RESUMO
This chapter describes the methods of isolation of mouse periosteal progenitor cells. There are three basic methods utilized. The bone grafting method was developed utilizing the fracture healing process to expand the progenitor populations. Bone capping methods requires enzymatic digestion and purification of cells from the native periosteum, while the Egression/Explant method requires the least manipulation with placement of cortical bone fragments with attached periosteum in a culture dish. Various cell surface antibodies have been employed over the years to characterize periosteum derived progenitor cells, but the most consistent minimal criteria was recommended by the International Society for Cellular Therapy. Confirmation of the multipotent status of these isolated cells can be achieved by differentiation into the three basic mesodermal lineages in vitro.
Assuntos
Transplante Ósseo/métodos , Técnicas de Cultura de Células/métodos , Periósteo/crescimento & desenvolvimento , Células-Tronco/citologia , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Células-Tronco Mesenquimais/citologia , Camundongos , Osteogênese/genética , Periósteo/citologiaRESUMO
Ionizing radiation (IR) therapy is a major cancer treatment modality and an indispensable auxiliary treatment for primary and metastatic cancers, but invariably results in debilitating organ dysfunctions. IR-induced depletion of neural stem/progenitor cells in the subgranular zone of the dentate gyrus in the hippocampus where neurogenesis occurs is considered largely responsible for deficiencies such as learning, memory, and spatial information processing in patients subjected to cranial irradiation. Similarly, IR therapy-induced intestinal injuries such as diarrhea and malabsorption are common side effects in patients with gastrointestinal tumors and are believed to be caused by intestinal stem cell drop out. Hematopoietic stem cell transplantation is currently used to reinstate blood production in leukemia patients and pre-clinical treatments show promising results in other organs such as the skin and kidney, but ethical issues and logistic problems make this route difficult to follow. An alternative way to restore the injured tissue is to preserve the stem cell pool located in that specific tissue/organ niche, but stem cell response to ionizing radiation is inadequately understood at the molecular mechanistic level. Although embryonic and fetal hypersensity to IR has been very well known for many decades, research on embryonic stem cell models in culture concerning molecular mechanisms have been largely inconclusive and often in contradiction of the in vivo observations. This review will summarize the latest discoveries on stem cell radiosensitivity, highlighting the possible molecular and epigenetic mechanism(s) involved in DNA damage response and programmed cell death after ionizing radiation therapy specific to normal stem cells. Finally, we will analyze the possible contribution of stem cell-specific chromatin's epigenetic constitution in promoting normal stem cell radiosensitivity.
RESUMO
Unintended outcomes of cancer therapy include ionizing radiation (IR)-induced stem cell depletion, diminished regenerative capacity, and accelerated aging. Stem cells exhibit attenuated DNA damage response (DDR) and are hypersensitive to IR, as compared to differentiated non-stem cells. We performed genomic discovery research to compare stem cells to differentiated cells, which revealed Phosphoprotein phosphatase 2A (PP2A) as a potential contributor to susceptibility in stem cells. PP2A dephosphorylates pATM, γH2AX, pAkt etc. and is believed to play dual role in regulating DDR and apoptosis. Although studied widely in cancer cells, the role of PP2A in normal stem cell radiosensitivity is unknown. Here we demonstrate that constitutively high expression and radiation induction of PP2A in stem cells plays a role in promoting susceptibility to irradiation. Transient inhibition of PP2A markedly restores DNA repair, inhibits apoptosis, and enhances survival of stem cells, without affecting differentiated non-stem and cancer cells. PP2Ai-mediated stem cell radioprotection was demonstrated in murine embryonic, adult neural, intestinal, and hematopoietic stem cells.
Assuntos
Inibidores Enzimáticos/farmacologia , Oxazóis/farmacologia , Proteína Fosfatase 2/antagonistas & inibidores , Tolerância a Radiação/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Células-Tronco/efeitos da radiação , Animais , Apoptose , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Reparo do DNA , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/enzimologia , Células-Tronco Hematopoéticas/patologia , Células-Tronco Hematopoéticas/efeitos da radiação , Humanos , Masculino , Toxinas Marinhas , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/enzimologia , Células-Tronco Embrionárias Murinas/patologia , Células-Tronco Embrionárias Murinas/efeitos da radiação , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/enzimologia , Células-Tronco Neurais/patologia , Células-Tronco Neurais/efeitos da radiação , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Células-Tronco/enzimologia , Células-Tronco/patologia , Fatores de Tempo , Técnicas de Cultura de TecidosRESUMO
Dynamic spatiotemporal modification of chromatin around DNA damage is vital for efficient DNA repair. Normal stem cells exhibit an attenuated DNA damage response (DDR), inefficient DNA repair, and high radiosensitivity. The impact of unique chromatin characteristics of stem cells in DDR regulation is not yet recognized. We demonstrate that murine embryonic stem cells (ES) display constitutively elevated acetylation of histone H3 lysine 9 (H3K9ac) and low H3K9 tri-methylation (H3K9me3). DNA damage-induced local deacetylation of H3K9 was abrogated in ES along with the subsequent H3K9me3. Depletion of H3K9ac in ES by suppression of monocytic leukemia zinc finger protein (MOZ) acetyltransferase improved ATM activation, DNA repair, diminished irradiation-induced apoptosis, and enhanced clonogenic survival. Simultaneous suppression of the H3K9 methyltransferase Suv39h1 abrogated the radioprotective effect of MOZ inhibition, suggesting that high H3K9ac promoted by MOZ in ES cells obstructs local upregulation of H3K9me3 and contributes to muted DDR and increased radiosensitivity.
Assuntos
Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/efeitos da radiação , Histonas/metabolismo , Lisina/metabolismo , Tolerância a Radiação , Radiação Ionizante , Acetilação , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quebras de DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Regulação para Baixo/efeitos da radiação , Histona Acetiltransferases/metabolismo , Metilação , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/efeitos da radiação , Tolerância a Radiação/efeitos da radiação , Regulação para Cima/genética , Regulação para Cima/efeitos da radiaçãoRESUMO
Normal tissue injury resulting from cancer radiotherapy is often associated with diminished regenerative capacity. We examined the relative radiosensitivity of normal stem cell populations compared with non-stem cells within several radiosensitive tissue niches and culture models. We found that these stem cells are highly radiosensitive, in contrast to their isogenic differentiated progeny. Of interest, they also exhibited a uniquely attenuated DNA damage response (DDR) and muted DNA repair. Whereas stem cells exhibit reduced ATM activation and ionizing radiation-induced foci, they display apoptotic pannuclear H2AX-S139 phosphorylation (γH2AX), indicating unique radioresponses. We also observed persistent phosphorylation of H2AX-Y142 along the DNA breaks in stem cells, which promotes apoptosis while inhibiting DDR signaling. In addition, down-regulation of constitutively elevated histone-3 lysine-56 acetylation (H3K56ac) in stem cells significantly decreased their radiosensitivity, restored DDR function, and increased survival, signifying its role as a key contributor to stem cell radiosensitivity. These results establish that unique epigenetic landscapes affect cellular heterogeneity in radiosensitivity and demonstrate the nonubiquitous nature of radiation responses. We thus elucidate novel epigenetic rheostats that promote ionizing radiation hypersensitivity in various normal stem cell populations, identifying potential molecular targets for pharmacological radioprotection of stem cells and hopefully improving the efficacy of future cancer treatment.
Assuntos
Histonas/metabolismo , Células-Tronco/metabolismo , Células-Tronco/efeitos da radiação , Acetilação , Animais , Apoptose/efeitos da radiação , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Regulação para Baixo/efeitos dos fármacos , Epigênese Genética , Fator de Crescimento de Hepatócito/metabolismo , Lisina/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Fosforilação , Proteínas Proto-Oncogênicas/metabolismo , Tolerância a Radiação , Radiação Ionizante , Células-Tronco/patologiaRESUMO
BACKGROUND: Histone post-translational modifications are critical determinants of chromatin structure and function, impacting multiple biological processes including DNA transcription, replication, and repair. The post-translational acetylation of histone H4 at lysine 16 (H4K16ac) was initially identified in association with dosage compensation of the Drosophila male X chromosome. However, in mammalian cells, H4K16ac is not associated with dosage compensation and the genomic distribution of H4K16ac is not precisely known. Therefore, we have mapped the genome-wide H4K16ac distribution in human cells. RESULTS: We performed H4K16ac chromatin immunoprecipitation from human embryonic kidney 293 (HEK293) cells followed by hybridization to whole-genome tiling arrays and identified 25,893 DNA regions (false discovery rate <0.005) with average length of 692 nucleotides. Interestingly, although a majority of H4K16ac sites localized within genes, only a relatively small fraction (~10%) was found near promoters, in contrast to the distribution of the acetyltransferase, MOF, responsible for acetylation at K16 of H4. Using differential gene expression profiling data, 73 genes (> ±1.5-fold) were identified as potential H4K16ac-regulated genes. Seventeen transcription factor-binding sites were significantly associated with H4K16ac occupancy (p < 0.0005). In addition, a consensus 12-nucleotide guanine-rich sequence motif was identified in more than 55% of the H4K16ac peaks. CONCLUSIONS: The results suggest that H4K16 acetylation has a limited effect on transcription regulation in HEK293 cells, whereas H4K16ac has been demonstrated to have critical roles in regulating transcription in mouse embryonic stem cells. Thus, H4K16ac-dependent transcription regulation is likely a cell type specific process.
RESUMO
Synthetic lethality is a powerful approach to study selective cell killing based on genotype. We show that loss of Rad52 function is synthetically lethal with breast cancer 2, early onset (BRCA2) deficiency, whereas there was no impact on cell growth and viability in BRCA2-complemented cells. The frequency of both spontaneous and double-strand break-induced homologous recombination and ionizing radiation-induced Rad51 foci decreased by 2-10 times when Rad52 was depleted in BRCA2-deficient cells, with little to no effect in BRCA2-complemented cells. The absence of both Rad52 and BRCA2 resulted in extensive chromosome aberrations, especially chromatid-type aberrations. Ionizing radiation-induced and S phase-associated Rad52-Rad51 foci form equally well in the presence or absence of BRCA2, indicating that Rad52 can respond to DNA double-strand breaks and replication stalling independently of BRCA2. Rad52 thus is an independent and alternative repair pathway of homologous recombination and a target for therapy in BRCA2-deficient cells.
Assuntos
Proteína BRCA2/genética , Regulação Neoplásica da Expressão Gênica , Rad51 Recombinase/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Instabilidade Cromossômica , Aberrações Cromossômicas , Dano ao DNA , Teste de Complementação Genética , Células HeLa , Humanos , Microscopia de Fluorescência/métodos , Sais de Tetrazólio/farmacologia , Tiazóis/farmacologiaRESUMO
DNA double-strand breaks (DSBs) are generated by the recombination activating gene (RAG) endonuclease in all developing lymphocytes as they assemble antigen receptor genes. DNA cleavage by RAG occurs only at the G1 phase of the cell cycle and generates two hairpin-sealed DNA (coding) ends that require nucleolytic opening before their repair by classical non-homologous end-joining (NHEJ). Although there are several cellular nucleases that could perform this function, only the Artemis nuclease is able to do so efficiently. Here, in vivo, we show that in murine cells the histone protein H2AX prevents nucleases other than Artemis from processing hairpin-sealed coding ends; in the absence of H2AX, CtIP can efficiently promote the hairpin opening and resection of DNA ends generated by RAG cleavage. This CtIP-mediated resection is inhibited by γ-H2AX and by MDC-1 (mediator of DNA damage checkpoint 1), which binds to γ-H2AX in chromatin flanking DNA DSBs. Moreover, the ataxia telangiectasia mutated (ATM) kinase activates antagonistic pathways that modulate this resection. CtIP DNA end resection activity is normally limited to cells at post-replicative stages of the cell cycle, in which it is essential for homology-mediated repair. In G1-phase lymphocytes, DNA ends that are processed by CtIP are not efficiently joined by classical NHEJ and the joints that do form frequently use micro-homologies and show significant chromosomal deletions. Thus, H2AX preserves the structural integrity of broken DNA ends in G1-phase lymphocytes, thereby preventing these DNA ends from accessing repair pathways that promote genomic instability.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Fase G1 , Rearranjo Gênico do Linfócito B , Histonas/metabolismo , Linfócitos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Linhagem Celular Transformada , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endonucleases , Rearranjo Gênico do Linfócito B/genética , Instabilidade Genômica , Histonas/deficiência , Histonas/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Linfócitos/citologia , Camundongos , Proteínas Nucleares , Células Precursoras de Linfócitos B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Recombinação Genética/genética , Especificidade por Substrato , Proteínas Supressoras de Tumor/metabolismoRESUMO
The human MOF gene encodes a protein that specifically acetylates histone H4 at lysine 16 (H4K16ac). Here we show that reduced levels of H4K16ac correlate with a defective DNA damage response (DDR) and double-strand break (DSB) repair to ionizing radiation (IR). The defect, however, is not due to altered expression of proteins involved in DDR. Abrogation of IR-induced DDR by MOF depletion is inhibited by blocking H4K16ac deacetylation. MOF was found to be associated with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a protein involved in nonhomologous end-joining (NHEJ) repair. ATM-dependent IR-induced phosphorylation of DNA-PKcs was also abrogated in MOF-depleted cells. Our data indicate that MOF depletion greatly decreased DNA double-strand break repair by both NHEJ and homologous recombination (HR). In addition, MOF activity was associated with general chromatin upon DNA damage and colocalized with the synaptonemal complex in male meiocytes. We propose that MOF, through H4K16ac (histone code), has a critical role at multiple stages in the cellular DNA damage response and DSB repair.
Assuntos
Quebras de DNA de Cadeia Dupla , Dano ao DNA , Reparo do DNA/fisiologia , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Acetilação , Proteínas Mutadas de Ataxia Telangiectasia , Sequência de Bases , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Cromatina/metabolismo , Cromatina/efeitos da radiação , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Feminino , Células HL-60 , Histona Acetiltransferases/antagonistas & inibidores , Histona Acetiltransferases/genética , Inibidores de Histona Desacetilases/farmacologia , Histonas/química , Humanos , Lisina/química , Masculino , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Complexo Sinaptonêmico/metabolismo , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/metabolismoRESUMO
The Mre11-Rad50-Nbs1 (MRN) complex functions in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) at postreplicative stages of the cell cycle. During HR, the MRN complex functions directly in the repair of DNA DSBs and in the initiation of DSB responses through activation of the ataxia telangiectasia-mutated (ATM) serine-threonine kinase. Whether MRN functions in DNA damage responses before DNA replication in G0/G1 phase cells has been less clear. In developing G1-phase lymphocytes, DNA DSBs are generated by the Rag endonuclease and repaired during the assembly of antigen receptor genes by the process of V(D)J recombination. Mice and humans deficient in MRN function exhibit lymphoid phenotypes that are suggestive of defects in V(D)J recombination. We show that during V(D)J recombination, MRN deficiency leads to the aberrant joining of Rag DSBs and to the accumulation of unrepaired coding ends, thus establishing a functional role for MRN in the repair of Rag-mediated DNA DSBs. Moreover, these defects in V(D)J recombination are remarkably similar to those observed in ATM-deficient lymphocytes, suggesting that ATM and MRN function in the same DNA DSB response pathways during lymphocyte antigen receptor gene assembly.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromossomos de Mamíferos/metabolismo , Quebras de DNA de Cadeia Dupla , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas Nucleares/metabolismo , Hidrolases Anidrido Ácido , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/antagonistas & inibidores , Enzimas Reparadoras do DNA/deficiência , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/deficiência , Ativação Enzimática , Humanos , Proteína Homóloga a MRE11 , Camundongos , Proteínas Nucleares/deficiência , Proteínas Oncogênicas v-abl/metabolismo , Células Precursoras de Linfócitos B/enzimologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Recombinação Genética/genética , Retroviridae , Timo/citologia , Proteínas Supressoras de Tumor/antagonistas & inibidores , Éxons VDJ/genéticaRESUMO
HP1 proteins are thought to be modulators of chromatin organization in all mammals, yet their exact physiological function remains unknown. In a first attempt to elucidate the function of these proteins in vivo, we disrupted the murine Cbx1 gene, which encodes the HP1-beta isotype, and show that the Cbx1(-/-) -null mutation leads to perinatal lethality. The newborn mice succumbed to acute respiratory failure, whose likely cause is the defective development of neuromuscular junctions within the endplate of the diaphragm. We also observe aberrant cerebral cortex development in Cbx1(-/-) mutant brains, which have reduced proliferation of neuronal precursors, widespread cell death, and edema. In vitro cultures of neurospheres from Cbx1(-/-) mutant brains reveal a dramatic genomic instability. Our results demonstrate that HP1 proteins are not functionally redundant and that they are likely to regulate lineage-specific changes in heterochromatin organization.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Diafragma/metabolismo , Heterocromatina/metabolismo , Placa Motora/metabolismo , Neocórtex/metabolismo , Animais , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Diafragma/embriologia , Diafragma/patologia , Instabilidade Genômica/genética , Heterocromatina/genética , Heterocromatina/patologia , Humanos , Camundongos , Camundongos Knockout , Placa Motora/embriologia , Placa Motora/patologia , Neocórtex/embriologia , Neocórtex/patologiaRESUMO
Single-strand DNA (ssDNA)-binding proteins (SSBs) are ubiquitous and essential for a wide variety of DNA metabolic processes, including DNA replication, recombination, DNA damage detection and repair. SSBs have multiple roles in binding and sequestering ssDNA, detecting DNA damage, stimulating nucleases, helicases and strand-exchange proteins, activating transcription and mediating protein-protein interactions. In eukaryotes, the major SSB, replication protein A (RPA), is a heterotrimer. Here we describe a second human SSB (hSSB1), with a domain organization closer to the archaeal SSB than to RPA. Ataxia telangiectasia mutated (ATM) kinase phosphorylates hSSB1 in response to DNA double-strand breaks (DSBs). This phosphorylation event is required for DNA damage-induced stabilization of hSSB1. Upon induction of DNA damage, hSSB1 accumulates in the nucleus and forms distinct foci independent of cell-cycle phase. These foci co-localize with other known repair proteins. In contrast to RPA, hSSB1 does not localize to replication foci in S-phase cells and hSSB1 deficiency does not influence S-phase progression. Depletion of hSSB1 abrogates the cellular response to DSBs, including activation of ATM and phosphorylation of ATM targets after ionizing radiation. Cells deficient in hSSB1 exhibit increased radiosensitivity, defective checkpoint activation and enhanced genomic instability coupled with a diminished capacity for DNA repair. These findings establish that hSSB1 influences diverse endpoints in the cellular DNA damage response.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica , Proteínas Mutadas de Ataxia Telangiectasia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Proteínas de Ciclo Celular/metabolismo , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Instabilidade Genômica/efeitos da radiação , Células HeLa , Humanos , Proteínas Mitocondriais , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico/efeitos da radiação , Radiação Ionizante , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Proteínas Supressoras de Tumor/metabolismoRESUMO
The mammalian ortholog of the Drosophila MOF (males absent on the first) gene product is a histone H4 lysine 16-specific acetyltransferase. Recent studies have shown that depletion of human MOF (hMOF) in human cell lines leads to genomic instability, spontaneous chromosomal aberrations, cell cycle defects, altered nuclear morphology, reduced transcription of certain genes, and defective DNA damage response to ionizing radiation (IR). Here we show that MOF plays an essential role in mammals during embryogenesis and oncogenesis. Ablation of the mouse Mof gene (mMof) by gene targeting resulted in early embryonic lethality and cell death. Lethality correlated with the loss of H4 lysine 16 acetylation (H4K16ac) and could not be rescued by concomitant inactivation of ATM or p53. In comparison to primary cells or normal tissue, all immortalized human normal and tumor cell lines and primary tumors demonstrated similar or elevated hMOF and H4K16ac levels. Accordingly, MOF overexpression correlated with increased cellular proliferation, oncogenic transformation, and tumor growth. Thus, these data reveal that the acetylation of histone H4 at K16 by MOF is an epigenetic signature of cellular proliferation common to both embryogenesis and oncogenesis and that MOF is an essential factor for embryogenesis and oncogenesis.
Assuntos
Transformação Celular Neoplásica/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Acetilação , Animais , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Células Cultivadas , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Perda do Embrião/genética , Perda do Embrião/metabolismo , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Deleção de Genes , Regulação da Expressão Gênica , Histona Acetiltransferases/genética , Masculino , Camundongos , Proteínas Nucleares/genética , RNA Mensageiro/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Ataxia-telangiectasia mutated (ATM)-deficient lymphocytes exhibit defects in coding joint formation during V(D)J recombination in vitro. Similar defects in vivo should affect both T and B cell development, yet the lymphoid phenotypes of ATM deficiency are more pronounced in the T cell compartment. In this regard, ATM-deficient mice exhibit a preferential T lymphopenia and have an increased incidence of nontransformed and transformed T cells with T cell receptor alpha/delta locus translocations. We demonstrate that there is an increase in the accumulation of unrepaired coding ends during different steps of antigen receptor gene assembly at both the immunoglobulin and T cell receptor loci in developing ATM-deficient B and T lymphocytes. Furthermore, we show that the frequency of ATM-deficient alphabeta T cells with translocations involving the T cell receptor alpha/delta locus is directly related to the number of T cell receptor alpha rearrangements that these cells can make during development. Collectively, these findings demonstrate that ATM deficiency leads to broad defects in coding joint formation in developing B and T lymphocytes in vivo, and they provide a potential molecular explanation as to why the developmental impact of these defects could be more pronounced in the T cell compartment.
Assuntos
Linfócitos B/metabolismo , Proteínas de Ligação a DNA/deficiência , Região de Junção de Imunoglobulinas/fisiologia , Proteínas Serina-Treonina Quinases/deficiência , Receptores de Antígenos de Linfócitos T/biossíntese , Recombinação Genética/fisiologia , Linfócitos T/metabolismo , Proteínas Supressoras de Tumor/deficiência , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Southern Blotting , Proteínas de Ciclo Celular , Citometria de Fluxo , Região de Junção de Imunoglobulinas/biossíntese , Região de Junção de Imunoglobulinas/genética , Camundongos , Reação em Cadeia da Polimerase/métodos , Receptores de Antígenos de Linfócitos T/genética , Recombinação Genética/imunologiaRESUMO
Homeobox genes encode transcription factors which function in body axis patterning in the developing embryo. Recent evidence suggests that the maintenance of specific HOX expression patterns is necessary for regulating the homeostasis of adult tissues as well. In this study, HOXB7 transformed human mammary epithelial cells, MCF10A, to grow in minimally supplemented medium, to form colonies in Matrigel, and display resistance to ionizing radiation. Searching for protein partners of HOXB7 that might contribute to resistance to ionizing radiation, we identified four HOXB7-binding proteins by GST pull-down/affinity chromatography and confirmed their interactions by coimmunoprecipitation in vivo. Interestingly, all four HOXB7-binding proteins shared functions as genomic caretakers and included members of the DNA-dependent protein kinase holoenzyme (Ku70, Ku80, DNA-PK(cs)) responsible for DNA double-strand break repair by nonhomologous end joining pathway and poly(ADP) ribose polymerase. Exogenous and endogenous expression of HOXB7 enhanced nonhomologous end joining and DNA repair functions in vitro and in vivo, which were reversed by silencing HOXB7. This is the first mechanistic study providing definitive evidence for the involvement of any HOX protein in DNA double-strand break repair.
Assuntos
Reparo do DNA/fisiologia , Proteínas de Homeodomínio/fisiologia , Sequência de Aminoácidos , Antígenos Nucleares/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Reparo do DNA/genética , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Humanos , Autoantígeno Ku , Dados de Sequência Molecular , Tolerância a Radiação/fisiologiaRESUMO
The ATM (ataxia-telangiectasia mutated) protein kinase mediates early cellular responses to DNA double-strand breaks (DSBs) generated during metabolic processes or by DNA-damaging agents. ATM deficiency leads to ataxia-telangiectasia, a disease marked by lymphopenia, genomic instability and an increased predisposition to lymphoid malignancies with chromosomal translocations involving lymphocyte antigen receptor loci. ATM activates cell-cycle checkpoints and can induce apoptosis in response to DNA DSBs. However, defects in these pathways of the DNA damage response cannot fully account for the phenotypes of ATM deficiency. Here, we show that ATM also functions directly in the repair of chromosomal DNA DSBs by maintaining DNA ends in repair complexes generated during lymphocyte antigen receptor gene assembly. When coupled with the cell-cycle checkpoint and pro-apoptotic activities of ATM, these findings provide a molecular explanation for the increase in lymphoid tumours with translocations involving antigen receptor loci associated with ataxia-telangiectasia.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Rearranjo Gênico do Linfócito B/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia , Linfócitos B/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular , Quebra Cromossômica/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Camundongos , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Células-Tronco/metabolismo , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genéticaRESUMO
The protein products of several rad checkpoint genes of Schizosaccharomyces pombe (rad1+, rad3+, rad9+, rad17+, rad26+, and hus1+) play crucial roles in sensing changes in DNA structure, and several function in the maintenance of telomeres. When the mammalian homologue of S. pombe Rad9 was inactivated, increases in chromosome end-to-end associations and frequency of telomere loss were observed. This telomere instability correlated with enhanced S- and G2-phase-specific cell killing, delayed kinetics of gamma-H2AX focus appearance and disappearance, and reduced chromosomal repair after ionizing radiation (IR) exposure, suggesting that Rad9 plays a role in cell cycle phase-specific DNA damage repair. Furthermore, mammalian Rad9 interacted with Rad51, and inactivation of mammalian Rad9 also resulted in decreased homologous recombinational (HR) repair, which occurs predominantly in the S and G2 phases of the cell cycle. Together, these findings provide evidence of roles for mammalian Rad9 in telomere stability and HR repair as a mechanism for promoting cell survival after IR exposure.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/genética , Reparo do DNA/genética , Recombinação Genética , Telômero/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Ciclo Celular/efeitos da radiação , Proteínas de Ciclo Celular/genética , Sobrevivência Celular/genética , Quinase do Ponto de Checagem 2 , Aberrações Cromossômicas , DNA/genética , DNA/metabolismo , DNA/efeitos da radiação , Dano ao DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fase G2/genética , Fase G2/efeitos da radiação , Histonas/genética , Histonas/metabolismo , Histonas/efeitos da radiação , Humanos , Mamíferos , Mutação , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Radiação Ionizante , Fase S/genética , Fase S/efeitos da radiação , Proteínas de Schizosaccharomyces pombe , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/metabolismo , Telômero/efeitos da radiação , Proteína 2 de Ligação a Repetições Teloméricas , Proteínas Supressoras de Tumor/metabolismoRESUMO
The exact duplication of chromosomal DNA during each cell cycle ensures the correct inheritance of genetic material from mother to daughter cells. In eukaryotic cells, DNA replication can occur only when the origin of DNA replication is accurately marked by a group of proteins termed licensing proteins. One such protein is Cdt1, which is recruited first to the origin of DNA replication followed by cell division cycle 6 (Cdc6) and mini-chromosome maintenance proteins (Mcms). We previously reported that NIH3T3 cells overexpressing Cdt1 readily formed tumors in mice. To further investigate its oncogenic mechanism, we generated transgenic mice expressing Cdt1 in thymocytes. Our studies demonstrated that T-cell-directed Cdt1 transgenic mice showed normal T-cell development. However, such transgenic mice developed thymic lymphoblastic lymphoma when crossed with p53 null mice. Furthermore, tumor cells derived from NIH3T3 cells overexpressing Cdt1 displayed numerical and structural chromosomal aberrations in the form of ploidy, double minutes, translocation, inversion, chromosome end-to-end fusion and robertsonian mutation. Collectively, our studies suggest that Cdt1 overexpression most likely contributes to tumorigenecity by causing genomic instability.
Assuntos
Proteínas de Ciclo Celular/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteína Supressora de Tumor p53/deficiência , Envelhecimento , Animais , Apoptose , Sequência de Bases , Primers do DNA , Feminino , Hormônio do Crescimento Humano/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Regiões Promotoras Genéticas , Linfócitos T , TransfecçãoRESUMO
Ataxia-telangiectasia (A-T) is a multisystem, cancer-predisposing genetic disorder caused by deficiency of the ATM protein. To dissect the A-T phenotype, we augmented specific features of the human disease by generating mouse strains that combine Atm deficiency with dysfunction of other proteins. Increasing oxidative stress by combining deficiencies in Atm and superoxide dismutase 1 (Sod1) exacerbated growth retardation and markedly reduced the mean survival time following ionizing radiation. In contrast, increasing genomic instability by combining deficiencies of Atm and the mismatch repair protein Mlh1 caused a moderate increase in radiation sensitivity and dramatic increase in aggressive lymphomas, compared with thes Atm-/- single knockout. Remarkably, Atm, Mlh1 or Mlh1/Atm single or double heterozygosity did not significantly affect the life span of the various genotypes. Mlh1/Atm double null tumors were polyclonal, whereas the tumors in other genotypes were mono- or oligoclonal, demonstrating the high predisposition of thymocytes with this genotype to become malignant. Chromosomal aberrations in the tumors were localized mainly in chromosomes 12 and 15. The genomic region on chromosome 15, which contains the gene for the c-Myc oncoprotein, was commonly amplified, and elevated levels of the c-Myc protein were subsequently observed in the tumors. Our data suggest that impaired genomic instability is an important contributing factor to cancer predisposition in A-T, whereas oxidative stress is more important in the radiation sensitivity and growth retardation facets of this disease.
Assuntos
Ataxia Telangiectasia/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Instabilidade Genômica/genética , Linfoma/genética , Estresse Oxidativo , Proteínas Serina-Treonina Quinases/genética , Tolerância a Radiação/genética , Neoplasias do Timo/genética , Proteínas Supressoras de Tumor/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Aberrações Cromossômicas , Amplificação de Genes , Predisposição Genética para Doença/genética , Genótipo , Camundongos , Camundongos Knockout , Repetições de Microssatélites/genética , Mutação , Superóxido Dismutase/genética , Superóxido Dismutase-1RESUMO
We have determined that hMOF, the human ortholog of the Drosophila MOF gene (males absent on the first), encoding a protein with histone acetyltransferase activity, interacts with the ATM (ataxia-telangiectasia-mutated) protein. Cellular exposure to ionizing radiation (IR) enhances hMOF-dependent acetylation of its target substrate, lysine 16 (K16) of histone H4 independently of ATM function. Blocking the IR-induced increase in acetylation of histone H4 at K16, either by the expression of a dominant negative mutant DeltahMOF or by RNA interference-mediated hMOF knockdown, resulted in decreased ATM autophosphorylation, ATM kinase activity, and the phosphorylation of downstream effectors of ATM and DNA repair while increasing cell killing. In addition, decreased hMOF activity was associated with loss of the cell cycle checkpoint response to DNA double-strand breaks. The overexpression of wild-type hMOF yielded the opposite results, i.e., a modest increase in cell survival and enhanced DNA repair after IR exposure. These results suggest that hMOF influences the function of ATM.