RESUMO
Current techniques for examining the global creation and repair of DNA double-strand breaks are restricted in their sensitivity, and such techniques mask any site-dependent variations in breakage and repair rate or fidelity. We present here a system for analyzing the fate of documented DNA breaks, using the MLL gene as an example, through application of ligation-mediated PCR. Here, a simple asymmetric double-stranded DNA adapter molecule is ligated to experimentally induced DNA breaks and subjected to seminested PCR using adapter and gene-specific primers. The rate of appearance and loss of specific PCR products allow detection of both the break and its repair. Using the additional technique of inverse PCR, the presence of misrepaired products (translocations) can be detected at the same site, providing information on the fidelity of the ligation reaction in intact cells. Such techniques may be adapted for the analysis of DNA breaks and rearrangements introduced into any identifiable genomic location. We have also applied parallel sequencing for the high-throughput analysis of inverse PCR products to facilitate the unbiased recording of all rearrangements located at a specific genomic location.
Assuntos
Cromossomos/genética , Quebras de DNA de Cadeia Dupla , DNA/genética , Reação em Cadeia da Polimerase/métodos , Translocação Genética , Apoptose/genética , DNA Ligases , Primers do DNA , Reparo do DNA , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Histona-Lisina N-Metiltransferase/genética , Humanos , Proteína de Leucina Linfoide-Mieloide/genética , Fluxo de TrabalhoRESUMO
Current techniques for examining the global creation and repair of DNA double-strand breaks are restricted in their sensitivity, and such techniques mask any site-dependent variations in breakage and repair rate or fidelity. We present here a system for analyzing the fate of documented DNA breaks, using the MLL gene as an example, through application of ligation-mediated PCR. Here, a simple asymmetric double-stranded DNA adapter molecule is ligated to experimentally induced DNA breaks and subjected to seminested PCR using adapter and gene-specific primers. The rate of appearance and loss of specific PCR products allows detection of both the break and its repair. Using the additional technique of inverse PCR, the presence of misrepaired products (translocations) can be detected at the same site, providing information on the fidelity of the ligation reaction in intact cells. Such techniques may be adapted for the analysis of DNA breaks introduced into any identifiable genomic location.
Assuntos
Quebra Cromossômica , Reparo do DNA , DNA/análise , Reação em Cadeia da Polimerase/métodos , Translocação Genética , Células Cultivadas , DNA/efeitos dos fármacos , DNA/efeitos da radiação , Dano ao DNA , Primers do DNA/química , HumanosRESUMO
Activation of apoptosis introduces a site-specific break within intron 11 of the MLL gene. Using the CD95 apoptotic signaling pathway in human lymphoblastoid cells, the 5' fragment of MLL undergoes translocation to intron 4 of AF9 and the proleukemogenic MLL-AF9 fusion gene created is transcribed. Both the breaks in MLL and transcription of the MLL-AF9 fusion gene are suppressed in the presence of the broad spectrum caspase inhibitor, zVAD.fmk. Duplicate cells containing sequence identical MLL-AF9 fusion junctions were identified within a cell population that had recovered from apoptosis. This indicated that cells harboring a translocation initiated by apoptotic cleavage had divided. These data are consistent with a novel pathogenic role for the apoptotic program where translocations with leukemogenic potential are created within cells that have the capacity to divide.
Assuntos
Apoptose/genética , Proteínas de Ligação a DNA/genética , Proteínas Nucleares/genética , Proto-Oncogenes , Fatores de Transcrição , Translocação Genética , Divisão Celular/genética , Linhagem Celular , Quebra Cromossômica , Proteínas de Ligação a DNA/biossíntese , Histona-Lisina N-Metiltransferase , Humanos , Íntrons , Linfócitos/citologia , Linfócitos/metabolismo , Linfócitos/fisiologia , Proteína de Leucina Linfoide-Mieloide , Proteínas Nucleares/biossíntese , Proteínas de Fusão Oncogênica/biossíntese , Proteínas de Fusão Oncogênica/genética , Reação em Cadeia da Polimerase/métodos , Transdução de Sinais/fisiologia , Transcrição Gênica , Receptor fas/fisiologiaRESUMO
Purpose The rejoining of fragmented nuclear DNA caused by ionizing radiation may lead to lethal chromosome rearrangements, such as rings or dicentrics. The clinically useful linear quadratic relationship between dose and cell survival has been interpreted as the generation of lethal lesions secondary to damage occurring in two separate chromosomes simultaneously (α component), or as potentially repairable separate events (ß component). Here, the generation of such lesions is discussed, synthesizing existing knowledge with new insights gleaned from spatial proximity data made possible by high-throughput sequencing of chromosome conformation capture experiments. Over a range of several Mbp, the linear DNA strand is organized as a fractal globule generating multiple sites of contact that may facilitate deletions or inversions if the points of contact are damaged. On a larger scale, transcriptionally active euchromatin occupies a physically identifiable space separate from inactive areas and is preferentially susceptible to free radical attack after irradiation. Specific transcriptional programs link genomic locations within that space, potentially enhancing their interaction if subject to simultaneous fragmentation by a single radiation event. Conclusions High throughput spatial analysis of the factors that control chromosome proximity has the potential to better describe the formation of the lethal chromosome aberrations that kill irradiated cells.
Assuntos
Apoptose/genética , Núcleo Celular/efeitos da radiação , Aberrações Cromossômicas/efeitos da radiação , Dano ao DNA/fisiologia , DNA/efeitos da radiação , Modelos Genéticos , Animais , Apoptose/efeitos da radiação , Núcleo Celular/fisiologia , Simulação por Computador , DNA/genética , Relação Dose-Resposta à Radiação , Medicina Baseada em Evidências , Humanos , Doses de RadiaçãoRESUMO
Current techniques for examining the global creation and repair of DNA double-strand breaks are restricted in their sensitivity, and such techniques mask any site-dependent variations in breakage and repair rate or fidelity. We present here a system for analyzing the fate of documented DNA breaks, using the MLL gene as an example, through application of ligation-mediated PCR. Here, a simple asymmetric double-stranded DNA adapter molecule is ligated to experimentally induced DNA breaks and subjected to seminested PCR using adapter and gene-specific primers. The rate of appearance and loss of specific PCR products allows detection of both the break and its repair. Using the additional technique of inverse PCR, the presence of misrepaired products (translocations) can be detected at the same site, providing information on the fidelity of the ligation reaction in intact cells. Such techniques may be adapted for the analysis of DNA breaks introduced into any identifiable genomic location.
Assuntos
Quebra Cromossômica/genética , Dano ao DNA , Reação em Cadeia da Polimerase/métodos , Translocação Genética/genética , Animais , DNA/análise , DNA/genética , Proteínas de Ligação a DNA/genética , Histona-Lisina N-Metiltransferase , Humanos , Proteína de Leucina Linfoide-Mieloide , Proto-Oncogenes/genética , Fatores de Transcrição/genéticaRESUMO
Epidemiological data have linked birth control formulations to an increased risk of infant acute leukemia involving MLL rearrangements. Reverse transcription polymerase chain reaction (RT-PCR) studies showed that 10 nM estradiol enhanced MLL transcription in addition to its common translocation partners, MLLT2 (AF4) and MLLT3 (AF9). The same concentration of estradiol triggered MLL and MLLT3 co-localization without affecting the interaction of genes located on the same chromosomes. Estradiol also stimulated the generation of MLL-MLLT3 fusion transcripts as seen by RT-PCR. RNAi knockdown of activation-induced cytidine deaminase (AICDA) suppressed the induction of MLL-MLLT3 fusion transcript formation observed with estradiol. Additionally, chromatin immunoprecipitation (ChIP) analysis showed estradiol dependent localization of AICDA in MLL intron 11, upstream of a hotspot for both DNA cleavage and rearrangement, but not downstream within intron 12. Combined, these studies show that levels of estradiol consistent with that observed during pregnancy have the potential to initiate MLL fusions through an AICDA-mediated mechanism.
Assuntos
Citidina Desaminase/metabolismo , Estradiol/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Fusão Oncogênica/genética , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Loci Gênicos , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Ligação Proteica , Transporte Proteico , Transcrição Gênica , Fatores de Elongação da TranscriçãoRESUMO
SIRT3 is a key NAD+-dependent protein deacetylase in the mitochondria of mammalian cells, functioning to prevent cell aging and transformation via regulation of mitochondrial metabolic homeostasis. However, SIRT3 is also found to express in some human tumors; its role in these SIRT3-expressing tumor cells needs to be elucidated. This study demonstrated that the expression of SIRT3 was elevated in a group of gastric cancer cells compared to normal gastric epithelial cells. Although SIRT3 expression levels were increased in the gastric tumor tissues compared to the adjacent non-tumor tissues, SIRT3 positive cancer cells were more frequently detected in the intestinal type gastric cancers than the diffuse type gastric cancers, indicating that SIRT3 is linked with subtypes of gastric cancer. Overexpression of SIRT3 promoted cell proliferation and enhanced ATP generation, glucose uptake, glycogen formation, MnSOD activity and lactate production, which were inhibited by SIRT3 knockdown, indicating that SIRT3 plays a role in reprogramming the bioenergetics in gastric tumor cells. Further analysis revealed that SIRT3 interacted with and deacetylated the lactate dehydrogenase A (LDHA), a key protein in regulating anaerobic glycolysis, enhancing LDHA activity. In consistence, a cluster of glycolysis-associated genes was upregulated in the SIRT3-overexpressing gastric tumor cells. Thus, in addition to the well-documented SIRT3-mediated mitochondrial homeostasis in normal cells, SIRT3 may enhance glycolysis and cell proliferation in SIRT3-expressing cancer cells.
Assuntos
Glicólise , Sirtuína 3/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Acetilação , Animais , Linhagem Celular Tumoral , Proliferação de Células , Metabolismo Energético , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos , Homeostase , Humanos , Camundongos Nus , Modelos Biológicos , Invasividade Neoplásica , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Gástricas/genética , Superóxido Dismutase/metabolismo , Regulação para Cima/genéticaRESUMO
Tumor adaptive resistance to therapeutic radiation remains a barrier for further improvement of local cancer control. SIRT3, a member of the sirtuin family of NAD(+)-dependent protein deacetylases in mitochondria, promotes metabolic homeostasis through regulation of mitochondrial protein deacetylation and plays a key role in prevention of cell aging. Here, we demonstrate that SIRT3 expression is induced in an array of radiation-treated human tumor cells and their corresponding xenograft tumors, including colon cancer HCT-116, glioblastoma U87, and breast cancer MDA-MB231 cells. SIRT3 transcriptional activation is due to SIRT3 promoter activation controlled by the stress transcription factor NF-κB. Posttranscriptionally, SIRT3 enzymatic activity is further enhanced via Thr150/Ser159 phosphorylation by cyclin B1-CDK1, which is also induced by radiation and relocated to mitochondria together with SIRT3. Cells expressing Thr150Ala/Ser159Ala-mutant SIRT3 show a reduction in mitochondrial protein lysine deacetylation, Δψm, MnSOD activity, and mitochondrial ATP generation. The clonogenicity of Thr150Ala/Ser159Ala-mutant transfectants is lower and significantly decreased under radiation. Tumors harboring Thr150Ala/Ser159Ala-mutant SIRT3 show inhibited growth and increased sensitivity to in vivo local irradiation. These results demonstrate that enhanced SIRT3 transcription and posttranslational modifications in mitochondria contribute to adaptive radioresistance in tumor cells. CDK1-mediated SIRT3 phosphorylation is a potential effective target to sensitize tumor cells to radiotherapy.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Mitocôndrias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Tolerância a Radiação/genética , Sirtuína 3/genética , Ativação Transcricional , Acetilação , Animais , Proteína Quinase CDC2 , Linhagem Celular Tumoral , Modelos Animais de Doenças , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Mitocôndrias/efeitos da radiação , Proteínas Mitocondriais/metabolismo , Mutação , NF-kappa B/metabolismo , Neoplasias/patologia , Neoplasias/radioterapia , Fosforilação , Sirtuína 3/metabolismo , Transcrição GênicaRESUMO
Mammalian cells are able to sense environmental oxidative and genotoxic conditions such as the environmental low-dose ionizing radiation (LDIR) present naturally on the earth's surface. The stressed cells then can induce a so-called radioadaptive response with an enhanced cellular homeostasis and repair capacity against subsequent similar genotoxic conditions such as a high dose radiation. Manganese superoxide dismutase (MnSOD), a primary mitochondrial antioxidant in mammals, has long been known to play a crucial role in radioadaptive protection by detoxifying O2(â¢-) generated by mitochondrial oxidative phosphorylation. In contrast to the well-studied mechanisms of SOD2 gene regulation, the mechanisms underlying posttranslational regulation of MnSOD for radioprotection remain to be defined. Herein, we demonstrate that cyclin D1/cyclin-dependent kinase 4 (CDK4) serves as the messenger to deliver the stress signal to mitochondria to boost mitochondrial homeostasis in human skin keratinocytes under LDIR-adaptive radioprotection. Cyclin D1/CDK4 relocates to mitochondria at the same time as MnSOD enzymatic activation peaks without significant changes in total MnSOD protein level. The mitochondrial-localized CDK4 directly phosphorylates MnSOD at serine-106 (S106), causing enhanced MnSOD enzymatic activity and mitochondrial respiration. Expression of mitochondria-targeted dominant negative CDK4 or the MnSOD-S106 mutant reverses LDIR-induced mitochondrial enhancement and adaptive protection. The CDK4-mediated MnSOD activation and mitochondrial metabolism boost are also detected in skin tissues of mice receiving in vivo whole-body LDIR. These results demonstrate a unique CDK4-mediated mitochondrial communication that allows cells to sense environmental genotoxic stress and boost mitochondrial homeostasis by enhancing phosphorylation and activation of MnSOD.
Assuntos
Quinase 4 Dependente de Ciclina/genética , Queratinócitos/efeitos da radiação , Mitocôndrias/efeitos da radiação , Superóxido Dismutase/genética , Adaptação Fisiológica , Animais , Linhagem Celular , Ciclina D1/genética , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Relação Dose-Resposta à Radiação , Regulação da Expressão Gênica , Humanos , Queratinócitos/citologia , Queratinócitos/enzimologia , Potencial da Membrana Mitocondrial/efeitos da radiação , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/enzimologia , Fosforilação Oxidativa , Fosforilação/efeitos dos fármacos , Tolerância a Radiação , Radiação Ionizante , Transdução de Sinais , Superóxido Dismutase/metabolismo , Irradiação Corporal TotalRESUMO
Rearrangements of the MLL gene involve multiple partners and are implicated in both therapy related acute leukemia [tAL] and infant acute leukemia. For these diseases, recently compiled clinical data confirms an elevated frequency of such breakpoints within a 4 kb tract between exon 11 and a region of structural instability adjacent to exon 12. Linked primarily to cases of tAL, interference with topoisomerase II activity may either contribute to the initial DNA lesion directly or indirectly by, for example, providing a physical block to transcription progression. Alternatively, sites of fragmentation may be mis-repaired, guided by intergenic spliced transcripts of the participating genes. Co-transcription of MLL and potential fusion partners may provide the localization that enhances the probability of gene interaction. An indirect role for the leukemogenic activity of topoisomerase II inhibitors would imply that the negative consequences of their use may be separated from their therapeutic effects.
Assuntos
Regulação Leucêmica da Expressão Gênica , Leucemia Aguda Bifenotípica/genética , Proteína de Leucina Linfoide-Mieloide/genética , Segunda Neoplasia Primária/genética , Proteínas de Fusão Oncogênica/genética , Adulto , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Epistasia Genética , Éxons , Histona-Lisina N-Metiltransferase , Humanos , Lactente , Íntrons , Leucemia Aguda Bifenotípica/metabolismo , Leucemia Aguda Bifenotípica/patologia , Proteína de Leucina Linfoide-Mieloide/metabolismo , Segunda Neoplasia Primária/induzido quimicamente , Segunda Neoplasia Primária/metabolismo , Segunda Neoplasia Primária/patologia , Proteínas de Fusão Oncogênica/metabolismo , Inibidores da Topoisomerase/efeitos adversos , Translocação GenéticaRESUMO
Current techniques for examining the global creation and repair of DNA double-strand breaks are restricted in their sensitivity, and such techniques mask any site-dependent variations in breakage and repair rate or fidelity. We present here a system for analyzing the fate of documented DNA breaks, using the MLL gene as an example, through application of ligation-mediated PCR. Here, a simple asymmetric double-stranded DNA adapter molecule is ligated to experimentally induced DNA breaks and subjected to seminested PCR using adapter- and gene-specific primers. The rate of appearance and loss of specific PCR products allows detection of both the break and its repair. Using the additional technique of inverse PCR, the presence of misrepaired products (translocations) can be detected at the same site, providing information on the fidelity of the ligation reaction in intact cells. Such techniques may be adapted for the analysis of DNA breaks and rearrangements introduced into any identifiable genomic location. We have also applied parallel sequencing for the high-throughput analysis of inverse PCR products to facilitate the unbiased recording of all rearrangements located at a specific genomic location.
Assuntos
Quebras de DNA de Cadeia Dupla , Análise Mutacional de DNA , Translocação Genética , Sequência de Bases , Linhagem Celular , Primers do DNA/genética , Reparo do DNA , Humanos , Reação em Cadeia da PolimeraseRESUMO
Calcitriol (1,25(OH)(2)D3) is cytostatic for prostate cancer (CaP), but had limited therapeutic utility due to hypercalcemia-related toxicities, leading to the development of low-calcemic calcitriol analogs. We show that one analog, 1-α-Hydroxyvitamin-D5 (1α(OH)D5), induced apoptosis in castration-sensitive LNCaP prostate cancer cells, but unlike calcitriol, did not increase androgen receptor (AR) transcriptional activity. LNCaP-AI, a castrate-resistant (CRCaP) LNCaP subline, was resistant to 1α(OH)D5 in the presence of androgens; however, androgen withdrawal (AWD), although ineffective by itself, sensitized LNCaP-AI cells to 1α(OH)D5. Investigation of the mechanism revealed that the vitamin D receptor (VDR), which mediates the effects of 1α(OH)D5, is downregulated in LNCaP-AI cells compared to LNCaP in the presence of androgens, whereas AWD restored VDR expression. Since LNCaP-AI cells expressed higher AR compared to LNCaP and AWD decreased AR, this indicated an inverse relationship between VDR and AR. Further, AR stimulation (by increased androgen) suppressed VDR, while AR downregulation (by ARsiRNA) stimulated VDR levels and sensitized LNCaP-AI cells to 1α(OH)D5 similar to AWD. Another cell line, pRNS-1-1, although isolated from a normal prostate, had lost AR expression in culture and adapted to androgen-independent growth. These cells expressed the VDR and were sensitive to 1α(OH)D5, but restoration of AR expression suppressed VDR levels and induced resistance to 1α(OH)D5 treatment. Taken together, these results demonstrate negative regulation of VDR by AR in CRCaP cells. This effect is likely mediated by prohibitin (PHB), which was inhibited by AR transcriptional activity and stimulated VDR in CRCaP, but not castrate-sensitive cells. Therefore, in castration sensitive cells, although the AR negatively regulates PHB, this does not affect VDR expression, whereas in CRCaP cells, negative regulation of PHB by the AR results in concomitant negative regulation of the VDR by the AR. These data demonstrate a novel mechanism by which 1α(OH)D5 prolong the effectiveness of AWD in CaP cells.