variation (CV) of NPM1 pixel intensities within each nucleus. These CV values were then normalized to the average CV for an untreated control and plotted by treatment group (Fig. 1, E and F). A lower CV value indicates more broadly distributed NPM1 in the nucleoplasm and more extensive nucleolar stress. We determined that significant NPM1 translocation from the nucleolus is observed by 4 h of treatment with oxaliplatin and that the translocation becomes more extensive with time (Fig. 1, D and F, Fig. S3). By contrast, at the same treatment concentration and time period, only a minor amount of NPM1 translocation occurs upon cisplatin treatment (Fig. 1, C and E). After oxaliplatin treatment, we observed rounding of the nucleoli as previously described (15Sutton E.C. McDevitt C.E. Prochnau J.Y. Yglesias M.V. Mroz A.M. Yang M.C. Cunningham R.M. Hendon C.H. DeRose V.J. Nucleolar stress induction by oxaliplatin and derivatives.J. Am. Chem. Soc. 2019; 141: 18411-18415Crossref PubMed Scopus (10) Google Scholar). Also of note is the appearance of bulging regions of NPM1 around the periphery of many of the rounded nucleoli after oxaliplatin treatment (Fig. 1D, white arrows). These features resemble "nucleolar caps" or structures that have been reported to form after exposure to ribosomal DNA damage and other nucleolar stressors (17Burger K. Mühl B. Harasim T. Rohrmoser M. Malamoussi A. Orban M. Kellner M. Gruber-Eber A. Kremmer E. Hölzel M. Eick D. Chemotherapeutic drugs inhibit ribosome biogenesis at various levels.J. Biol. Chem. 2010; 285: 12416-12425Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 24Shav-Tal Y. Blechman J. Darzacq X. Montagna C. Dye B.T. Patton J.G. Singer R.H. Zipori D. Dynamic sorting of nuclear components into distinct nucleolar caps during transcriptional inhibition.Mol. Biol. Cell. 2005; 16: 2395-2413Crossref PubMed Scopus (242) Google Scholar). Interestingly, NPM1 is not among the proteins previously reported to comprise nucleolar caps, with some studies expressly stating that NPM1 does not compartmentalize into nucleolar caps (24Shav-Tal Y. Blechman J. Darzacq X. Montagna C. Dye B.T. Patton J.G. Singer R.H. Zipori D. Dynamic sorting of nuclear components into distinct nucleolar caps during transcriptional inhibition.Mol. Biol. Cell. 2005; 16: 2395-2413Crossref PubMed Scopus (242) Google Scholar, 25Brodská B. Holoubek A. Otevřelová P. Kuželová K. Low-dose actinomycin-D induces redistribution of wild-type and mutated nucleophosmin followed by cell death in leukemic cells: Mutant NPM and ActD-induced apoptosis.J. Cell. Biochem. 2016; 117: 1319-1329Crossref PubMed Scopus (16) Google Scholar). This rapid observable response with oxaliplatin treatment that includes NPM1 redistribution, morphological features such as nucleolar rounding, and the appearance of putative cap-like structures all raise interesting questions about the molecular processes behind them. NPM1 redistribution is just one marker of nucleolar stress, and while it is a robust and ubiquitous marker, observation of this redistribution with accompanying morphological changes does not address the molecular mechanisms by which Pt(II) compounds might be inducing nucleolar stress. Several known triggers of nucleolar stress include inhibition of any of the stages of ribosome biogenesis, DNA damage, or direct perturbation of nucleolar structure (26Yang K. Wang M. Zhao Y. Sun X. Yang Y. Li X. Zhou A. Chu H. Zhou H. Xu J. Wu M. Yang J. Yi J. A redox mechanism underlying nucleolar stress sensing by nucleophosmin.Nat. Commun. 2016; 7: 13599Crossref PubMed Scopus (54) Google Scholar, 27Rubbi C.P. Milner J. Disruption of the nucleolus mediates stabilization of p53 in response to DNA damage and other stresses.EMBO J. 2003; 22: 6068-6077Crossref PubMed Scopus (607) Google Scholar, 28Boulon S. Westman B.J. Hutten S. Boisvert F.-M.M. Lamond A.I. The nucleolus under stress.Mol. Cell. 2010; 40: 216-227Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar). We sought to narrow down the potential causes of oxaliplatin-induced NPM1 redistribution by examining three processes known to induce nucleolar stress—inhibition of rRNA synthesis, inhibition of rRNA processing, and DNA damage. It has been established that inhibition of rRNA synthesis and processing are linked to nucleolar stress and NPM1 redistribution (28Boulon S. Westman B.J. Hutten S. Boisvert F.-M.M. Lamond A.I. The nucleolus under stress.Mol. Cell. 2010; 40: 216-227Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar). Prior studies on the influences of cisplatin and oxaliplatin on rRNA synthesis have yielded mixed results. Using pulse-chase approaches, it has been shown that both cisplatin and oxaliplatin can inhibit rRNA transcription but that neither affects rRNA processing. However, in these studies, cisplatin causes inhibition between 40 and 100 μM, doses above the IC-50 values in the cell lines used and also thought to be above clinical relevance (17Burger K. Mühl B. Harasim T. Rohrmoser M. Malamoussi A. Orban M. Kellner M. Gruber-Eber A. Kremmer E. Hölzel M. Eick D. Chemotherapeutic drugs inhibit ribosome biogenesis at various levels.J. Biol. Chem. 2010; 285: 12416-12425Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 22Hamdane N. Herdman C. Mars J.-C. Stefanovsky V. Tremblay M.G. Moss T. Depletion of the cisplatin targeted HMGB-box factor UBF selectively induces p53-independent apoptotic death in transformed cells.Oncotarget. 2015; 6: 27519-27536Crossref PubMed Scopus (16) Google Scholar). Other studies have shown stark differences between cisplatin and oxaliplatin with regard to their ability to affect rRNA transcription. Using RT-qPCR, Bruno et al. observed decreases in pre-rRNA levels after 30 min of treatment with oxaliplatin and ActD, followed by marked increases between 1 and 6 h of continuous treatment (14Bruno P.M. Liu Y. Park G.Y. Murai J. Koch C.E. Eisen T.J. Pritchard J.R. Pommier Y. Lippard S.J. Hemann M.T. A subset of platinum-containing chemotherapeutic agents kills cells by inducing ribosome biogenesis stress.Nat. Med. 2017; 23: 461-471Crossref PubMed Scopus (199) Google Scholar). Treatment with cisplatin did not demonstrate this trend in pre-rRNA levels. These data were used to support the conclusion that oxaliplatin acts as a ribosome biogenesis inhibitor like ActD, while cisplatin works by a different mechanism (namely DNA damage). To clarify the influence of cisplatin and oxaliplatin on rRNA transcription and processing and to determine the relationship of NPM1 redistribution to these effects, we conducted pulse-chase radiolabeling experiments using a method previously described (Fig. 2A) (17Burger K. Mühl B. Harasim T. Rohrmoser M. Malamoussi A. Orban M. Kellner M. Gruber-Eber A. Kremmer E. Hölzel M. Eick D. Chemotherapeutic drugs inhibit ribosome biogenesis at various levels.J. Biol. Chem. 2010; 285: 12416-12425Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). After treatment of A549 cells with compounds for a fixed period, cells were incubated in Pt(II)-free media containing 32P-labeled phosphate, which would be incorporated into any newly synthe

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