{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T02:45:17Z","timestamp":1768445117851,"version":"3.49.0"},"reference-count":37,"publisher":"Ovid Technologies (Wolters Kluwer Health)","issue":"6","license":[{"start":{"date-parts":[[2015,9,1]],"date-time":"2015-09-01T00:00:00Z","timestamp":1441065600000},"content-version":"tdm","delay-in-days":457,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["lww.com","ovid.com"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[2014,6]]},"abstract":"<jats:sec>\n            <jats:title\/>\n            <jats:p>Reversal of DNA hypermethylation and associated gene silencing is an emerging cancer therapy approach. Here we addressed the impact of epigenetic alterations and cellular context on functional and transcriptional reprogramming of hepatocellular carcinoma (HCC) cells. Our strategy employed a 3-day treatment of established and primary human HCC-derived cell lines grown as a monolayer at various cell densities with the DNMT1 inhibitor zebularine (ZEB) followed by a 3D culture to identify cells endowed with self-renewal potential. Differences in self-renewal, gene expression, tumorigenicity, and metastatic potential of spheres at generations G1-G5 were examined. Transient ZEB exposure produced differential cell density-dependent responses. In cells grown at low density, ZEB caused a remarkable increase in self-renewal and tumorigenicity associated with long-lasting gene expression changes characterized by a stable overexpression of cancer stem cell-related and key epithelial-mesenchymal transition genes. These effects persisted after restoration of DNMT1 expression. In contrast, at high cell density, ZEB caused a gradual decrease in self-renewal and tumorigenicty, and up-regulation of apoptosis- and differentiation-related genes. A permanent reduction of DNMT1 protein using short hairpin RNA (shRNA)-mediated DNMT1 silencing rendered HCC cells insensitive both to cell density and ZEB effects. Similarly, WRL68 and HepG2 hepatoblastoma cells expressing low DNMT1 basal levels also possessed a high self-renewal, irrespective of cell density or ZEB exposure. Spheres formed by low-density cells treated with ZEB or sh<jats:italic toggle=\"yes\">DNMT1<\/jats:italic> displayed a high molecular similarity which was sustained through consecutive generations, confirming the essential role of DNMT1 depletion in the enhancement of cancer stem cell properties. <jats:italic toggle=\"yes\">Conclusion<\/jats:italic>: These results identify DNA methylation as a key epigenetic regulatory mechanism determining the pool of cancer stem cells in liver cancer and possibly other solid tumors. (Hepatology 2014;59:2251\u20132262)<\/jats:p>\n          <\/jats:sec>","DOI":"10.1002\/hep.27026","type":"journal-article","created":{"date-parts":[[2014,1,21]],"date-time":"2014-01-21T10:24:15Z","timestamp":1390299855000},"page":"2251-2262","update-policy":"https:\/\/doi.org\/10.1097\/lww.0000000000001000","source":"Crossref","is-referenced-by-count":74,"title":["Epigenetic reprogramming modulates malignant properties of human liver cancer"],"prefix":"10.1097","volume":"59","author":[{"given":"Chiara","family":"Raggi","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Valentina M.","family":"Factor","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Daekwan","family":"Seo","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Agnes","family":"Holczbauer","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Matthew C.","family":"Gillen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jens U.","family":"Marquardt","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jesper B.","family":"Andersen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marian","family":"Durkin","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Snorri S.","family":"Thorgeirsson","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"276","reference":[{"key":"R1-26-20251014","doi-asserted-by":"crossref","first-page":"277302","DOI":"10.1146\/annurev-pathol-020712-163923","article-title":"Cellular heterogeneity and molecular evolution in cancer.","volume":"8","author":"Almendro","year":"2013","journal-title":"Annu Rev Pathol"},{"key":"R2-26-20251014","doi-asserted-by":"crossref","first-page":"283296","DOI":"10.1016\/j.ccr.2012.03.003","article-title":"Cancer stem cells: impact, heterogeneity, and uncertainty.","volume":"21","author":"Magee","year":"2012","journal-title":"Cancer Cell"},{"key":"R3-26-20251014","doi-asserted-by":"crossref","first-page":"920","DOI":"10.1016\/j.ccr.2012.06.008","article-title":"Cancer genetics and epigenetics: two sides of the same coin?","volume":"22","author":"You","year":"2012","journal-title":"Cancer Cell"},{"key":"R4-26-20251014","doi-asserted-by":"crossref","first-page":"2133","DOI":"10.1038\/nrg1748","article-title":"The epigenetic progenitor origin of human cancer.","volume":"7","author":"Feinberg","year":"2006","journal-title":"Nat Rev Genet"},{"key":"R5-26-20251014","doi-asserted-by":"crossref","first-page":"683692","DOI":"10.1016\/j.cell.2007.01.029","article-title":"The epigenomics of cancer.","volume":"128","author":"Jones","year":"2007","journal-title":"Cell"},{"key":"RU6-26-20251014","doi-asserted-by":"crossref","first-page":"341350","DOI":"10.1016\/j.str.2008.01.004","article-title":"Mammalian DNA methyltransferases: a structural perspective.","volume":"16","author":"Cheng","year":"2008","journal-title":"Structure"},{"key":"R7-26-20251014","first-page":"484492","article-title":"Functions of DNA methylation: islands, start sites, gene bodies and beyond.","volume":"13","author":"Jones","year":"2012","journal-title":"Nat Rev Genet"},{"key":"R8-26-20251014","first-page":"4955","article-title":"Dissecting direct reprogramming through integrative genomic analysis.","volume":"454","author":"Mikkelsen","year":"2008","journal-title":"Nature"},{"key":"R9-26-20251014","doi-asserted-by":"crossref","first-page":"920926","DOI":"10.1634\/stemcells.2007-0649","article-title":"Pluripotency associated genes are reactivated by chromatinmodifying agents in neurosphere cells.","volume":"26","author":"Ruau","year":"2008","journal-title":"Stem Cells"},{"key":"R10-26-20251014","doi-asserted-by":"crossref","first-page":"10311042","DOI":"10.1002\/hep.24454","article-title":"Human hepatic cancer stem cells are characterized by common stemness traits and diverse oncogenic pathways.","volume":"54","author":"Marquardt","year":"2011","journal-title":"Hepatology"},{"key":"R11-26-20251014","doi-asserted-by":"crossref","first-page":"609617","DOI":"10.1016\/j.tcb.2010.08.003","article-title":"DNA methylation and cellular reprogramming.","volume":"20","author":"De Carvalho","year":"2010","journal-title":"Trends Cell Biol"},{"key":"R12-26-20251014","doi-asserted-by":"crossref","first-page":"711718","DOI":"10.1038\/ncb2074","article-title":"Actin and serum response factor transduce physical cues from the microenvironment to regulate epidermal stem cell fate decisions.","volume":"12","author":"Connelly","year":"2010","journal-title":"Nat Cell Biol"},{"key":"R13-26-20251014","doi-asserted-by":"crossref","first-page":"1563715638","DOI":"10.1073\/pnas.0808457105","article-title":"Reprogramming stem cells is a microenvironmental task.","volume":"105","author":"Bissell","year":"2008","journal-title":"Proc Natl Acad Sci U S A"},{"key":"R14-26-20251014","doi-asserted-by":"crossref","first-page":"11921205","DOI":"10.1002\/hep.24108","article-title":"Matrix stiffness modulates proliferation, chemotherapeutic response, and dormancy in hepatocellular carcinoma cells.","volume":"53","author":"Schrader","year":"2011","journal-title":"Hepatology"},{"key":"R15-26-20251014","first-page":"642649","article-title":"Extracellularmatrix tethering regulates stemcell fate.","volume":"11","author":"Trappmann","year":"2012","journal-title":"Nat Mater"},{"key":"R16-26-20251014","doi-asserted-by":"crossref","first-page":"e27259","DOI":"10.1371\/journal.pone.0027259","article-title":"Shapeinduced terminal differentiation of human epidermal stem cells requires p38 and is regulated by histone acetylation.","volume":"6","author":"Connelly","year":"2011","journal-title":"PLoS One"},{"key":"R17-26-20251014","first-page":"30663075","article-title":"Cell shape regulates global histone acetylation in human mammary epithelial cells.","volume":"313","author":"Le Beyec","year":"2007","journal-title":"Exp Cell Res"},{"key":"R18-26-20251014","doi-asserted-by":"crossref","first-page":"167182","DOI":"10.1002\/mc.21844","article-title":"In vitro threedimensional (3D) models in cancer research: an update.","volume":"52","author":"Kimlin","year":"2013","journal-title":"Mol Carcinog"},{"key":"R19-26-20251014","doi-asserted-by":"crossref","first-page":"55065511","DOI":"10.1158\/0008-5472.CAN-05-0626","article-title":"Isolation and in vitro propagation of tumorigenic breast cancer cells with stemprogenitor cell properties.","volume":"65","author":"Ponti","year":"2005","journal-title":"Cancer Res"},{"key":"R20-26-20251014","doi-asserted-by":"crossref","first-page":"704715","DOI":"10.1016\/j.cell.2008.03.027","article-title":"The epithelialmesenchymal transition generates cells with properties of stem cells.","volume":"133","author":"Mani","year":"2008","journal-title":"Cell"},{"key":"RU21-26-20251014","doi-asserted-by":"crossref","first-page":"593598","DOI":"10.1038\/nature07567","article-title":"Efficient tumour formation by single human melanoma cells.","volume":"456","author":"Quintana","year":"2008","journal-title":"Nature"},{"key":"R22-26-20251014","first-page":"173179","article-title":"A bootstrap test for the analysis of microarray experiments with a very small number of replications.","volume":"5","author":"Neuhauser","year":"2006","journal-title":"Appl Bioinformatics"},{"key":"RU23-26-20251014","doi-asserted-by":"crossref","first-page":"14011409","DOI":"10.1002\/hep.23488","article-title":"Progenitorderived hepatocellular carcinoma model in the rat.","volume":"51","author":"Andersen","year":"2010","journal-title":"Hepatology"},{"key":"R24-26-20251014","doi-asserted-by":"crossref","first-page":"54ra77","DOI":"10.1126\/scitranslmed.3001338","article-title":"An integrated genomic and epigenomic approach predicts therapeutic response to zebularine in human liver cancer.","volume":"2","author":"Andersen","year":"2010","journal-title":"Sci Transl Med"},{"key":"R25-26-20251014","doi-asserted-by":"crossref","first-page":"5063","DOI":"10.1016\/j.stem.2011.06.005","article-title":"CD24() liver tumorinitiating cells drive selfrenewal and tumor initiation through STAT3mediated NANOG regulation.","volume":"9","author":"Lee","year":"2011","journal-title":"Cell Stem Cell"},{"key":"RU26-26-20251014","doi-asserted-by":"crossref","first-page":"25422556","DOI":"10.1053\/j.gastro.2007.04.025","article-title":"Identification and characterization of tumorigenic liver cancer stemprogenitor cells.","volume":"132","author":"Ma","year":"2007","journal-title":"Gastroenterology"},{"key":"R27-26-20251014","doi-asserted-by":"crossref","first-page":"16351644","DOI":"10.1002\/hep.23544","article-title":"Epigenetic regulation of cancer stem cell marker CD133 by transforming growth factorbeta.","volume":"51","author":"You","year":"2010","journal-title":"Hepatology"},{"key":"R28-26-20251014","doi-asserted-by":"crossref","first-page":"10371046","DOI":"10.1038\/cr.2008.270","article-title":"Promoter hypomethylation regulates CD133 expression in human gliomas.","volume":"18","author":"Tabu","year":"2008","journal-title":"Cell Res"},{"key":"R29-26-20251014","first-page":"599605","article-title":"Methylation of the prominin 1 TATAless main promoters and tissue specificity of their transcript content.","volume":"1779","author":"Pleshkan","year":"2008","journal-title":"Biochim Biophys Acta"},{"key":"R30-26-20251014","first-page":"42054211","article-title":"The radioresistance biological function of the SCFkit signaling pathway is mediated by the zincfinger transcription factor Slug.","volume":"22","author":"PerezLosada","year":"2003","journal-title":"Oncogene"},{"key":"R31-26-20251014","first-page":"480488","article-title":"Transcriptomal profiling of the cellular response to DNA damage mediated by Slug (Snai2).","volume":"98","author":"PerezCaro","year":"2008","journal-title":"Br J Cancer"},{"key":"R32-26-20251014","doi-asserted-by":"crossref","first-page":"151158","DOI":"10.1016\/j.ccr.2004.06.023","article-title":"Preferential response of cancer cells to zebularine.","volume":"6","author":"Cheng","year":"2004","journal-title":"Cancer Cell"},{"key":"R33-26-20251014","doi-asserted-by":"crossref","first-page":"871874","DOI":"10.1038\/nature04431","article-title":"The Polycomb group protein EZH2 directly controls DNA methylation.","volume":"439","author":"Vire","year":"2006","journal-title":"Nature"},{"key":"R34-26-20251014","doi-asserted-by":"crossref","first-page":"35413549","DOI":"10.1158\/0008-5472.CAN-05-2481","article-title":"Silenced tumor suppressor genes reactivated by DNA demethylation do not return to a fully euchromatic chromatin state.","volume":"66","author":"McGarvey","year":"2006","journal-title":"Cancer Res"},{"key":"R35-26-20251014","doi-asserted-by":"crossref","first-page":"1124","DOI":"10.1016\/j.ccr.2011.11.025","article-title":"Selfrenewal does not predict tumor growth potential in mouse models of highgrade glioma.","volume":"21","author":"Barrett","year":"2012","journal-title":"Cancer Cell"},{"key":"R36-26-20251014","doi-asserted-by":"crossref","first-page":"871890","DOI":"10.1016\/j.cell.2009.11.007","article-title":"Epithelialmesenchymal transitions in development and disease.","volume":"139","author":"Thiery","year":"2009","journal-title":"Cell"},{"key":"R37-26-20251014","doi-asserted-by":"crossref","first-page":"430446","DOI":"10.1016\/j.ccr.2011.12.029","article-title":"Transient low doses of DNAdemethylating agents exert durable antitumor effects on hematological and epithelial tumor cells.","volume":"21","author":"Tsai","year":"2012","journal-title":"Cancer Cell"}],"container-title":["Hepatology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/api.wiley.com\/onlinelibrary\/tdm\/v1\/articles\/10.1002%2Fhep.27026","content-type":"unspecified","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/journals.lww.com\/01515467-201406000-00026","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,14]],"date-time":"2025-10-14T15:54:24Z","timestamp":1760457264000},"score":1,"resource":{"primary":{"URL":"https:\/\/journals.lww.com\/01515467-201406000-00026"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2014,6]]},"references-count":37,"journal-issue":{"issue":"6","published-print":{"date-parts":[[2014]]}},"URL":"https:\/\/doi.org\/10.1002\/hep.27026","relation":{},"ISSN":["0270-9139"],"issn-type":[{"value":"0270-9139","type":"print"}],"subject":[],"published":{"date-parts":[[2014,6]]}}}