RESUMO
Replication origins, fragile sites, and rDNA have been implicated as sources of chromosomal instability. However, the defining genomic features of replication origins and fragile sites are among the least understood elements of eukaryote genomes. Here, we map sites of replication initiation and breakage in primary cells at high resolution. We find that replication initiates between transcribed genes within nucleosome-depleted structures established by long asymmetrical poly(dA:dT) tracts flanking the initiation site. Paradoxically, long (>20 bp) (dA:dT) tracts are also preferential sites of polar replication fork stalling and collapse within early-replicating fragile sites (ERFSs) and late-replicating common fragile sites (CFSs) and at the rDNA replication fork barrier. Poly(dA:dT) sequences are fragile because long single-strand poly(dA) stretches at the replication fork are unprotected by the replication protein A (RPA). We propose that the evolutionary expansion of poly(dA:dT) tracts in eukaryotic genomes promotes replication initiation, but at the cost of chromosome fragility.
Assuntos
Replicação do DNA , DNA Ribossômico/química , Nucleossomos/metabolismo , Poli dA-dT/química , Origem de Replicação , Motivos de Aminoácidos , Animais , Linhagem Celular , Imunoprecipitação da Cromatina , Instabilidade Cromossômica , Sítios Frágeis do Cromossomo , Fragilidade Cromossômica , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Saccharomyces cerevisiae , Schizosaccharomyces , Sítio de Iniciação de Transcrição , Transcrição GênicaRESUMO
In this study, we show that evolutionarily conserved chromosome loop anchors bound by CCCTC-binding factor (CTCF) and cohesin are vulnerable to DNA double strand breaks (DSBs) mediated by topoisomerase 2B (TOP2B). Polymorphisms in the genome that redistribute CTCF/cohesin occupancy rewire DNA cleavage sites to novel loop anchors. While transcription- and replication-coupled genomic rearrangements have been well documented, we demonstrate that DSBs formed at loop anchors are largely transcription-, replication-, and cell-type-independent. DSBs are continuously formed throughout interphase, are enriched on both sides of strong topological domain borders, and frequently occur at breakpoint clusters commonly translocated in cancer. Thus, loop anchors serve as fragile sites that generate DSBs and chromosomal rearrangements. VIDEO ABSTRACT.
Assuntos
Fragilidade Cromossômica , Quebras de DNA de Cadeia Dupla , Neoplasias/genética , Animais , Linfócitos B/metabolismo , Fator de Ligação a CCCTC , Linhagem Celular Tumoral , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Ligação a Poli-ADP-Ribose , Proteínas Repressoras/metabolismoRESUMO
53BP1 activity drives genome instability and lethality in BRCA1-deficient mice by inhibiting homologous recombination (HR). The anti-recombinogenic functions of 53BP1 require phosphorylation-dependent interactions with PTIP and RIF1/shieldin effector complexes. While RIF1/shieldin blocks 5'-3' nucleolytic processing of DNA ends, it remains unclear how PTIP antagonizes HR. Here, we show that mutation of the PTIP interaction site in 53BP1 (S25A) allows sufficient DNA2-dependent end resection to rescue the lethality of BRCA1Δ11 mice, despite increasing RIF1 "end-blocking" at DNA damage sites. However, double-mutant cells fail to complete HR, as excessive shieldin activity also inhibits RNF168-mediated loading of PALB2/RAD51. As a result, BRCA1Δ1153BP1S25A mice exhibit hallmark features of HR insufficiency, including premature aging and hypersensitivity to PARPi. Disruption of shieldin or forced targeting of PALB2 to ssDNA in BRCA1D1153BP1S25A cells restores RNF168 recruitment, RAD51 nucleofilament formation, and PARPi resistance. Our study therefore reveals a critical function of shieldin post-resection that limits the loading of RAD51.
Assuntos
Recombinação Homóloga/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Envelhecimento/efeitos dos fármacos , Envelhecimento/genética , Animais , Proteína BRCA1/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Instabilidade Genômica/efeitos dos fármacos , Instabilidade Genômica/genética , Recombinação Homóloga/efeitos dos fármacos , Camundongos , Mutação/efeitos dos fármacos , Mutação/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Rad51 Recombinase/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
BRCA1 functions at two distinct steps during homologous recombination (HR). Initially, it promotes DNA end resection, and subsequently it recruits the PALB2 and BRCA2 mediator complex, which stabilizes RAD51-DNA nucleoprotein filaments. Loss of 53BP1 rescues the HR defect in BRCA1-deficient cells by increasing resection, suggesting that BRCA1's downstream role in RAD51 loading is dispensable when 53BP1 is absent. Here we show that the E3 ubiquitin ligase RNF168, in addition to its canonical role in inhibiting end resection, acts in a redundant manner with BRCA1 to load PALB2 onto damaged DNA. Loss of RNF168 negates the synthetic rescue of BRCA1 deficiency by 53BP1 deletion, and it predisposes BRCA1 heterozygous mice to cancer. BRCA1+/-RNF168-/- cells lack RAD51 foci and are hypersensitive to PARP inhibitor, whereas forced targeting of PALB2 to DNA breaks in mutant cells circumvents BRCA1 haploinsufficiency. Inhibiting the chromatin ubiquitin pathway may, therefore, be a synthetic lethality strategy for BRCA1-deficient cancers.
Assuntos
Proteína BRCA1/genética , Cromatina/enzimologia , Fibroblastos/enzimologia , Haploinsuficiência , Neoplasias/enzimologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Animais , Proteína BRCA2/genética , Linhagem Celular Tumoral , Cromatina/genética , Dano ao DNA , Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , Proteína do Grupo de Complementação N da Anemia de Fanconi/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genéticaRESUMO
Topoisomerase II (TOP2) relieves torsional stress by forming transient cleavage complex intermediates (TOP2ccs) that contain TOP2-linked DNA breaks (DSBs). While TOP2ccs are normally reversible, they can be "trapped" by chemotherapeutic drugs such as etoposide and subsequently converted into irreversible TOP2-linked DSBs. Here, we have quantified etoposide-induced trapping of TOP2ccs, their conversion into irreversible TOP2-linked DSBs, and their processing during DNA repair genome-wide, as a function of time. We find that while TOP2 chromatin localization and trapping is independent of transcription, it requires pre-existing binding of cohesin to DNA. In contrast, the conversion of trapped TOP2ccs to irreversible DSBs during DNA repair is accelerated 2-fold at transcribed loci relative to non-transcribed loci. This conversion is dependent on proteasomal degradation and TDP2 phosphodiesterase activity. Quantitative modeling shows that only two features of pre-existing chromatin structure-namely, cohesin binding and transcriptional activity-can be used to predict the kinetics of TOP2-induced DSBs.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo II/química , DNA/genética , Complexos Multiproteicos/química , Proteínas de Ligação a Poli-ADP-Ribose/química , Quebra Cromossômica , Cromossomos/genética , DNA/química , Reparo do DNA/genética , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Etoposídeo/química , Conversão Gênica/genética , Células HCT116 , Humanos , Cinética , Complexos Multiproteicos/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/farmacologia , Torção Mecânica , Transcrição Gênica , Translocação Genética/genéticaRESUMO
Little research has taken place on the effect of euthanasia methods on biophysical and biochemical changes at the time of euthanasia in fish. These changes are used in multiple species to determine stress levels before death. Koi (Cyprinus carpio) are an important fish species often used in laboratory research, kept in backyard ponds, and managed in zoological and aquarium collections. The current study evaluated euthanasia of koi by immersion in 0.5 g/L tricaine methanesulfonate (MS-222) (n = 10), 0.5 g/L clove oil (n = 8), 1 g/L clove oil (n = 10), and CO2 (n = 7) on time to cessation of opercular movement, plasma lactate levels, and plasma cortisol levels. CO2 had the longest mean time to cessation of opercular movement, and MS-222 had the shortest (mean CO2: 24.9 min, range 13.18-31.35 min; MS-222: 2.68 min, range 1.33-4.5 min). The difference was not significant between any of the groups for plasma cortisol or lactate levels. MS-222 demonstrated the highest cortisol levels, and CO2 had the lowest (mean CO2: 108.7 ng/ml, range 33.9-195.8 ng/ml; MS-222: 650.6 ng/ml, range 77.3-2374.9 ng/ml). Average lactate levels were highest for 1 g/L clove oil and lowest for 0.5 g/L clove oil (mean 0.5 g/L clove oil: 5.1 mmol/L, range 1.8-8.1 mmol/L; 1 g/L clove oil: 7.4 mmol/L, range 5.6-10.5 mmol/L).
Assuntos
Aminobenzoatos , Carpas , Ácido Láctico , Animais , Dióxido de Carbono , Óleo de Cravo/farmacologia , Hidrocortisona , Água , Imersão , Anestésicos Locais , Ésteres , MesilatosRESUMO
BACKGROUND: Breast cancer is a heterogenous disease with several histological and molecular subtypes. Models that represent these subtypes are essential for translational research aimed at improving clinical strategy for targeted therapeutics. METHODS: Different combinations of genetic aberrations (Brca1 and Trp53 loss, and inhibition of proteins of the Rb family) were induced in the mammary gland by injection of adenovirus expressing Cre recombinase into the mammary ducts of adult genetically engineered mice. Mammary tumors with different genetic aberrations were classified into molecular subtypes based on expression of molecular markers and RNAseq analysis. In vitro potency assays and Western blots were used to examine their drug sensitivities. RESULTS: Induction of Brca1 and Trp53 loss in mammary ductal epithelium resulted in development of basal-like hormone receptor (HR)-negative mammary tumors. Inhibition of Rb and Trp53 loss or the combination of Rb, Trp53 and Brca1 aberrations resulted in development of luminal ductal carcinoma positive for ER, PR, and Her2 expression. HR positivity in tumors with Rb, Trp53 and Brca1 aberrations indicated that functionality of the Rb pathway rather than Brca1 status affected HR status in these models. Mammary tumor gene expression profiles recapitulated human basal-like or luminal B breast cancer signatures, but HR-positive luminal cancer models were endocrine resistant and exhibited upregulation of PI3K signaling and sensitivity to this pathway inhibition. Furthermore, both tumor subtypes were resistant to CDK4/6 inhibition. CONCLUSIONS: Examination of molecular expression profiles and drug sensitivities of tumors indicate that these breast cancer models can be utilized as a translational platform for evaluation of targeted combinations to improve chemotherapeutic response in patients that no longer respond to hormone therapy or that are resistant to CDK4/6 inhibition.
Assuntos
Neoplasias da Mama , Glândulas Mamárias Humanas , Neoplasias Mamárias Animais , Camundongos , Animais , Humanos , Feminino , Glândulas Mamárias Humanas/metabolismo , Fosfatidilinositol 3-Quinases , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias Mamárias Animais/patologia , Epitélio/metabolismo , Hormônios , Proteína BRCA1/genéticaRESUMO
Cells deficient in the Brca1 and Brca2 genes have reduced capacity to repair DNA double-strand breaks by homologous recombination and consequently are hypersensitive to DNA-damaging agents, including cisplatin and poly(ADP-ribose) polymerase (PARP) inhibitors. Here we show that loss of the MLL3/4 complex protein, PTIP, protects Brca1/2-deficient cells from DNA damage and rescues the lethality of Brca2-deficient embryonic stem cells. However, PTIP deficiency does not restore homologous recombination activity at double-strand breaks. Instead, its absence inhibits the recruitment of the MRE11 nuclease to stalled replication forks, which in turn protects nascent DNA strands from extensive degradation. More generally, acquisition of PARP inhibitors and cisplatin resistance is associated with replication fork protection in Brca2-deficient tumour cells that do not develop Brca2 reversion mutations. Disruption of multiple proteins, including PARP1 and CHD4, leads to the same end point of replication fork protection, highlighting the complexities by which tumour cells evade chemotherapeutic interventions and acquire drug resistance.
Assuntos
Replicação do DNA/fisiologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Deleção de Genes , Genes BRCA1 , Genes BRCA2 , Neoplasias/patologia , Proteínas Nucleares/deficiência , Animais , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Cisplatino/farmacologia , DNA/biossíntese , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , DNA Helicases/genética , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Enzimas Reparadoras do DNA/antagonistas & inibidores , Enzimas Reparadoras do DNA/metabolismo , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Feminino , Recombinação Homóloga , Proteína Homóloga a MRE11 , Camundongos , Neoplasias/genética , Proteínas Nucleares/genética , Poli(ADP-Ribose) Polimerase-1 , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/genéticaRESUMO
OBJECTIVE: To determine the cumulative incidence and natural history of axillary web syndrome (AWS) and its related postoperative risk for physical impairments in a cohort of women followed for 5 years post breast cancer surgery. DESIGN: Prospective, longitudinal study. SETTING: Academic health center. PARTICIPANTS: Women (N=36) with and without AWS after breast cancer surgery with sentinel node biopsy or axillary lymph node dissection. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Participants were assessed for AWS, shoulder goniometric flexion and abduction range of motion, function (Disability of the Arm, Shoulder, and Hand), lymphedema (bioimpedance spectroscopy, girth measures, tissue dielectric constant), and pain (visual analog scale) at 2, 4, 12, and 78 weeks and 5 years after breast cancer surgery. Analysis of variance compared range of motion, function, lymphedema, and pain in women identified with AWS with those without AWS across visits. Univariate logistic regression assessed if AWS was a risk factor for physical impairment at 5 years. RESULTS: The cumulative incidence of AWS was 57%. Fifty percent (14/28) of the women who completed all study visits had signs of AWS at 5 years. Abduction active range of motion was significantly lower in women with AWS at 2 and 4 weeks post surgery. AWS was identified as a risk factor for reduced shoulder motion at 5 years. Regardless of AWS, 75% of the women experienced 1 or more upper extremity physical impairments at 5 years, which is an increase from 66% at 78 weeks in the same cohort. CONCLUSIONS: AWS is associated with reduced shoulder range of motion in the early postoperative time period, can persist for 5 years after breast cancer surgery, and increases the risk of long-term reduced shoulder range of motion. Long-term physical issues are apparent after breast cancer surgery regardless of AWS.
Assuntos
Neoplasias da Mama , Linfedema , Axila/patologia , Axila/cirurgia , Feminino , Humanos , Incidência , Estudos Longitudinais , Excisão de Linfonodo/efeitos adversos , Linfedema/epidemiologia , Linfedema/etiologia , Dor , Estudos Prospectivos , Amplitude de Movimento Articular , Sobreviventes , Extremidade SuperiorRESUMO
Self-renewal is the hallmark feature both of normal stem cells and cancer stem cells. Since the regenerative capacity of normal haematopoietic stem cells is limited by the accumulation of reactive oxygen species and DNA double-strand breaks, we speculated that DNA damage might also constrain leukaemic self-renewal and malignant haematopoiesis. Here we show that the histone methyl-transferase MLL4, a suppressor of B-cell lymphoma, is required for stem-cell activity and an aggressive form of acute myeloid leukaemia harbouring the MLL-AF9 oncogene. Deletion of MLL4 enhances myelopoiesis and myeloid differentiation of leukaemic blasts, which protects mice from death related to acute myeloid leukaemia. MLL4 exerts its function by regulating transcriptional programs associated with the antioxidant response. Addition of reactive oxygen species scavengers or ectopic expression of FOXO3 protects MLL4(-/-) MLL-AF9 cells from DNA damage and inhibits myeloid maturation. Similar to MLL4 deficiency, loss of ATM or BRCA1 sensitizes transformed cells to differentiation, suggesting that myeloid differentiation is promoted by loss of genome integrity. Indeed, we show that restriction-enzyme-induced double-strand breaks are sufficient to induce differentiation of MLL-AF9 blasts, which requires cyclin-dependent kinase inhibitor p21(Cip1) (Cdkn1a) activity. In summary, we have uncovered an unexpected tumour-promoting role of genome guardians in enforcing the oncogene-induced differentiation blockade in acute myeloid leukaemia.
Assuntos
Dano ao DNA , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/patologia , Mielopoese , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Transformação Celular Neoplásica , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Feminino , Regulação Neoplásica da Expressão Gênica , Genes BRCA1 , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Histona-Lisina N-Metiltransferase/deficiência , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Masculino , Camundongos , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Piwi-interacting RNAs (piRNAs) fulfill a critical, conserved role in defending the genome against foreign genetic elements. In many organisms, piRNAs appear to be derived from processing of a long, polycistronic RNA precursor. Here, we establish that each Caenorhabditis elegans piRNA represents a tiny, autonomous transcriptional unit. Remarkably, the minimal C. elegans piRNA cassette requires only a 21 nucleotide (nt) piRNA sequence and an â¼50 nt upstream motif with limited genomic context for expression. Combining computational analyses with a novel, in vivo transgenic system, we demonstrate that this upstream motif is necessary for independent expression of a germline-enriched, Piwi-dependent piRNA. We further show that a single nucleotide position within this motif directs differential germline enrichment. Accordingly, over 70% of C. elegans piRNAs are selectively expressed in male or female germline, and comparison of the genes they target suggests that these two populations have evolved independently. Together, our results indicate that C. elegans piRNA upstream motifs act as independent promoters to specify which sequences are expressed as piRNAs, how abundantly they are expressed, and in what germline. As the genome encodes well over 15,000 unique piRNA sequences, our study reveals that the number of transcriptional units encoding piRNAs rivals the number of mRNA coding genes in the C. elegans genome.
Assuntos
Células Germinativas/metabolismo , Motivos de Nucleotídeos/genética , RNA Interferente Pequeno , Sequências Reguladoras de Ácido Ribonucleico/genética , Animais , Caenorhabditis elegans/genética , Feminino , Regulação da Expressão Gênica , Genoma , Masculino , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
Treatment of Mycoplasma spp. pneumonia has rarely been described in domestic ferrets (Mustela putorius furo). A 10-month-old, 0.53 kg, female spayed domestic ferret was presented for oxygen-dependent, chronic dyspnea of one-month's duration. Physical examination findings included dyspnea, tachypnea, increased bronchovesicular sounds bilaterally, and an intermittent non-productive cough. Bloodwork abnormalities included a mild leukocytosis (8.6×103/µL), mild neutrophilia (4.0×103/µL), mild hypoalbuminemia (2.7 g/dL), mild hyperglobulinemia (3.3 g/dL), mild hyponatremia (147 mEq/L), and mild hypochloremia (111.4 mEq/L). Radiographs revealed a marked diffuse bronchial pattern with peribronchial cuffing, a mild main pulmonary artery bulge, distended caudal lobar pulmonary arteries, and decreased serosal detail within the abdomen. An echocardiogram revealed indications of moderate pulmonary hypertension and systolic anterior motion of the mitral valve. Polymerase chain reaction testing for Mycoplasma spp. was positive, and treatment was initiated with doxycycline (10 mg/kg PO q 12 h for 16 weeks), prednisolone (0.4 mg/kg PO q 12 h for 13 weeks, tapered to 0.2 mg/kg PO q 12 h for two weeks, then eventually increased to 0.7 mg/kg PO q 12 h until further notice), sildenafil (0.3 mg/kg PO q 24 h for 13 weeks), and oxygen supplementation via an oxygen cage for six weeks. On repeat echocardiogram eleven weeks after initiation of doxycycline therapy, the pulmonary hypertension had resolved. At follow up six months later, the ferret was stable on previously prescribed medications and did not require oxygen supplementation. Mycoplasma spp. and pulmonary hypertension should be considered in cases of respiratory distress in ferrets.
Assuntos
Antibacterianos , Furões , Hipertensão Pulmonar , Animais , Feminino , Hipertensão Pulmonar/veterinária , Antibacterianos/uso terapêutico , Pneumonia por Mycoplasma/veterinária , Pneumonia por Mycoplasma/tratamento farmacológico , Pneumonia por Mycoplasma/diagnóstico , Doxiciclina/uso terapêutico , Prednisolona/uso terapêutico , Citrato de Sildenafila/uso terapêuticoRESUMO
MET pathway activation is one of the most common mechanisms of resistance to osimertinib in EGFR-mutant non-small cell lung cancer (NSCLC). We previously demonstrated spatial and temporal heterogeneity in MET pathway activation upon osimertinib resistance in EGFR-mutant NSCLC; however, the functional relevance of these findings is unclear. Here, we generated 19 patient-derived xenografts (PDX) from 9 patients with multi-region and temporal sampling of osimertinib-resistant tumor tissue from patients with EGFR-mutant NSCLC. MET pathway activation was a putative mechanism of osimertinib resistance in 66% (n = 6/9) patients from whom PDXs were generated. Significant spatial and temporal heterogeneity in MET pathway activation was evident. Osimertinib-resistant PDXs with MET amplification by FISH (defined as MET/CEP7 ratio ≥2.0 or mean MET ≥ 6.0 copies/cell) and high-level phospho-MET, but not c-MET expression, had better responses to osimertinib and savolitinib combination than to osimertinib alone. MET polysomy tumors by FISH from both PDXs and patients had evidence of subclonal phospho-MET expression. Select MET polysomy PDX tumors with phospho-MET expression responded better to osimertinib and savolitinib combination than MET polysomy PDX tumors without phospho-MET expression. Our results suggest osimertinib and savolitinib combination is most effective for osimertinib-resistant EGFR-mutant tumors with MET pathway activation as evidenced by phospho-MET. As subclonal MET amplification may be evident in MET polysomy tumor progression, MET polysomy warrants close clinical follow-up with phospho-MET IHC in parallel with FISH diagnostic. SIGNIFICANCE: Using a novel cohort of in vivo PDX models of MET pathway activation with acquired resistance to osimertinib in EGFR-mutant lung cancer, we demonstrate that phospho-MET may be a clinically relevant assay to guide treatment selection with osimertinib and savolitinib combination. In addition, our work shows that patients with MET polysomy tumors may have subclonal MET amplification and therefore require close follow up for the use of osimertinib and savolitinib combination.
Assuntos
Acrilamidas , Compostos de Anilina , Carcinoma Pulmonar de Células não Pequenas , Indóis , Neoplasias Pulmonares , Pirimidinas , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Mutação , Proteínas Proto-Oncogênicas c-met/genética , Receptores ErbB/genética , Inibidores de Proteínas Quinases/farmacologia , Resistencia a Medicamentos Antineoplásicos/genéticaRESUMO
Meiotic recombination is initiated by SPO11-induced double-strand breaks (DSBs). In most mammals, the methyltransferase PRDM9 guides SPO11 targeting, and the ATM kinase controls meiotic DSB numbers. Following MRE11 nuclease removal of SPO11, the DSB is resected and loaded with DMC1 filaments for homolog invasion. Here, we demonstrate the direct detection of meiotic DSBs and resection using END-seq on mouse spermatocytes with low sample input. We find that DMC1 limits both minimum and maximum resection lengths, whereas 53BP1, BRCA1 and EXO1 play surprisingly minimal roles. Through enzymatic modifications to END-seq, we identify a SPO11-bound meiotic recombination intermediate (SPO11-RI) present at all hotspots. We propose that SPO11-RI forms because chromatin-bound PRDM9 asymmetrically blocks MRE11 from releasing SPO11. In Atm-/- spermatocytes, trapped SPO11 cleavage complexes accumulate due to defective MRE11 initiation of resection. Thus, in addition to governing SPO11 breakage, ATM and PRDM9 are critical local regulators of mammalian SPO11 processing.
Assuntos
Endodesoxirribonucleases/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Recombinação Homóloga/fisiologia , Meiose/fisiologia , Espermatócitos/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Feminino , Histona-Lisina N-Metiltransferase/genética , Proteína Homóloga a MRE11/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Ligação a Fosfato/genética , Proteínas de Ligação a Fosfato/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genéticaRESUMO
PURPOSE: Submaximal exercise tests, such as the 6-min walk test (6MWT), are used to assess and determine changes in cardiorespiratory fitness in cardiopulmonary rehabilitation (CR) programs. However, other modalities such as recumbent steppers are frequently utilized in rehabilitation settings and suit individuals of all ability levels; yet, no self-paced submaximal test has been developed for this exercise modality. The aim of this study was to produce a self-paced, submaximal 6-min recumbent stepper test (6MRST) that is comparable with the 6MWT and can be used as pre- and post-CR assessments. METHODS: Seventy subjects participating in CR performed a 6WMT and a 6MRST at the beginning and end of their CR program. Distance exercised, blood pressure, heart rate, and perceived exertion were measured to determine whether the 6MRST was comparable with the 6MWT. RESULTS: Significant correlations were found between the distance exercised in the 6MWT and the 6MRST during both pre- and post-CR testing (r = 0.540 and r = 0.700, respectively; P < .001). The distance for the 6MRST increased from 1599 to 2101 ft after CR. Heart rate and rating of perceived exertion showed strong correlations between the 6MWT and the 6MRST at both testing time points. CONCLUSIONS: The 6MRST was found to produce similar results to that of the 6MWT in a CR setting. The 6MRST could therefore serve as an alternative to the 6MWT in those patients who cannot adequately perform a 6MWT, in a facility where space may be too limited, and a walking track is not available or simply due to patient or staff preference.
Assuntos
Reabilitação Cardíaca/métodos , Teste de Esforço/métodos , Avaliação de Resultados em Cuidados de Saúde/métodos , Teste de Caminhada/métodos , Determinação da Pressão Arterial , Feminino , Cardiopatias/reabilitação , Frequência Cardíaca , Humanos , Masculino , Pessoa de Meia-Idade , Consumo de Oxigênio , Fatores Sexuais , Resultado do TratamentoRESUMO
Plasma levels of beta-2 microglobulin (beta2M), a subunit of the human leukocyte antigen-class I (HLA-I) molecule, correlate negatively with outcome in non-Hodgkin's lymphoma (NHL) and Hodgkin's disease (HD). We examined the clinical relevance of soluble HLA-I (sHLA-I) levels in NHL and HD. Sera from consecutive NHL (n=65) and HD (n=37) patients were analyzed in a blinded manner. NHL and HD patients had significantly higher levels of sHLA-1 and beta2M than control subjects. In NHL patients, sHLA-I levels correlated with clinical behavior in a fashion similar to that of beta2M. However, multivariate analysis incorporating beta2M, sHLA-I, and international prognostic index (IPI) indicated that NHL patients with elevated (>312.6mug/100mL) sHLA-I levels had significantly shorter survival, independent of IPI score as well as beta2M. In HD patients, beta2M but not sHLA-I levels were associated with clinical behavior. These findings not only establish the role of sHLA-I as an independent tumor marker in NHL that can be used to stratify patients, but also suggest that beta2M and sHLA-I may reflect different biological processes in HD and NHL. Further studies are needed to assess whether the immunomodulatory properties of sHLA-I may be responsible for its divergence from beta2M as an indicator of clinical behavior in HD.
Assuntos
Biomarcadores Tumorais/sangue , Antígenos HLA/sangue , Doença de Hodgkin/sangue , Linfoma não Hodgkin/sangue , Microglobulina beta-2/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Doença de Hodgkin/terapia , Humanos , Linfoma não Hodgkin/terapia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Estadiamento de Neoplasias , Valor Preditivo dos Testes , Prognóstico , Solubilidade , Taxa de Sobrevida , Fatores de Tempo , Resultado do TratamentoRESUMO
Among the various subtypes of acute myeloid leukemia (AML), those with chromosomal rearrangements of the MLL oncogene (AML-MLL) have a poor prognosis. AML-MLL tumor cells are resistant to current genotoxic therapies because of an attenuated response by p53, a protein that induces cell cycle arrest and apoptosis in response to DNA damage. In addition to chemicals that damage DNA, efforts have focused on targeting DNA repair enzymes as a general chemotherapeutic approach to cancer treatment. Here, we found that inhibition of the kinase ATR, which is the primary sensor of DNA replication stress, induced chromosomal breakage and death of mouse AML(MLL) cells (with an MLL-ENL fusion and a constitutively active N-RAS independently of p53. Moreover, ATR inhibition as a single agent exhibited antitumoral activity, both reducing tumor burden after establishment and preventing tumors from growing, in an immunocompetent allograft mouse model of AML(MLL) and in xenografts of a human AML-MLL cell line. We also found that inhibition of ATM, a kinase that senses DNA double-strand breaks, also promoted the survival of the AML(MLL) mice. Collectively, these data indicated that ATR or ATM inhibition represent potential therapeutic strategies for the treatment of AML, especially MLL-driven leukemias.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia , Rearranjo Gênico , Histona-Lisina N-Metiltransferase , Leucemia Mieloide Aguda , Proteína de Leucina Linfoide-Mieloide , Neoplasias Experimentais , Animais , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Camundongos , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/enzimologia , Neoplasias Experimentais/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
La antigen (Sjögren's syndrome antigen B) is a phosphoprotein associated with nascent precursor tRNAs and other RNAs, and it is targeted by autoantibodies in patients with Sjögren's syndrome, systemic lupus erythematosus, and neonatal lupus. Increased levels of La are associated with leukemias and other cancers, and various viruses usurp La to promote their replication. Yeast cells (Saccharomyces cerevisiae and Schizosaccharomyces pombe) genetically depleted of La grow and proliferate, whereas deletion from mice causes early embryonic lethality, raising the question of whether La is required by mammalian cells generally or only to surpass a developmental stage. We developed a conditional La allele and used it in mice that express Cre recombinase in either B cell progenitors or the forebrain. B cell Mb1(Cre) La-deleted mice produce no B cells. Consistent with αCamKII Cre, which induces deletion in hippocampal CA1 cells in the third postnatal week and later throughout the neocortex, brains develop normally in La-deleted mice until â¼5 weeks and then lose a large amount of forebrain cells and mass, with evidence of altered pre-tRNA processing. The data indicate that La is required not only in proliferating cells but also in nondividing postmitotic cells. Thus, La is essential in different cell types and required for normal development of various tissue types.
Assuntos
Autoantígenos/imunologia , Linfócitos B/imunologia , Lobo Frontal/imunologia , Neurônios/imunologia , Ribonucleoproteínas/imunologia , Animais , Autoantígenos/genética , Autoantígenos/metabolismo , Linfócitos B/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/imunologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Lobo Frontal/metabolismo , Lobo Frontal/patologia , Hipocampo/imunologia , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neurônios/metabolismo , RNA/genética , RNA/imunologia , RNA/metabolismo , Precursores de RNA/genética , Precursores de RNA/imunologia , Precursores de RNA/metabolismo , RNA de Transferência/genética , RNA de Transferência/imunologia , RNA de Transferência/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Síndrome de Sjogren/genética , Síndrome de Sjogren/imunologia , Síndrome de Sjogren/metabolismo , Fatores de Tempo , Antígeno SS-BRESUMO
Tissue homeostasis is controlled by the availability of growth factors, which sustain exogenous nutrient uptake and prevent apoptosis. Although autophagy can provide an alternate intracellular nutrient source to support essential basal metabolism of apoptosis-resistant growth factor-withdrawn cells, antiapoptotic Bcl-2 family proteins can suppress autophagy in some settings. Thus, the role of autophagy and interactions between autophagy and apoptosis in growth factor-withdrawn cells expressing Bcl-2 or Bcl-xL were unclear. Here we show autophagy was rapidly induced in hematopoietic cells upon growth factor withdrawal regardless of Bcl-2 or Bcl-xL expression and led to increased mitochondrial lipid oxidation. Deficiency in autophagy-essential gene expression, however, did not lead to metabolic catastrophe and rapid death of growth factor-deprived cells. Rather, inhibition of autophagy enhanced survival of cells with moderate Bcl-2 expression for greater than 1 wk, indicating that autophagy promoted cell death in this time frame. Cell death was not autophagic, but apoptotic, and relied on Chop-dependent induction of the proapoptotic Bcl-2 family protein Bim. Therefore, although ultimately important, autophagy-derived nutrients appear initially nonessential after growth factor withdrawal. Instead, autophagy promotes tissue homeostasis by sensitizing cells to apoptosis to ensure only the most apoptosis-resistant cells survive long-term using autophagy-derived nutrients when growth factor deprived.