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1.
DNA Repair (Amst) ; 7(8): 1233-49, 2008 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-18502190

RESUMO

Telomeres play an important role in protecting the ends of chromosomes and preventing chromosome fusion. We have previously demonstrated that double-strand breaks near telomeres in mammalian cells result in either the addition of a new telomere at the site of the break, termed chromosome healing, or sister chromatid fusion that initiates chromosome instability. In the present study, we have investigated the role of telomerase in chromosome healing and the importance of chromosome healing in preventing chromosome instability. In embryonic stem cell lines that are wild type for the catalytic subunit of telomerase (TERT), chromosome healing at I-SceI-induced double-strand breaks near telomeres accounted for 22 of 35 rearrangements, with the new telomeres added directly at the site of the break in all but one instance. In contrast, in two TERT-knockout embryonic stem cell lines, chromosome healing accounted for only 1 of 62 rearrangements, with a 23 bp insertion at the site of the sole chromosome-healing event. However, in a third TERT-knockout embryonic stem cell line, 10PTKO-A, chromosome healing was a common event that accounted for 20 of 34 rearrangements. Although this chromosome healing also occurred at the I-SceI site, differences in the microhomology at the site of telomere addition demonstrated that the mechanism was distinct from that in wild-type embryonic stem cell lines. In addition, the newly added telomeres in 10PTKO-A shortened with time in culture, eventually resulting in either telomere elongation through a telomerase-independent mechanism or loss of the subtelomeric plasmid sequences entirely. The combined results demonstrate that chromosome healing can occur through both telomerase-dependent and -independent mechanisms, and that although both mechanisms can prevent degradation and sister chromatid fusion, neither mechanism is efficient enough to prevent sister chromatid fusion from occurring in many cells experiencing double-strand breaks near telomeres.


Assuntos
Cromossomos , Células-Tronco Embrionárias/ultraestrutura , Telomerase/metabolismo , Animais , Sequência de Bases , Primers do DNA , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Telomerase/genética , Telomerase/fisiologia
2.
Mol Cell Biol ; 26(5): 1865-78, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16479005

RESUMO

Reversible transcriptional silencing of genes located near telomeres, termed the telomere position effect (TPE), is well characterized in Saccharomyces cerevisiae. TPE has also been observed in human tumor cell lines, but its function remains unknown. To investigate TPE in normal mammalian cells, we developed clones of mouse embryonic stem (ES) cells that contain single-copy marker genes integrated adjacent to different telomeres. Analysis of these telomeric transgenes demonstrated that they were expressed at very low levels compared to the same transgenes integrated at interstitial sites. Similar to the situation in yeast, but in contrast to studies with human tumor cell lines, TPE in mouse ES cells was not reversed with trichostatin A. Prolonged culturing without selection resulted in extensive DNA methylation and complete silencing of telomeric transgenes, which could be reversed by treatment with 5-azacytidine. Thus, complete silencing of the telomeric transgenes appears to involve a two-step process in which the initial repression is reinforced by DNA methylation. Extensive methylation of the telomeric transgenes was also observed in various tissues and embryonic fibroblasts isolated from transgenic mice. In contrast, telomeric transgenes were not silenced in ES cell lines isolated from 3-day-old preimplantation embryos, consistent with the hypothesis that TPE plays a role in the development of the embryo.


Assuntos
Inativação Gênica , Telômero , Transgenes/genética , Animais , Células Cultivadas , Metilação de DNA , Regulação da Expressão Gênica , Ordem dos Genes , Engenharia Genética/métodos , Ácidos Hidroxâmicos/farmacologia , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Células-Tronco/fisiologia , Telômero/efeitos dos fármacos
3.
Stem Cells ; 25(12): 3085-92, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17823235

RESUMO

In addition to their role in protecting the ends of chromosomes, telomeres also influence the expression of adjacent genes, a process called telomere-position effect. We previously reported that the neo and HSV-tk transgenes located adjacent to telomeres in mouse embryonic stem cells are initially expressed at low levels and then become gradually silenced upon passage in culture through a process involving DNA methylation. We also reported extensive DNA methylation in these telomeric transgenes in three different tissues isolated from mice generated from one of these embryonic stem cell clones. In the present study, we demonstrate that embryo fibroblasts isolated from two different mouse strains show extensive DNA methylation and silencing of the telomeric transgenes. Consistent with this observation, we also demonstrate little or no detectable expression of the HSV-tk telomeric transgene in somatic tissues using whole body imaging. In contrast, both telomeric transgenes are expressed at low levels and have little DNA methylation in embryonic stem cell lines isolated from these same mouse strains. Our results demonstrate that telomere-position effect in mammalian cells can be observed either as a low level of expression in embryonic stem cells in the preimplantation embryo or as complete silencing and DNA methylation in differentiated cells and somatic tissues. This pattern of expression of the telomeric transgenes demonstrates that subtelomeric regions, like much of the genome, are epigenetically reprogrammed in the preimplantation embryo, a process that has been proposed to be important in early embryonic development. Disclosure of potential conflicts of interest is found at the end of this article.


Assuntos
Células-Tronco Embrionárias/enzimologia , Fibroblastos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Inativação Gênica , Telômero/genética , Timidina Quinase/antagonistas & inibidores , Timidina Quinase/genética , Transgenes , Fatores Etários , Envelhecimento/genética , Animais , Células-Tronco Embrionárias/citologia , Fibroblastos/citologia , Fibroblastos/enzimologia , Masculino , Camundongos , Camundongos Transgênicos , Simplexvirus/enzimologia , Simplexvirus/genética , Telômero/enzimologia , Timidina Quinase/biossíntese , Distribuição Tecidual/genética
4.
Mol Cell Biol ; 22(13): 4836-50, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12052890

RESUMO

Telomeres are essential for protecting the ends of chromosomes and preventing chromosome fusion. Telomere loss has been proposed to play an important role in the chromosomal rearrangements associated with tumorigenesis. To determine the relationship between telomere loss and chromosome instability in mammalian cells, we investigated the events resulting from the introduction of a double-strand break near a telomere with I-SceI endonuclease in mouse embryonic stem cells. The inactivation of a selectable marker gene adjacent to a telomere as a result of the I-SceI-induced double-strand break involved either the addition of a telomere at the site of the break or the formation of inverted repeats and large tandem duplications on the end of the chromosome. Nucleotide sequence analysis demonstrated large deletions and little or no complementarity at the recombination sites involved in the formation of the inverted repeats. The formation of inverted repeats was followed by a period of chromosome instability, characterized by amplification of the subtelomeric region, translocation of chromosomal fragments onto the end of the chromosome, and the formation of dicentric chromosomes. Despite this heterogeneity, the rearranged chromosomes eventually acquired telomeres and were stable in most of the cells in the population at the time of analysis. Our observations are consistent with a model in which broken chromosomes that do not regain a telomere undergo sister chromatid fusion involving nonhomologous end joining. Sister chromatid fusion is followed by chromosome instability resulting from breakage-fusion-bridge cycles involving the sister chromatids and rearrangements with other chromosomes. This process results in highly rearranged chromosomes that eventually become stable through the addition of a telomere onto the broken end. We have observed similar events after spontaneous telomere loss in a human tumor cell line, suggesting that chromosome instability resulting from telomere loss plays a role in chromosomal rearrangements associated with tumor cell progression.


Assuntos
Dano ao DNA , DNA , Células-Tronco/fisiologia , Telômero/genética , Animais , Clonagem Molecular , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Embrião de Mamíferos/citologia , Feminino , Rearranjo Gênico , Marcadores Genéticos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Plasmídeos/genética , Sequências Repetitivas de Ácido Nucleico , Proteínas de Saccharomyces cerevisiae , Simplexvirus/genética , Timidina Quinase/genética
5.
Nat Commun ; 6: 5990, 2015 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-25606712

RESUMO

The exquisite sensitivity of mitotic cancer cells to ionizing radiation (IR) underlies an important rationale for the widely used fractionated radiation therapy. However, the mechanism for this cell cycle-dependent vulnerability is unknown. Here we show that treatment with IR leads to mitotic chromosome segregation errors in vivo and long-lasting aneuploidy in tumour-derived cell lines. These mitotic errors generate an abundance of micronuclei that predispose chromosomes to subsequent catastrophic pulverization thereby independently amplifying radiation-induced genome damage. Experimentally suppressing whole-chromosome missegregation reduces downstream chromosomal defects and significantly increases the viability of irradiated mitotic cells. Further, orthotopically transplanted human glioblastoma tumours in which chromosome missegregation rates have been reduced are rendered markedly more resistant to IR, exhibiting diminished markers of cell death in response to treatment. This work identifies a novel mitotic pathway for radiation-induced genome damage, which occurs outside of the primary nucleus and augments chromosomal breaks. This relationship between radiation treatment and whole-chromosome missegregation can be exploited to modulate therapeutic response in a clinically relevant manner.


Assuntos
Neoplasias Encefálicas/genética , Instabilidade Cromossômica , Glioblastoma/genética , Neoplasias/radioterapia , Aneuploidia , Animais , Neoplasias Encefálicas/radioterapia , Ciclo Celular , Morte Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Quebra Cromossômica , Segregação de Cromossomos , Glioblastoma/radioterapia , Células HCT116 , Humanos , Masculino , Camundongos , Camundongos Nus , Testes para Micronúcleos , Mitose/genética , Transplante de Neoplasias , Radiação Ionizante
6.
DNA Repair (Amst) ; 10(5): 536-44, 2011 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-21466975

RESUMO

We have previously demonstrated that double-strand breaks (DSBs) in regions near telomeres are much more likely to result in large deletions, gross chromosome rearrangements, and chromosome instability than DSBs at interstitial sites within chromosomes. In the present study, we investigated whether this response of subtelomeric regions to DSBs is a result of a deficiency in DSB repair by comparing the frequency of homologous recombination repair (HRR) and nonhomologous end joining (NHEJ) at interstitial and telomeric sites following the introduction of DSBs by I-SceI endonuclease. We also monitored the frequency of small deletions, which have been shown to be the most common mutation at I-SceI-induced DSBs at interstitial sites. We observed no difference in the frequency of small deletions or HRR at interstitial and subtelomeric DSBs. However, the frequency of NHEJ was significantly lower at DSBs near telomeres compared to interstitial sites. The frequency of NHEJ was also lower at DSBs occurring at interstitial sites containing telomeric repeat sequences. We propose that regions near telomeres are deficient in classical NHEJ as a result of the presence of cis-acting telomere-binding proteins that cause DSBs to be processed as though they were telomeres, resulting in excessive resection, telomere loss, and eventual chromosome rearrangements by alternative NHEJ.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Telômero/genética , Telômero/metabolismo , Linhagem Celular Tumoral , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Deleção de Genes , Ordem dos Genes , Humanos
7.
DNA Repair (Amst) ; 10(11): 1164-73, 2011 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-21945094

RESUMO

Telomerase serves to maintain telomeric repeat sequences at the ends of chromosomes. However, telomerase can also add telomeric repeat sequences at DNA double-strand breaks (DSBs), a process called chromosome healing. Here, we employed a method of inducing DSBs near telomeres to query the role of two proteins, PIF1 and NBS1, in chromosome healing in mammalian cells. PIF1 was investigated because the PIF1 homolog in Saccharomyces cerevisiae inhibits chromosome healing, as shown by a 1000-fold increase in chromosome in PIF1-deficient cells. NBS1 was investigated because the functional homolog of NBS1 in S. cerevisiae, Xrs2, is part of the Mre11/Rad50/Xrs2 complex that is required for chromosome healing due to its role in the processing of DSBs and recruitment of telomerase. We found that disruption of mPif1 had no detectable effect on the frequency of chromosome healing at DSBs near telomeres in murine embryonic stem cells. Moreover, the Nbs1(ΔB) hypomorph, which is defective in the processing of DSBs, also had no detectable effect on the frequency of chromosome healing, DNA degradation, or gross chromosome rearrangements (GCRs) that result from telomeric DSBs. Although we cannot rule out small changes in chromosome healing using this system, it is clear from our results that knockout of PIF1 or the Nbs1(ΔB) hypomorph does not result in large differences in chromosome healing in murine cells. These results represent the first genetic assessment of the role of these proteins in chromosome healing in mammals, and suggest that murine cells have evolved mechanisms to ensure the functional redundancy of Pif1 or Nbs1 in the regulation of chromosome healing.


Assuntos
Proteínas de Ciclo Celular/genética , Aberrações Cromossômicas , Quebras de DNA de Cadeia Dupla , DNA Helicases/genética , Células-Tronco Embrionárias/metabolismo , Proteínas Nucleares/genética , Telômero/metabolismo , Animais , Sequência de Bases , Proteínas de Ciclo Celular/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA , Feminino , Técnicas de Inativação de Genes , Masculino , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Alinhamento de Sequência , Telomerase/genética , Telomerase/metabolismo
8.
Sci Transl Med ; 3(94): 94ra70, 2011 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-21813754

RESUMO

Identifying new targeted therapies that kill tumor cells while sparing normal tissue is a major challenge of cancer research. Using a high-throughput chemical synthetic lethal screen, we sought to identify compounds that exploit the loss of the von Hippel-Lindau (VHL) tumor suppressor gene, which occurs in about 80% of renal cell carcinomas (RCCs). RCCs, like many other cancers, are dependent on aerobic glycolysis for ATP production, a phenomenon known as the Warburg effect. The dependence of RCCs on glycolysis is in part a result of induction of glucose transporter 1 (GLUT1). Here, we report the identification of a class of compounds, the 3-series, exemplified by STF-31, which selectively kills RCCs by specifically targeting glucose uptake through GLUT1 and exploiting the unique dependence of these cells on GLUT1 for survival. Treatment with these agents inhibits the growth of RCCs by binding GLUT1 directly and impeding glucose uptake in vivo without toxicity to normal tissue. Activity of STF-31 in these experimental renal tumors can be monitored by [(18)F]fluorodeoxyglucose uptake by micro-positron emission tomography imaging, and therefore, these agents may be readily tested clinically in human tumors. Our results show that the Warburg effect confers distinct characteristics on tumor cells that can be selectively targeted for therapy.


Assuntos
Antineoplásicos/uso terapêutico , Carcinoma de Células Renais/tratamento farmacológico , Transportador de Glucose Tipo 1/metabolismo , Neoplasias Renais/tratamento farmacológico , Trifosfato de Adenosina/biossíntese , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose , Carcinoma de Células Renais/metabolismo , Glucose/metabolismo , Glicólise , Humanos , Neoplasias Renais/metabolismo
9.
DNA Repair (Amst) ; 8(8): 886-900, 2009 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-19540174

RESUMO

We previously reported that a single DNA double-strand break (DSB) near a telomere in mouse embryonic stem cells can result in chromosome instability. We have observed this same type of instability as a result of spontaneous telomere loss in human tumor cell lines, suggesting that a deficiency in the repair of DSBs near telomeres has a role in chromosome instability in human cancer. We have now investigated the frequency of the chromosome instability resulting from DSBs near telomeres in the EJ-30 human bladder carcinoma cell line to determine whether subtelomeric regions are sensitive to DSBs, as previously reported in yeast. These studies involved determining the frequency of large deletions, chromosome rearrangements, and chromosome instability resulting from I-SceI endonuclease-induced DSBs at interstitial and telomeric sites. As an internal control, we also analyzed the frequency of small deletions, which have been shown to be the most common type of mutation resulting from I-SceI-induced DSBs at interstitial sites. The results demonstrate that although the frequency of small deletions is similar at interstitial and telomeric DSBs, the frequency of large deletions and chromosome rearrangements is much greater at telomeric DSBs. DSB-induced chromosome rearrangements at telomeric sites also resulted in prolonged periods of chromosome instability. Telomeric regions in mammalian cells are therefore highly sensitive to DSBs, suggesting that spontaneous or ionizing radiation-induced DSBs at these locations may be responsible for many of the chromosome rearrangements that are associated with human cancer.


Assuntos
Quebras de DNA de Cadeia Dupla , Telômero/metabolismo , Sequência de Bases , Southern Blotting , Linhagem Celular Tumoral , Instabilidade Cromossômica/efeitos dos fármacos , Cromossomos Humanos/genética , Células Clonais , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Ganciclovir/farmacologia , Rearranjo Gênico/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Humanos , Dados de Sequência Molecular , Plasmídeos/genética , Reação em Cadeia da Polimerase , Recombinação Genética/efeitos dos fármacos , Deleção de Sequência/efeitos dos fármacos , Simplexvirus/enzimologia , Timidina Quinase/genética
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