HMGB1 and HMGB2 proteins are abundantly expressed in human embryonic stem cells (hESCs) and hESC-derived progenitor cells (neuroectodermal cells, hNECs), though their functional roles in pluripotency and the mechanisms underlying their differentiation in response to the anticancer drug etoposide remain to be elucidated. Here, we show that HMGB1 and/or HMGB2 knockdown (KD) by shRNA in hESCs did not affect the cell stemness/pluripotency regardless of etoposide treatments, while in hESC-derived neuroectodermal cells, treatment resulted in differential effects on cell survival and the generation of rosette structures. The objective of this work was to determine whether HMGB1/2 proteins could modulate the sensitivity of hESCs and hESC-derived progenitor cells (hNECs) to etoposide. We observed that HMGB1 KD knockdown (KD) and, to a lesser extent, HMGB2 KD enhanced the sensitivity of hESCs to etoposide. Enhanced accumulation of 53BP1 on telomeres was detected by confocal microscopy in both untreated and etoposide-treated HMGB1 KD hESCs and hNECs, indicating that the loss of HMGB1 could destabilize telomeres. On the other hand, decreased accumulation of 53BP1 on telomeres in etoposide-treated HMGB2 KD hESCs (but not in HMGB2 KD hNECs) suggested that the loss of HMGB2 promoted the stability of telomeres. Etoposide treatment of hESCs resulted in a significant enhancement of telomerase activity, with the highest increase observed in the HMGB2 KD cells. Interestingly, no changes in telomerase activity were found in etoposide-treated control hNECs, but HMGB2 KD (unlike HMGB1 KD) markedly decreased telomerase activity in these cells. Changes in telomerase activity in the etoposide-treated HMGB2 KD hESCs or hNECs coincided with the appearance of DNA damage markers and could already be observed before the onset of apoptosis. Collectively, we have demonstrated that HMGB1 or HMGB2 differentially modulate the impact of etoposide treatment on human embryonic stem cells and their progenitor cells, suggesting possible strategies for the enhancement of the efficacy of this anticancer drug.

• Klíčová slova
• HMGB1 and HMGB2, apoptosis, etoposide, human embryonic stem cells, neuroectodermal cells, telomerase,
• MeSH
• apoptóza účinky léků   MeSH
• buněčná diferenciace genetika   MeSH
• etoposid farmakologie   MeSH
• kmenové buňky účinky léků   MeSH
• lidé MeSH
• lidské embryonální kmenové buňky MeSH
• malá interferující RNA MeSH
• nádorové kmenové buňky účinky léků   metabolismus   MeSH
• nádory farmakoterapie   genetika   patologie   MeSH
• protein HMGB1 antagonisté a inhibitory   genetika   MeSH
• protein HMGB2 antagonisté a inhibitory   genetika   MeSH
• protinádorové látky farmakologie   MeSH
• regulace genové exprese u nádorů genetika   MeSH
• telomerasa genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• etoposid MeSH
• HMGB1 protein, human MeSH Prohlížeč
• malá interferující RNA MeSH
• protein HMGB1 MeSH
• protein HMGB2 MeSH
• protinádorové látky MeSH
• telomerasa MeSH

High-mobility group box (HMGB)1 and HMGB2 proteins are the subject of intensive research because of their involvement in DNA replication, repair, transcription, differentiation, proliferation, cell signaling, inflammation, and tumor migration. Using inducible, stably transfected human embryonic stem cells (hESCs) capable of the short hairpin RNA-mediated knockdown (KD) of HMGB1 and HMGB2, we provide evidence that deregulation of HMGB1 or HMGB2 expression in hESCs and their differentiated derivatives (neuroectodermal cells) results in distinct modulation of telomere homeostasis. Whereas HMGB1 enhances telomerase activity, HMGB2 acts as a negative regulator of telomerase activity in the cell. Stimulation of telomerase activity in the HMGB2-deficient cells may be related to activation of the PI3K/protein kinase B/ glycogen synthase kinase-3β/β-catenin signaling pathways by HMGB1, augmented TERT/telomerase RNA subunit transcription, and possibly also because of changes in telomeric repeat-containing RNA (TERRA) and TERRA-polyA+ transcription. The impact of HMGB1/2 KD on telomerase transcriptional regulation observed in neuroectodermal cells is partially masked in hESCs by their pluripotent state. Our findings on differential roles of HMGB1 and HMGB2 proteins in regulation of telomerase activity may suggest another possible outcome of HMGB1 targeting in cells, which is currently a promising approach aiming at increasing the anticancer activity of cytotoxic agents.-Kučírek, M., Bagherpoor, A. J., Jaroš, J., Hampl, A., Štros, M. HMGB2 is a negative regulator of telomerase activity in human embryonic stem and progenitor cells.

• Klíčová slova
• HMGB1, hESCs, neuroectodermal cells, telomeres,
• MeSH
• buněčná diferenciace MeSH
• genetická transkripce MeSH
• kmenové buňky cytologie   enzymologie   MeSH
• lidé MeSH
• lidské embryonální kmenové buňky cytologie   enzymologie   MeSH
• protein HMGB1 genetika   MeSH
• protein HMGB2 genetika   fyziologie   MeSH
• telomerasa metabolismus   MeSH
• transfekce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• HMGB1 protein, human MeSH Prohlížeč
• protein HMGB1 MeSH
• protein HMGB2 MeSH
• telomerasa MeSH

HMGB1 and HMGB2 proteins have been implicated in numerous cellular processes, including proliferation, differentiation, apoptosis, and tumor growth. It is unknown whether they are involved in regulating the typical functions of pluripotent human embryonic stem cells (hESCs) and/or those of the differentiated derivatives of hESCs. Using inducible, stably transfected hESCs capable of shRNA-mediated knockdown of HMGB1 and HMGB2, we provide evidence that downregulation of HMGB1 and/or HMGB2 in undifferentiated hESCs does not affect the stemness of cells and induces only minor changes to the proliferation rate, cell-cycle profile, and apoptosis. After differentiation is induced, however, the downregulation of those proteins has important effects on proliferation, apoptosis, telomerase activity, and the efficiency of differentiation toward the neuroectodermal lineage. Furthermore, those processes are affected only when one, but not both, of the two proteins is downregulated; the knockdown of both HMGB1 and HMGB2 results in a normal phenotype. Those results advance our knowledge of regulation of hESC and human neuroectodermal cell differentiation and illustrate the distinct roles of HMGB1 and HMGB2 during early human development.

• Klíčová slova
• HMGB1, HMGB2, differentiation, human embryonic stem cells, neuroectodermal cells,
• MeSH
• apoptóza genetika   MeSH
• buněčná diferenciace * MeSH
• buněčná sebeobnova genetika   MeSH
• buněčné linie MeSH
• buněčný cyklus genetika   MeSH
• buněčný rodokmen genetika   MeSH
• down regulace genetika   MeSH
• histony metabolismus   MeSH
• lidé MeSH
• lidské embryonální kmenové buňky cytologie   metabolismus   MeSH
• neurální ploténka cytologie   MeSH
• proliferace buněk genetika   MeSH
• protein HMGB1 metabolismus   MeSH
• protein HMGB2 metabolismus   MeSH
• telomerasa metabolismus   MeSH
• transfekce MeSH
• tvar buňky genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• histony MeSH
• protein HMGB1 MeSH
• protein HMGB2 MeSH
• telomerasa MeSH

Telomere repeats are added onto chromosome ends by telomerase, consisting of two main core components: a catalytic protein subunit (telomerase reverse trancriptase, TERT), and an RNA subunit (telomerase RNA, TR). Here, we report for the first time evidence that HMGB1 (a chromatin-associated protein in mammals, acting as a DNA chaperone in transcription, replication, recombination, and repair) can modulate cellular activity of mammalian telomerase. Knockout of the HMGB1 gene (HMGB1 KO) in mouse embryonic fibroblasts (MEFs) results in chromosomal abnormalities, enhanced colocalization of γ-H2AX foci at telomeres, and a moderate shortening of telomere lengths. HMGB1 KO MEFs also exhibit significantly (>5-fold) lower telomerase activity than the wild-type MEFs. Correspondingly, enhanced telomerase activity is observed upon overexpression of HMGB1 in MEFs. HMGB1 physically interacts with both TERT and TR, as well as with active telomerase complex in vitro. However, direct interaction of HMGB1 with telomerase is most likely not accountable for the observed higher telomerase activity in HMGB1-containing cells, as revealed from the inability of purified HMGB1 protein to stimulate telomerase activity in vitro. While no transcriptional silencing of TERT is observed in HMGB1 KO MEFs, levels of TR are diminished (~3-fold), providing possible explanation for the observed lower telomerase activity in HMGB1 KO cells. Interestingly, knockout of the HMGB2 gene elevates telomerase activity (~3-fold) in MEFs, suggesting that the two closely related proteins of the HMGB family, HMGB1 and HMGB2, have opposite effects on telomerase activity in the cell. The ability of HMGB1 to modulate cellular activity of telomerase and to maintain telomere integrity can help to understand some aspects of the protein involvement in chromosome stability and cancer.

• MeSH
• buněčné linie MeSH
• chromozomální aberace MeSH
• down regulace MeSH
• fibroblasty cytologie   metabolismus   MeSH
• fluorescenční mikroskopie MeSH
• fragmentace DNA MeSH
• genový knockout * MeSH
• histony genetika   metabolismus   MeSH
• hybridizace in situ fluorescenční MeSH
• myši MeSH
• poškození DNA MeSH
• protein HMGB1 genetika   metabolismus   MeSH
• protein HMGB2 genetika   metabolismus   MeSH
• replikace DNA MeSH
• RNA genetika   metabolismus   MeSH
• telomerasa genetika   metabolismus   MeSH
• telomery metabolismus   patologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• gamma-H2AX protein, mouse MeSH Prohlížeč
• histony MeSH
• protein HMGB1 MeSH
• protein HMGB2 MeSH
• RNA MeSH
• telomerasa MeSH
• telomerase RNA MeSH Prohlížeč
• Tert protein, mouse MeSH Prohlížeč

Topoisomerase IIalpha (topo IIalpha) is a nuclear enzyme involved in several critical processes, including chromosome replication, segregation and recombination. Previously we have shown that chromosomal protein HMGB1 interacts with topo IIalpha, and stimulates its catalytic activity. Here we show the effect of HMGB1 on the activity of the human topo IIalpha gene promoter in different cell lines. We demonstrate that HMGB1, but not a mutant of HMGB1 incapable of DNA bending, up-regulates the activity of the topo IIalpha promoter in human cells that lack functional retinoblastoma protein pRb. Transient over-expression of pRb in pRb-negative Saos-2 cells inhibits the ability of HMGB1 to activate the topo IIalpha promoter. The involvement of HMGB1 and its close relative, HMGB2, in modulation of activity of the topo IIalpha gene is further supported by knock-down of HMGB1/2, as evidenced by significantly decreased levels of topo IIalpha mRNA and protein. Our experiments suggest a mechanism of up-regulation of cellular expression of topo IIalpha by HMGB1/2 in pRb-negative cells by modulation of binding of transcription factor NF-Y to the topo IIalpha promoter, and the results are discussed in the framework of previously observed pRb-inactivation, and increased levels of HMGB1/2 and topo IIalpha in tumors.

• MeSH
• aktivace transkripce MeSH
• antigeny nádorové biosyntéza   genetika   MeSH
• DNA vazebné proteiny biosyntéza   genetika   MeSH
• DNA-topoisomerasy typu II biosyntéza   genetika   MeSH
• DNA chemie   metabolismus   MeSH
• faktor vázající CCAAT metabolismus   MeSH
• lidé MeSH
• mutageneze MeSH
• nádorové buněčné linie MeSH
• promotorové oblasti (genetika) MeSH
• protein HMGB1 chemie   genetika   metabolismus   MeSH
• protein HMGB2 metabolismus   MeSH
• retinoblastomový protein metabolismus   MeSH
• senioři MeSH
• upregulace * MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny nádorové MeSH
• DNA vazebné proteiny MeSH
• DNA-topoisomerasy typu II MeSH
• DNA MeSH
• faktor vázající CCAAT MeSH
• protein HMGB1 MeSH
• protein HMGB2 MeSH
• retinoblastomový protein MeSH

The recently cloned gene p73 is a close homologue of p53, which is a crucial tumor suppressor gene for preventing the malignant transformation of cells by inducing cell cycle arrest and apoptosis. Previous reports have shown that architectural DNA-bending/looping chromosomal proteins HMGB1 and HMGB2 (formerly known as HMG1 and HMG2), which function in a number of biological processes including transcription and DNA repair, interact in vitro with p53 and stimulate p53 binding to DNA containing p53 consensus sites. Here, we report that HMGB1 physically interacts with two splicing variants of p73, alpha and beta (pull-down assay), and enhances binding of p73 to specific cognate DNA sites (gel-shift assay). Both HMG box domains of HMGB1, A and B, interact with p73alpha. Association of HMGB1 with p73, like the demonstrated ability of HMGB1 to stimulate p73 binding to different p53-responsive elements, requires the oligomerization region and/or region between DNA-binding domain and oligomerization domain of p73 (residues 312-381). Transient transfections revealed that ectopically expressed or endogenous HMGB1 and HMGB2 (antisense strategy) significantly inhibit in vivo both p73alpha/beta- and p53-dependent transactivation from the Bax gene promoter (and much less from Mdm2 and p21(waf1) promoters) in p53-deficient SAOS-2 cells. In contrast, HMGB1 and HGMB2 stimulate p73- or p53-dependent transactivation in p53-deficient H1299 cells, irrespective of the promoter used. Our results suggest that ubiquitously expressed HMGB1 and HMGB2 have potential to cell- and promoter-specifically down- or up-regulate in vivo transcriptional activity of different members of the p53 family. A possible mechanism of HMGB1-mediated modulation of p73- and p53-dependent transactivation is discussed.

• MeSH
• aktivace transkripce * MeSH
• alternativní sestřih MeSH
• buněčné linie MeSH
• DNA vazebné proteiny chemie   genetika   MeSH
• down regulace * MeSH
• geny p53 genetika   MeSH
• glutathiontransferasa metabolismus   MeSH
• jaderné proteiny chemie   genetika   MeSH
• lidé MeSH
• luciferasy metabolismus   MeSH
• nádorové supresorové proteiny MeSH
• plazmidy metabolismus   MeSH
• promotorové oblasti (genetika) MeSH
• protein HMGB1 metabolismus   MeSH
• protein HMGB2 metabolismus   MeSH
• protein p73 MeSH
• protein X asociovaný s bcl-2 MeSH
• proteosyntéza MeSH
• protoonkogenní proteiny c-bcl-2 * MeSH
• protoonkogenní proteiny c-mdm2 MeSH
• protoonkogenní proteiny genetika   metabolismus   MeSH
• terciární struktura proteinů MeSH
• transfekce MeSH
• transkripční faktor Sp1 metabolismus   MeSH
• tumor supresorové geny MeSH
• upregulace MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• BAX protein, human MeSH Prohlížeč
• DNA vazebné proteiny MeSH
• glutathiontransferasa MeSH
• jaderné proteiny MeSH
• luciferasy MeSH
• MDM2 protein, human MeSH Prohlížeč
• nádorové supresorové proteiny MeSH
• protein HMGB1 MeSH
• protein HMGB2 MeSH
• protein p73 MeSH
• protein X asociovaný s bcl-2 MeSH
• protoonkogenní proteiny c-bcl-2 * MeSH
• protoonkogenní proteiny c-mdm2 MeSH
• protoonkogenní proteiny MeSH
• TP73 protein, human MeSH Prohlížeč
• transkripční faktor Sp1 MeSH