title: Thymosin beta-4 knockdown in IEC-6 normal intestinal epithelial cells induces DNA re-replication via downregulating Emi1.
abstract: Thymosin β4 (Tβ4 ) is a multifunctional protein already used clinically to treat various diseases; however, the promoting effect of this protein on tumor malignancy should not be neglected. Here, we assessed whether Tβ4 alteration influences normal intestinal epithelial cells because Tβ4 is deemed a novel target for treating colorectal cancer (CRC). For this purpose, we examined the consequences of shRNA-mediated knockdown of Tβ4 in IEC-6 normal rat small intestinal cells and found that inhibiting Tβ4 expression significantly suppressed their growth and induced apoptosis in some cells. Flow cytometric analysis further revealed a marked decrease of G0/G1 population but a drastic increase of polyploid ones in these cells. The increase of polyploidy likely resulted from DNA re-replication because not only the de novo DNA synthesis was greatly increased but also the expression levels of Cdc6 (a replication-licensing factor), cyclin A, and phosphorylated-checkpoint kinase 1 were all dramatically elevated. Moreover, marked reductions in both RNA and protein levels of Emi1 (early mitotic inhibitor 1) were also detected in Tβ4 -downregulated IEC-6 cells which might be accounted by the downregulation of E2F1, a transcription factor capable of inducing Emi1 expression, mediated by glycogen synthase-3β (GSK-3β). To our best knowledge, this is the first report showing that inhibiting Tβ4 expression triggers DNA re-replication in normal intestinal epithelial cells, suggesting that this G-actin sequester may play a crucial role in maintaining genome stability in these cells. More importantly, clinical oncologists should take this novel activity into consideration when design CRC therapy based on targeting Tβ4 .
authors.affiliation: Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan; Division of Hematology and Oncology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan.
authors.forename: Ta-Chung
authors.initials: TC
authors.lastname: Chao
authors.forename: Ke-Jay
authors.initials: KJ
authors.lastname: Chen
authors.forename: Mei-Chuan
authors.initials: MC
authors.lastname: Tang
authors.forename: Li-Chuan
authors.initials: LC
authors.lastname: Chan
authors.forename: Po-Min
authors.initials: PM
authors.lastname: Chen
authors.forename: Cheng-Hwai
authors.initials: CH
authors.lastname: Tzeng
authors.forename: Yeu
authors.initials: Y
authors.lastname: Su
mesh_terms: D000818:Animals; D059447:Cell Cycle Checkpoints; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D049109:Cell Proliferation; D004249:DNA Damage; D004261:DNA Replication; D015536:Down-Regulation; D050687:E2F1 Transcription Factor / Q000378:metabolism; D020895:Enterocytes / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006657:Histones / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D011123:Polyploidy; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D013947:Thymosin / Q000378:metabolism*; D000099201:Thymosin beta(4)
chemical_list: D018797:Cell Cycle Proteins; D050687:E2F1 Transcription Factor; D038362:Glycogen Synthase Kinase 3; D000071679:Glycogen Synthase Kinase 3 beta; D006657:Histones; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D013947:Thymosin; D000099201:Thymosin beta(4); C498962:Fbxo5 protein, rat; C577575:gamma-H2AX protein, rat; C000605508:Gsk3b protein, rat