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

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