RESUMO
Resveratrol is a natural product associated with wide-ranging effects in animal and cellular models, including lifespan extension. To identify the genetic target of resveratrol in human cells, we conducted genome-wide CRISPR-Cas9 screens to pinpoint genes that confer sensitivity or resistance to resveratrol. An extensive network of DNA damage response and replicative stress genes exhibited genetic interactions with resveratrol and its analog pterostilbene. These genetic profiles showed similarity to the response to hydroxyurea, an inhibitor of ribonucleotide reductase that causes replicative stress. Resveratrol, pterostilbene, and hydroxyurea caused similar depletion of nucleotide pools, inhibition of replication fork progression, and induction of replicative stress. The ability of resveratrol to inhibit cell proliferation and S phase transit was independent of the histone deacetylase sirtuin 1, which has been implicated in lifespan extension by resveratrol. These results establish that a primary impact of resveratrol on human cell proliferation is the induction of low-level replicative stress.
Assuntos
Proliferação de Células/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Resveratrol/farmacologia , Sistemas CRISPR-Cas , Linhagem Celular , Resistência a Medicamentos/genética , Humanos , Hidroxiureia/farmacologia , Células Jurkat , Nucleotídeos/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Sirtuína 1/metabolismo , Estilbenos/farmacologiaRESUMO
DNA interstrand cross-links (ICLs) are a form of DNA damage that requires the interplay of a number of repair proteins including those of the Fanconi anemia (FA) and the homologous recombination (HR) pathways. Pathogenic variants in the essential gene BRCA2/FANCD1, when monoallelic, predispose to breast and ovarian cancer, and when biallelic, result in a severe subtype of Fanconi anemia. BRCA2 function in the FA pathway is attributed to its role as a mediator of the RAD51 recombinase in HR repair of programmed DNA double-strand breaks (DSB). BRCA2 and RAD51 functions are also required to protect stalled replication forks from nucleolytic degradation during response to hydroxyurea (HU). While RAD51 has been shown to be necessary in the early steps of ICL repair to prevent aberrant nuclease resection, the role of BRCA2 in this process has not been described. Here, based on the analysis of BRCA2 DNA-binding domain (DBD) mutants (c.8488-1G>A and c.8524C>T) discovered in FA patients presenting with atypical FA-like phenotypes, we establish that BRCA2 is necessary for the protection of DNA at ICLs. Cells carrying BRCA2 DBD mutations are sensitive to ICL-inducing agents but resistant to HU treatment consistent with relatively high HR repair in these cells. BRCA2 function at an ICL protects against DNA2-WRN nuclease-helicase complex and not the MRE11 nuclease that is implicated in the resection of HU-induced stalled replication forks. Our results also indicate that unlike the processing at HU-induced stalled forks, the function of the SNF2 translocases (SMARCAL1, ZRANB3, or HLTF), implicated in fork reversal, are not an integral component of the ICL repair, pointing to a different mechanism of fork protection at different DNA lesions.
Assuntos
Proteína BRCA2/metabolismo , Anemia de Fanconi/genética , Anemia de Fanconi/fisiopatologia , Proteína BRCA2/genética , Linhagem Celular , DNA/química , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Replicação do DNA/efeitos dos fármacos , Recombinação Homóloga/genética , Humanos , Hidroxiureia/farmacologia , Mutação , Domínios Proteicos/genética , Rad51 Recombinase/metabolismoRESUMO
R-loops represent a major source of replication stress, but the mechanism by which these structures impede fork progression remains unclear. To address this question, we monitored fork progression, arrest, and restart in Saccharomyces cerevisiae cells lacking RNase H1 and H2, two enzymes responsible for degrading RNA:DNA hybrids. We found that while RNase H-deficient cells could replicate their chromosomes normally under unchallenged growth conditions, their replication was impaired when exposed to hydroxyurea (HU) or methyl methanesulfonate (MMS). Treated cells exhibited increased levels of RNA:DNA hybrids at stalled forks and were unable to generate RPA-coated single-stranded (ssDNA), an important postreplicative intermediate in resuming replication. Similar impairments in nascent DNA resection and ssDNA formation at HU-arrested forks were observed in human cells lacking RNase H2. However, fork resection was fully restored by addition of triptolide, an inhibitor of transcription that induces RNA polymerase degradation. Taken together, these data indicate that RNA:DNA hybrids not only act as barriers to replication forks, but also interfere with postreplicative fork repair mechanisms if not promptly degraded by RNase H.
Assuntos
Replicação do DNA , RNA , Humanos , RNA/genética , Ribonucleases/genética , DNA/metabolismo , Hidroxiureia/farmacologia , Ribonuclease H/genética , Ribonuclease H/metabolismoRESUMO
Nucleotide metabolism fuels normal DNA replication and is also primarily targeted by the DNA replication checkpoint when replication stalls. To reveal a comprehensive interconnection between genome maintenance and metabolism, we analyzed the metabolomic changes upon replication stress in the budding yeast S. cerevisiae. We found that upon treatment of cells with hydroxyurea, glucose is rapidly diverted to the oxidative pentose phosphate pathway (PPP). This effect is mediated by the AMP-dependent kinase, SNF1, which phosphorylates the transcription factor Mig1, thereby relieving repression of the gene encoding the rate-limiting enzyme of the PPP. Surprisingly, NADPH produced by the PPP is required for efficient recruitment of replication protein A (RPA) to single-stranded DNA, providing the signal for the activation of the Mec1/ATR-Rad53/CHK1 checkpoint signaling kinase cascade. Thus, SNF1, best known as a central energy controller, determines a fast mode of replication checkpoint activation through a redox mechanism. These findings establish that SNF1 provides a hub with direct links to cellular metabolism, redox, and surveillance of DNA replication in eukaryotes.
Assuntos
Replicação do DNA , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Replicação do DNA/efeitos dos fármacos , DNA de Cadeia Simples/metabolismo , Glucose/genética , Glucose/metabolismo , Glicólise/fisiologia , Hidroxiureia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , NADP/metabolismo , Via de Pentose Fosfato , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
ABSTRACT: After starting hydroxyurea treatment, Ugandan children with sickle cell anemia had 60% fewer severe or invasive infections, including malaria, bacteremia, respiratory tract infections, and gastroenteritis, than before starting hydroxyurea treatment (incidence rate ratio, 0.40 [95% confidence interval, 0.29-0.54]; P < .001).
Assuntos
Anemia Falciforme , Malária , Criança , Humanos , Hidroxiureia/uso terapêutico , Antidrepanocíticos/uso terapêutico , Uganda/epidemiologia , Anemia Falciforme/complicações , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/epidemiologia , Malária/complicações , Malária/tratamento farmacológico , Malária/epidemiologiaRESUMO
Transcription hinders replication fork progression and stability, and the Mec1/ATR checkpoint protects fork integrity. Examining checkpoint-dependent mechanisms controlling fork stability, we find that fork reversal and dormant origin firing due to checkpoint defects are rescued in checkpoint mutants lacking THO, TREX-2, or inner-basket nucleoporins. Gene gating tethers transcribed genes to the nuclear periphery and is counteracted by checkpoint kinases through phosphorylation of nucleoporins such as Mlp1. Checkpoint mutants fail to detach transcribed genes from nuclear pores, thus generating topological impediments for incoming forks. Releasing this topological complexity by introducing a double-strand break between a fork and a transcribed unit prevents fork collapse. Mlp1 mutants mimicking constitutive checkpoint-dependent phosphorylation also alleviate checkpoint defects. We propose that the checkpoint assists fork progression and stability at transcribed genes by phosphorylating key nucleoporins and counteracting gene gating, thus neutralizing the topological tension generated at nuclear pore gated genes.
Assuntos
Replicação do DNA , Poro Nuclear/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Quinase do Ponto de Checagem 2 , Quebras de DNA de Cadeia Dupla , Hidroxiureia/farmacologia , Mutação , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
DNA-RNA hybrids associated with R-loops promote DNA damage and genomic instability. The capacity of hybrids at different genomic sites to cause DNA damage was not known, and the mechanisms leading from hybrid to damage were poorly understood. Here, we adopt a new strategy to map and characterize the sites of hybrid-induced damage genome-wide in budding yeast. We show that hybrid removal is essential for life because persistent hybrids cause irreparable DNA damage and cell death. We identify that a subset of hybrids is prone to cause damage, and the chromosomal context of hybrids dramatically impacts their ability to induce damage. Furthermore, persistent hybrids affect the repair pathway, generating large regions of single-stranded DNA (ssDNA) by two distinct mechanisms, likely resection and re-replication. These damaged regions may act as potential precursors to gross chromosomal rearrangements like deletions and duplications that are associated with R-loops and cancers.
Assuntos
DNA de Cadeia Simples/genética , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Instabilidade Genômica , RNA/genética , Saccharomyces cerevisiae/genética , Clivagem do DNA , Dano ao DNA , DNA Helicases/genética , DNA Helicases/metabolismo , Replicação do DNA , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Hidroxiureia/farmacologia , Ácidos Indolacéticos/farmacologia , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , RNA/química , RNA/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
DNA replication is a fundamental cellular process that ensures the transfer of genetic information during cell division. Genome duplication takes place in S phase and requires a dynamic and highly coordinated recruitment of multiple proteins at replication forks. Various genotoxic stressors lead to fork instability and collapse, hence the need for DNA repair pathways. By identifying the multitude of protein interactions implicated in those events, we can better grasp the complex and dynamic molecular mechanisms that facilitate DNA replication and repair. Proximity-dependent biotin identification was used to identify associations with 17 proteins within four core replication components, namely the CDC45/MCM2-7/GINS helicase that unwinds DNA, the DNA polymerases, replication protein A subunits, and histone chaperones needed to disassemble and reassemble chromatin. We further investigated the impact of genotoxic stress on these interactions. This analysis revealed a vast proximity association network with 108 nuclear proteins further modulated in the presence of hydroxyurea; 45 being enriched and 63 depleted. Interestingly, hydroxyurea treatment also caused a redistribution of associations with 11 interactors, meaning that the replisome is dynamically reorganized when stressed. The analysis identified several poorly characterized proteins, thereby uncovering new putative players in the cellular response to DNA replication arrest. It also provides a new comprehensive proteomic framework to understand how cells respond to obstacles during DNA replication.
Assuntos
Replicação do DNA , Hidroxiureia , Proteômica , Hidroxiureia/farmacologia , Proteômica/métodos , Humanos , Dano ao DNA , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteoma/metabolismoRESUMO
The mutational processes dictating the accumulation of mutations in genomes are shaped by genetic background, environment and their interactions. Accurate quantification of mutation rates and spectra under drugs has important implications in disease treatment. Here, we used whole-genome sequencing and time-resolved growth phenotyping of yeast mutation accumulation lines to give a detailed view of the mutagenic effects of rapamycin and hydroxyurea on the genome and cell growth. Mutation rates depended on the genetic backgrounds but were only marginally affected by rapamycin. As a remarkable exception, rapamycin treatment was associated with frequent chromosome XII amplifications, which compensated for rapamycin induced rDNA repeat contraction on this chromosome and served to maintain rDNA content homeostasis and fitness. In hydroxyurea, a wide range of mutation rates were elevated regardless of the genetic backgrounds, with a particularly high occurrence of aneuploidy that associated with dramatic fitness loss. Hydroxyurea also induced a high T-to-G and low C-to-A transversion rate that reversed the common G/C-to-A/T bias in yeast and gave rise to a broad range of structural variants, including mtDNA deletions. The hydroxyurea mutation footprint was consistent with the activation of error-prone DNA polymerase activities and non-homologues end joining repair pathways. Taken together, our study provides an in-depth view of mutation rates and signatures in rapamycin and hydroxyurea and their impact on cell fitness, which brings insights for assessing their chronic effects on genome integrity.
Assuntos
Hidroxiureia , Saccharomyces cerevisiae , Humanos , Hidroxiureia/farmacologia , Saccharomyces cerevisiae/genética , Sirolimo/farmacologia , Mutação , Instabilidade Genômica/genética , DNA Ribossômico/genéticaRESUMO
Upon replication stress, budding yeast checkpoint kinase Mec1ATR triggers the downregulation of transcription, thereby reducing the level of RNA polymerase (RNAP) on chromatin to facilitate replication fork progression. Here, we identify a hydroxyurea-induced phosphorylation site on Mec1, Mec1-S1991, that contributes to the eviction of RNAPII and RNAPIII during replication stress. The expression of the non-phosphorylatable mec1-S1991A mutant reduces replication fork progression genome-wide and compromises survival on hydroxyurea. This defect can be suppressed by destabilizing chromatin-bound RNAPII through a TAP fusion to its Rpb3 subunit, suggesting that lethality in mec1-S1991A mutants arises from replication-transcription conflicts. Coincident with a failure to repress gene expression on hydroxyurea in mec1-S1991A cells, highly transcribed genes such as GAL1 remain bound at nuclear pores. Consistently, we find that nuclear pore proteins and factors controlling RNAPII and RNAPIII are phosphorylated in a Mec1-dependent manner on hydroxyurea. Moreover, we show that Mec1 kinase also contributes to reduced RNAPII occupancy on chromatin during an unperturbed S phase by promoting degradation of the Rpb1 subunit.
Assuntos
Replicação do DNA , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , RNA Polimerase III/genética , RNA Polimerase II/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Cromatina/química , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Galactoquinase/genética , Galactoquinase/metabolismo , Regulação Fúngica da Expressão Gênica , Hidroxiureia/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fosfoproteínas , Fosforilação , Proteínas Serina-Treonina Quinases/genética , RNA Polimerase II/metabolismo , RNA Polimerase III/metabolismo , Fase S/efeitos dos fármacos , Fase S/genética , Saccharomyces cerevisiae/genética , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Transcrição GênicaRESUMO
We tested the hypothesis that fixed oral moderate-dose hydroxyurea (20 mg/kg per day) for initial treatment of secondary stroke prevention results in an 80% relative risk reduction of stroke or death when compared with fixed oral low-dose hydroxyurea (10 mg/kg per day) in a phase 3 double-blind, parallel-group, randomized controlled trial in children with sickle cell anemia (SCA) living in Nigeria. A total of 101 participants were randomly allocated to low-dose (n = 49) and moderate-dose (n = 52) hydroxyurea treatment groups. The median participant follow-up was 1.6 years (interquartile range, 1.0-2.3), with a planned minimum follow-up of 3.0 years. A total of 6 recurrent strokes and 2 deaths vs 5 recurrent strokes and 3 deaths occurred in the low- and moderate-dose groups, respectively. The incidence rate ratio (IRR) of the primary outcome measure of stroke or death in the low- and moderate-dose hydroxyurea treatment groups was 0.98 (95% confidence interval [CI], 0.32-3.00; P = .97). The trial was stopped early owing to no clinical difference in the incidence rates of the primary outcome measure. The incidence rates of recurrent strokes were 7.1 and 6.0 per 100 person-years in the low- and moderate-dose groups, respectively, (IRR, 1.18; 95% CI, 0.30-4.88; P = .74). As a measure of adherence to the oral hydroxyurea therapy, the median percent of returned pills was 3.0% and 2.6% in the low- and moderate-dose groups, respectively. No participant had hydroxyurea therapy stopped for myelosuppression. For children with SCA in low-income settings without access to regular blood transfusion therapy, initial low-dose hydroxyurea is a minimum known efficacious dose for secondary stroke prevention.
Assuntos
Anemia Falciforme , Acidente Vascular Cerebral , Criança , Humanos , Hidroxiureia/uso terapêutico , Antidrepanocíticos/uso terapêutico , Nigéria , Anemia Falciforme/complicações , Acidente Vascular Cerebral/etiologia , Prevenção Secundária/métodosRESUMO
Polycythemia vera (PV) belongs to the BCR-ABL1-negative myeloproliferative neoplasms and is characterized by activating mutations in JAK2 and clinically presents with erythrocytosis, variable degrees of systemic and vasomotor symptoms, and an increased risk of both thromboembolic events and progression to myelofibrosis and acute myeloid leukemia (AML). Treatment selection is based on a patient's age and a history of thrombosis in patients with low-risk PV treated with therapeutic phlebotomy and aspirin alone, whereas cytoreductive therapy with either hydroxyurea or interferon alfa (IFN-α) is added for high-risk disease. However, other disease features such as significant disease-related symptoms and splenomegaly, concurrent thrombocytosis and leukocytosis, or intolerance of phlebotomy can constitute an indication for cytoreductive therapy in patients with otherwise low-risk disease. Additionally, recent studies demonstrating the safety and efficacy (ie, reduction in phlebotomy requirements and molecular responses) of ropegylated IFN-α2b support its use for patients with low-risk PV. Additionally, emerging data suggest that early treatment is associated with higher rates of molecular responses, which might eventually enable time-limited therapy. Nonetheless, longer follow-up is needed to assess whether molecular responses associate with clinically meaningful outcome measures such as thrombosis and progression to myelofibrosis or AML. In this article, we provide an overview of the current and evolving treatment landscape of PV and outline our vision for a patient-centered, phlebotomy-free, treatment approach using time-limited, disease-modifying treatment modalities early in the disease course, which could ultimately affect the natural history of the disease.
Assuntos
Leucemia Mieloide Aguda , Policitemia Vera , Mielofibrose Primária , Trombocitose , Trombose , Humanos , Policitemia Vera/complicações , Policitemia Vera/genética , Policitemia Vera/terapia , Mielofibrose Primária/tratamento farmacológico , Trombocitose/terapia , Hidroxiureia/uso terapêutico , Trombose/terapia , Trombose/induzido quimicamente , Interferon-alfa/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Janus Quinase 2/genéticaRESUMO
Realizing Effectiveness Across Continents with Hydroxyurea (REACH, NCT01966731) provides hydroxyurea at maximum tolerated dose (MTD) for children with sickle cell anemia (SCA) in sub-Saharan Africa. Beyond reducing SCA-related clinical events, documented treatment benefits include â¼50% reduction in malaria incidence. To identify associations and propose mechanisms by which hydroxyurea could be associated with lower malaria rates, infections were recorded across all clinical sites (Angola, Democratic Republic of Congo, Kenya, and Uganda). Hazard ratios (HR) with 95% confidence intervals (CIs) for baseline demographics, and time-varying laboratory and clinical parameters were estimated in a modified Cox gap-time model for repeated events. Over 3387 patient-years of hydroxyurea treatment, 717 clinical malaria episodes occurred in 336 of 606 study participants; over half were confirmed by blood smear and/or rapid diagnostic testing with 97.8% Plasmodium falciparum. In univariate analysis limited to 4 confirmed infections per child, malaria risk was significantly associated with absolute neutrophil count (ANC), splenomegaly, hemoglobin, and achieving MTD; age, malaria season, MTD dose, fetal hemoglobin, α-thalassemia, and glucose-6-phosphate dehydrogenase deficiency had no effect. In multivariable regression of confirmed infections, ANC was significant (HR, 1.37 per doubled value; 95% CI, 1.10-1.70; P = .0052), and ANC values <3.0 × 109/L were associated with lower malaria incidence. Compared with nonpalpable spleen, 1- to 4-cm splenomegaly also was associated with higher malaria risk (HR, 2.01; 95% CI, 1.41-2.85; P = .0001). Hydroxyurea at MTD is associated with lower malaria incidence in SCA through incompletely defined mechanisms, but treatment-associated mild myelosuppression with ANC <3.0 × 109/L is salutary. Splenomegaly is an unexplained risk factor for malaria infections among children with SCA in Africa.
Assuntos
Anemia Falciforme , Malária , Humanos , Criança , Hidroxiureia/efeitos adversos , Incidência , Esplenomegalia/epidemiologia , Esplenomegalia/tratamento farmacológico , Anemia Falciforme/complicações , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/epidemiologia , Malária/tratamento farmacológico , Malária/epidemiologia , Malária/prevenção & controle , África Subsaariana/epidemiologiaRESUMO
Mismatch repair (MMR) is a replication-coupled DNA repair mechanism and plays multiple roles at the replication fork. The well-established MMR functions include correcting misincorporated nucleotides that have escaped the proofreading activity of DNA polymerases, recognizing nonmismatched DNA adducts, and triggering a DNA damage response. In an attempt to determine whether MMR regulates replication progression in cells expressing an ultramutable DNA polymerase É (PolÉ), carrying a proline-to-arginine substitution at amino acid 286 (PolÉ-P286R), we identified an unusual MMR function in response to hydroxyurea (HU)-induced replication stress. PolÉ-P286R cells treated with hydroxyurea exhibit increased MRE11-catalyzed nascent strand degradation. This degradation by MRE11 depends on the mismatch recognition protein MutSα and its binding to stalled replication forks. Increased MutSα binding at replication forks is also associated with decreased loading of replication fork protection factors FANCD2 and BRCA1, suggesting blockage of these fork protection factors from loading to replication forks by MutSα. We find that the MutSα-dependent MRE11-catalyzed fork degradation induces DNA breaks and various chromosome abnormalities. Therefore, unlike the well-known MMR functions of ensuring replication fidelity, the newly identified MMR activity of promoting genome instability may also play a role in cancer avoidance by eliminating rogue cells.
Assuntos
Proteínas de Ligação a DNA , Hidroxiureia , Aminoácidos/genética , Arginina/genética , Adutos de DNA , Reparo de Erro de Pareamento de DNA , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Hidroxiureia/farmacologia , Proteína Homóloga a MRE11/genética , Proteína Homóloga a MRE11/metabolismo , Nucleotídeos/metabolismo , Prolina/genéticaRESUMO
Stem cell transplantation and genetic therapies offer potential cures for patients with sickle cell disease (SCD), but these options require advanced medical facilities and are expensive. Consequently, these treatments will not be available for many years to the majority of patients suffering from this disease. What is urgently needed now is an inexpensive oral drug in addition to hydroxyurea, the only drug approved by the FDA that inhibits sickle-hemoglobin polymerization. Here, we report the results of the first phase of our phenotypic screen of the 12,657 compounds of the Scripps ReFRAME drug repurposing library using a recently developed high-throughput assay to measure sickling times following deoxygenation to 0% oxygen of red cells from sickle trait individuals. The ReFRAME library is a very important collection because the compounds are either FDA-approved drugs or have been tested in clinical trials. From dose-response measurements, 106 of the 12,657 compounds exhibit statistically significant antisickling at concentrations ranging from 31 nM to 10 µM. Compounds that inhibit sickling of trait cells are also effective with SCD cells. As many as 21 of the 106 antisickling compounds emerge as potential drugs. This estimate is based on a comparison of inhibitory concentrations with free concentrations of oral drugs in human serum. Moreover, the expected therapeutic potential for each level of inhibition can be predicted from measurements of sickling times for cells from individuals with sickle syndromes of varying severity. Our results should motivate others to develop one or more of these 106 compounds into drugs for treating SCD.
Assuntos
Anemia Falciforme , Antidrepanocíticos , Antidrepanocíticos/farmacologia , Antidrepanocíticos/uso terapêutico , Reposicionamento de Medicamentos , Hemoglobina Falciforme , Humanos , Hidroxiureia/farmacologia , Oxigênio/uso terapêuticoRESUMO
Telomere length maintenance is crucial to cancer cell immortality. Up to 15% of cancers utilize a telomerase-independent, recombination-based mechanism termed alternative lengthening of telomeres (ALT). Currently, the primary ALT biomarker is the C-circle, a type of circular DNA with extrachromosomal telomere repeats (cECTRs). How C-circles form is not well characterized. We investigated C-circle formation in the human cen3tel cell line, a long-telomere, telomerase+ (LTT+) cell line with progressively hyper-elongated telomeres (up to â¼100 kb). cECTR signal was observed in 2D gels and C-circle assays but not t-circle assays, which also detect circular DNA with extrachromosomal telomere repeats. Telomerase activity and C-circle signal were not separable in the analysis of clonal populations, consistent with C-circle production occurring within telomerase+ cells. We observed similar cECTR results in two other LTT+ cell lines, HeLa1.3 (â¼23 kb telomeres) and HeLaE1 (â¼50 kb telomeres). In LTT+ cells, telomerase activity did not directly impact C-circle signal; instead, C-circle signal correlated with telomere length. LTT+ cell lines were less sensitive to hydroxyurea than ALT+ cell lines, suggesting that ALT status is a stronger contributor to replication stress levels than telomere length. Additionally, the DNA repair-associated protein FANCM did not suppress C-circles in LTT+ cells as it does in ALT+ cells. Thus, C-circle formation may be driven by telomere length, independently of telomerase and replication stress, highlighting limitations of C-circles as a stand-alone ALT biomarker.
Assuntos
DNA Circular , Telomerase , Telômero , Humanos , DNA Helicases/metabolismo , Telomerase/metabolismo , Telômero/genética , Telômero/metabolismo , Homeostase do Telômero , Linhagem Celular , Células HeLa , Replicação do DNA , Hidroxiureia , Reparo do DNARESUMO
Recent structural analysis of Fe-S centers in replication proteins and insights into the structure and function of DNA polymerase δ (DNA Pol δ) subunits have shed light on the key role played by this polymerase at replication forks under stress. The sequencing of cancer genomes reveals multiple point mutations that compromise the activity of POLD1, the DNA Pol δ catalytic subunit, whereas the loci encoding the accessory subunits POLD2 and POLD3 are amplified in a very high proportion of human tumors. Consistently, DNA Pol δ is key for the survival of replication stress and is involved in multiple long-patch repair pathways. Synthetic lethality arises from compromising the function and availability of the noncatalytic subunits of DNA Pol δ under conditions of replication stress, opening the door to novel therapies.
Assuntos
DNA Polimerase III/química , DNA Polimerase III/metabolismo , Replicação do DNA/fisiologia , Neoplasias/genética , Animais , DNA Polimerase III/genética , Reparo do DNA , Humanos , Hidroxiureia/química , Neoplasias/patologia , OncogenesRESUMO
Health-related quality of life (HRQoL) is an important outcome for patients with sickle cell disease (SCD). It is often poor compared with other chronic medical conditions or measured as a multidomain disease-specific construct. We previously reported outcomes in the Start Healing in Patients with Hydroxyurea (SHIP-HU) randomized controlled trial in adolescents and adults with SCD at six clinical sites. Besides the primary outcomes, we also measured HRQoL as a secondary outcome. Patients in the intervention arm were each assigned community health workers (CHWs) who provided case management services. CHW services were independent of medical management, and medical managers were blinded to the study arm. Patients in the control arm received only standard of care. We hypothesized that having a CHW would improve HRQoL in patients enrolled in SHIP-HU. We did not find significant differences between domains of HRQoL in the two study arms. Possible explanations include selection bias of enrolled versus unenrolled patients, selection bias of sites, medical providers and medical management, enforced blinding, and a lack of cooperation between medical managers and CHWs. The importance of CHWs and HRQoL is nonetheless recognized based on the literature. Future interventions on HRQoL in SCD should consider alternative study designs and multimodal interventions.
Assuntos
Anemia Falciforme , Hidroxiureia , Adolescente , Humanos , Adulto Jovem , Anemia Falciforme/complicações , Antidrepanocíticos/uso terapêutico , Agentes Comunitários de Saúde , Hidroxiureia/uso terapêutico , Qualidade de VidaRESUMO
Cyclic thrombocytopenia (CTP) is characterized by periodic platelet oscillation with substantial amplitude. Most CTP cases have a thrombocytopenic background and are often misdiagnosed as immune thrombocytopenia with erratically effective treatment choices. CTP also occurs during hydroxyurea treatment in patients with myeloproliferative diseases. While the aetiology of CTP remains uncertain, here we evaluate historical, theoretical and clinical findings to provide a framework for understanding CTP pathophysiology. CTP retains the intrinsic oscillatory factors defined by the homeostatic regulation of platelet count, presenting as reciprocal platelet/thrombopoietin oscillations and stable oscillation periodicity. Moreover, CTP patients possess pathogenic factors destabilizing the platelet homeostatic system thereby creating opportunities for external perturbations to initiate and sustain the exaggerated platelet oscillations. Beyond humoral and cell-mediated autoimmunity, we propose recently uncovered germline and somatic genetic variants, such as those of MPL, STAT3 or DNMT3A, as pathogenic factors in thrombocytopenia-related CTP. Likewise, the JAK2 V617F or BCR::ABL1 translocation that drives underlying myeloproliferative diseases may also play a pathogenic role in hydroxyurea-induced CTP, where hydroxyurea treatment can serve as both a trigger and a pathogenic factor of platelet oscillation. Elucidating the pathogenic landscape of CTP provides an opportunity for targeted therapeutic approaches in the future.
Assuntos
Neoplasias da Medula Óssea , Transtornos Mieloproliferativos , Trombocitopenia , Humanos , Hidroxiureia/uso terapêutico , Trombocitopenia/etiologia , Trombocitopenia/genética , Contagem de Plaquetas , Plaquetas , Transtornos Mieloproliferativos/genéticaRESUMO
We compared serum anti-Mullerian hormone (AMH) levels in women with sickle cell disease (SCD) (n = 152) to those of Black comparison women (n = 128) between the ages of 20 and 45 years and evaluated the impact of hydroxyurea (HU) and iron overload on ovarian reserve in those with SCD. SCD treatment was abstracted from medical records. Linear regression models were fit to examine the relationship between log(AMH) and SCD, adjusting for age. The analysis was repeated to account for HU use (current, previous, never) and iron overload (ferritin ≥1000 ng/mL vs. <1000 ng/mL). AMH estimates among women with SCD were lower than those among comparison women (2.23, 95% confidence interval [CI] 1.80-2.76 vs. 4.12, 95% CI 3.11-5.45, respectively). Women with SCD who were currently using HU had 63% lower (95% CI 43-76) AMH values than comparison women; those with SCD with prior or no HU use also had lower AMH estimates than comparison women, but the difference was less pronounced. There were no differences in predicted AMH values among women with SCD for those with and without iron overload. Women with SCD and low AMH may have a shorter reproductive window and may benefit from referral to a reproductive specialist.