RESUMEN
WEE1 kinase phosphorylates CDK1 and CDK2 to regulate origin firing and mitotic entry. Inhibition of WEE1 has become an attractive target for cancer therapy due to the simultaneous induction of replication stress and inhibition of the G2/M checkpoint. WEE1 inhibition in cancer cells with high levels of replication stress results in induction of replication catastrophe and mitotic catastrophe. To increase potential as a single agent chemotherapeutic, a better understanding of genetic alterations that impact cellular responses to WEE1 inhibition is warranted. Here, we investigate the impact of loss of the helicase, FBH1, on the cellular response to WEE1 inhibition. FBH1-deficient cells have a reduction in ssDNA and double strand break signaling indicating FBH1 is required for induction of replication stress response in cells treated with WEE1 inhibitors. Despite the defect in the replication stress response, FBH1-deficiency sensitizes cells to WEE1 inhibition by increasing mitotic catastrophe. We propose loss of FBH1 is resulting in replication-associated damage that requires the WEE1-dependent G2 checkpoint for repair.
Asunto(s)
Proteínas de Ciclo Celular , ADN Helicasas , Proteínas de Ciclo Celular/metabolismo , ADN Helicasas/metabolismo , Muerte Celular , Transducción de Señal , Mitosis , Línea Celular TumoralRESUMEN
The proper resolution of DNA damage during replication is essential for genome stability. FBH1, a UvrD, helicase plays crucial roles in the DNA damage response. FBH1 promotes double strand break formation and signaling in response to prolonged replication stress to initiate apoptosis. Human FBH1 regulates RAD51 to inhibit homologous recombination. A previous study suggested that mis-regulation of RAD51 may contribute to replication stress resistance in FBH1-deficient cells, but the underlying mechanism remains unknown. Here, we provide direct evidence that RAD51 promotes replication stress resistance in FBH1-deficient cells. We demonstrate inhibition of RAD51 using the small molecule, B02, partially rescues double strand break signaling in FBH1-deficient cells. We show that inhibition of only the strand exchange activity of RAD51 rescues double strand break signaling in FBH1 knockout cells. Finally, we show that depletion of UBC13, a E2 protein that promotes RAD51-dependent template switching, rescues double strand break formation and signaling sensitizing FBH1-deficient cells to replication stress. Our results suggest FBH1 regulates template switching to promote replication stress sensitivity.
RESUMEN
Autosomal dominant polycystic kidney disease (ADPKD) resulting from pathogenic variants in PKD1 and PKD2 is the most common form of PKD, but other genetic causes tied to primary cilia function have been identified. Biallelic pathogenic variants in the serine/threonine kinase NEK8 cause a syndromic ciliopathy with extra-kidney manifestations. Here we identify NEK8 as a disease gene for ADPKD in 12 families. Clinical evaluation was combined with functional studies using fibroblasts and tubuloids from affected individuals. Nek8 knockout mouse kidney epithelial (IMCD3) cells transfected with wild type or variant NEK8 were further used to study ciliogenesis, ciliary trafficking, kinase function, and DNA damage responses. Twenty-one affected monoallelic individuals uniformly exhibited cystic kidney disease (mostly neonatal) without consistent extra-kidney manifestations. Recurrent de novo mutations of the NEK8 missense variant p.Arg45Trp, including mosaicism, were seen in ten families. Missense variants elsewhere within the kinase domain (p.Ile150Met and p.Lys157Gln) were also identified. Functional studies demonstrated normal localization of the NEK8 protein to the proximal cilium and no consistent cilia formation defects in patient-derived cells. NEK8-wild type protein and all variant forms of the protein expressed in Nek8 knockout IMCD3 cells were localized to cilia and supported ciliogenesis. However, Nek8 knockout IMCD3 cells expressing NEK8-p.Arg45Trp and NEK8-p.Lys157Gln showed significantly decreased polycystin-2 but normal ANKS6 localization in cilia. Moreover, p.Arg45Trp NEK8 exhibited reduced kinase activity in vitro. In patient derived tubuloids and IMCD3 cells expressing NEK8-p.Arg45Trp, DNA damage signaling was increased compared to healthy passage-matched controls. Thus, we propose a dominant-negative effect for specific heterozygous missense variants in the NEK8 kinase domain as a new cause of PKD.
Asunto(s)
Enfermedades Renales Poliquísticas , Riñón Poliquístico Autosómico Dominante , Animales , Humanos , Recién Nacido , Ratones , Proteínas Portadoras/metabolismo , Cilios/patología , Riñón/metabolismo , Mutación , Quinasas Relacionadas con NIMA/genética , Quinasas Relacionadas con NIMA/metabolismo , Enfermedades Renales Poliquísticas/genética , Riñón Poliquístico Autosómico Dominante/patología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Serina/genética , Serina/metabolismo , Canales Catiónicos TRPP/genética , Canales Catiónicos TRPP/metabolismoRESUMEN
WEE1 kinase phosphorylates CDK1 and CDK2 to regulate origin firing and mitotic entry. Inhibition of WEE1 has become an attractive target for cancer therapy due to the simultaneous induction of replication stress and inhibition of the G2/M checkpoint. WEE1 inhibition in cancer cells with high levels of replication stress results in induction of replication catastrophe and mitotic catastrophe. To increase potential as a single agent chemotherapeutic, a better understanding of genetic alterations that impact cellular responses to WEE1 inhibition is warranted. Here, we investigate the impact of loss of the helicase, FBH1, on the cellular response to WEE1 inhibition. FBH1-deficient cells have a reduction in ssDNA and double strand break signaling indicating FBH1 is required for induction of replication stress response in cells treated with WEE1 inhibitors. Despite the defect in the replication stress response, FBH1-deficiency sensitizes cells to WEE1 inhibition by increasing mitotic catastrophe. We propose loss of FBH1 is resulting in replication-associated damage that requires the WEE1-dependent G2 checkpoint for repair.
RESUMEN
A significant proportion of genetic disease cases arise from truncation of proteins caused by premature termination codons. In eukaryotic cells some aminoglycosides cause readthrough of premature termination codons during protein translation. Inducing readthrough of these codons can potentially be of therapeutic value in the treatment of numerous genetic diseases. A significant drawback to the repeated use of aminoglycosides as treatments is the lack of balance between their readthrough efficacy and toxicity. The synthesis and biological testing of designer aminoglycoside compounds is documented herein. We disclose the implementation of a strategy to reduce cellular toxicity and maintain readthrough activity of a library of compounds by modification of the overall cationic charge of the aminoglycoside scaffold through ring I modifications.
RESUMEN
Innate immune defense mechanisms play a pivotal role in antitumor responses. Recent evidence suggests that antiviral innate immunity is regulated not only by exogenous non-self-RNA but also by host-derived pseudogene RNAs. A growing body of evidence also indicates a biological role for pseudogenes as gene expression regulators or immune modulators. Here, we report an important role for BRCA1P1, the pseudogene of the BRCA1 tumor-suppressor gene, in regulating innate immune defense mechanisms in breast cancer cells. BRCA1P1 expresses a long-noncoding RNA (lncRNA) in breast cancer cells through divergent transcription. Expression of lncRNA-BRCA1P1 is increased in breast tumors compared with normal breast tissues. Depletion of BRCA1P1 induces an antiviral defense-like program, including the expression of antiviral genes in breast cancer cells. Furthermore, BRCA1P1-deficient cancer cells mimic virus-infected cells by stimulating cytokines and inducing cell apoptosis. Accordingly, depletion of BRCA1P1 increases host innate immune responses and restricts virus replication. In converse, overexpression of BRCA1P1 reduces cytokine expression in breast cancer cells. Mechanistically, lncRNA-BRCA1P1 is localized in the nucleus, binds to the NF-κB subunit RelA, and negatively regulates antiviral gene expression. Finally, in a xenograft mouse model of breast cancer, depletion of BRCA1P1 stimulates cytokine expression and local immunity, and suppresses tumor growth. Our results suggest an important role for BRCA1P1 in innate immune defense mechanisms and antitumor responses. This mechanism of antiviral immunity regulated by a host-derived pseudogene RNA may guide the development of novel therapies targeting immune responses in breast cancer. SIGNIFICANCE: This study identifies a novel mechanism of innate immunity driven by a host pseudogene RNA that inhibits innate immune defense mechanisms and antitumor responses through regulation of antiviral gene expression.
Asunto(s)
Neoplasias de la Mama/genética , Carcinoma Ductal de Mama/genética , Seudogenes/fisiología , ARN Largo no Codificante/metabolismo , Escape del Tumor/genética , Animales , Mama/patología , Mama/cirugía , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Neoplasias de la Mama/cirugía , Carcinoma Ductal de Mama/inmunología , Carcinoma Ductal de Mama/patología , Carcinoma Ductal de Mama/cirugía , Línea Celular Tumoral , Núcleo Celular/genética , Citocinas/genética , Femenino , Regulación Neoplásica de la Expresión Génica/inmunología , Técnicas de Inactivación de Genes , Interacciones Microbiota-Huesped/genética , Interacciones Microbiota-Huesped/inmunología , Humanos , Inmunidad Innata/genética , Mastectomía , Ratones , Cultivo Primario de Células , ARN Largo no Codificante/genética , Infecciones por Respirovirus/inmunología , Infecciones por Respirovirus/virología , Virus Sendai/inmunología , Factor de Transcripción ReIA/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Domesticated dogs show unparalleled diversity in body size across breeds, but within breeds variation is limited by selective breeding. Many heritable diseases of dogs are found among breeds of similar sizes, suggesting that as in humans, alleles governing growth have pleiotropic effects. Here, we conducted independent genome-wide association studies in the small Shetland Sheepdog breed and discovered a locus on chromosome 9 that is associated with a dental abnormality called maxillary canine-tooth mesioversion (MCM) (P = 1.53 × 10-7) as well as two body size traits: height (P = 1.67 × 10-5) and weight (P = 1.16 × 10-7). Using whole-genome resequencing data, we identified variants in two proximal genes: FTSJ3, encoding an RNA methyltransferase, and GH1, encoding growth hormone. A substitution in FTSJ3 and a splice donor insertion in GH1 are strongly associated with MCM and reduced body size in Shetland Sheepdogs. We demonstrated in vitro that the GH1 variant leads to exon 3 skipping, predicting a mutant protein known to cause human pituitary dwarfism. Statistical modeling, however, indicates that the FTSJ3 variant is the stronger predictor of MCM and that each derived allele reduces body size by about 1 inch and 5 pounds. In a survey of 224 breeds, both FTSJ3 and GH1 variants are frequent among very small "toy" breeds and absent from larger breeds. Our findings indicate that a chromosome 9 locus harboring tightly linked variants in FTSJ3 and GH1 reduces growth in the Shetland Sheepdog and toy breed dogs and confers risk for MCM through vertical pleiotropy.
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Tamaño Corporal/genética , Estudio de Asociación del Genoma Completo , Hormona del Crecimiento/genética , Metiltransferasas/genética , Alelos , Animales , Peso Corporal , Cruzamiento , Perros , Exones , Genotipo , Haplotipos/genética , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
The central recombination enzyme RAD51 has been implicated in replication fork processing and restart in response to replication stress. Here, we use a separation-of-function allele of RAD51 that retains DNA binding, but not D-loop activity, to reveal mechanistic aspects of RAD51's roles in the response to replication stress. Here, we find that cells lacking RAD51's enzymatic activity protect replication forks from MRE11-dependent degradation, as expected from previous studies. Unexpectedly, we find that RAD51's strand exchange activity is not required to convert stalled forks to a form that can be degraded by DNA2. Such conversion was shown previously to require replication fork regression, supporting a model in which fork regression depends on a non-enzymatic function of RAD51. We also show RAD51 promotes replication restart by both strand exchange-dependent and strand exchange-independent mechanisms.
Asunto(s)
Replicación del ADN , Proteínas de Unión al ADN/metabolismo , ADN/química , Recombinasa Rad51/metabolismo , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/genética , Humanos , Modelos Genéticos , Mutación , Conformación de Ácido Nucleico , Recombinasa Rad51/genéticaRESUMEN
The rise of antibiotic resistance, coupled with increased expectations for mobility in later life, is creating a need for biofilm inhibitors and delivery systems that will reduce surgical implant infection. A limitation of some of these existing delivery approaches is toxicity exhibited toward host cells. Here, we report the application of a novel inhibitor of the enzyme, methylthioadenosine nucleosidase (MTAN), a key enzyme in bacterial metabolic pathways, which include S-adenosylmethionine catabolism and purine nucleotide recycling, in combination with a poly(vinyl alcohol)-tyramine-based (PVA-Tyr) hydrogel delivery system. We demonstrate that a lead MTAN inhibitor, selected from a screened library of 34 candidates, (2S)-2-(4-amino-5H-pyrrolo3,2-dpyrimidin-7-ylmethyl)aminoundecan-1-ol (31), showed a minimum biofilm inhibitory concentration of 2.2 ± 0.4 µM against a clinical staphylococcal species isolated from an infected implant. We observed that extracellular DNA, a key constituent of biofilms, is significantly reduced when treated with 10 µM compound 31, along with a decrease in biofilm thickness. Compound 31 was incorporated into a hydrolytically degradable photo-cross-linked PVA-Tyr hydrogel and the release profile was evaluated by HPLC studies. Compound 31 released from the PVA-hydrogel system significantly reduced biofilm formation (77.2 ± 8.4% biofilm inhibition). Finally, compound 31 released from PVA-Tyr showed no negative impact on human bone marrow stromal cell (MSC) viability, proliferation, or morphology. The results demonstrate the potential utility of MTAN inhibitors in treating infections caused by Gram-positive bacteria, and the development of a nontoxic release system that has potential for tunability for time scale of delivery.
RESUMEN
Telomeres serve as protective caps that help the cell differentiate between the naturally occurring ends of chromosomes and double-stranded breaks. When telomere capping function becomes compromised, chromosome ends are subjected to elevated rates of chromosome alterations. These effects can be particularly dramatic in the telomere-adjacent subtelomeric region. While the catastrophic impact of severe telomere dysfunction on genome stability has been well documented, the adaptive telomere failure hypothesis considers an alternative role telomere dysfunction may play in adaptive evolution. This hypothesis suggests that low levels of telomere failure, induced by certain environmental stresses, can lead to elevated subtelomeric recombination. Mutational loss, duplication, or modification of subtelomeric contingency genes could ultimately facilitate adaptation by generating novel mutants better able to survive environmental stress. In this perspective, we discuss recent work that examined mild telomere dysfunction and its role in altering the adaptive potential of subtelomeric genes.
Asunto(s)
Cromosomas Fúngicos , Evolución Molecular , Saccharomyces cerevisiae/genética , Telómero/fisiología , Roturas del ADN de Doble Cadena , Replicación del ADN , Inestabilidad Genómica , MutaciónRESUMEN
Subtelomeric regions have several unusual characteristics, including complex repetitive structures, increased rates of evolution, and enrichment for genes involved in niche adaptation. The adaptive telomere failure hypothesis suggests that certain environmental stresses can induce a low level of telomere failure, potentially leading to elevated subtelomeric recombination that could result in adaptive mutational changes within subtelomeric genes. Here, we tested a key prediction of the adaptive telomere failure hypothesis-that telomere dysfunction mild enough to have little or no overall effect on cell fitness could still lead to substantial increases in the mutation rates of subtelomeric genes. Our results show that a mutant of Kluyveromyces lactis with stably short telomeres produced a large increase in the frequency of mutations affecting the native subtelomeric ß-galactosidase (LAC4) gene. All lac4 mutants examined from strains with severe telomere dysfunction underwent terminal deletion/duplication events consistent with being due to break-induced replication. In contrast, although cells with mild telomere dysfunction also exhibited similar terminal deletion and duplication events, up to 50% of lac4 mutants from this background unexpectedly contained base changes within the LAC4 coding region. This mutational bias for producing base changes demonstrates that mild telomere dysfunction can be well suited as a force for altering the adaptive potential of subtelomeric genes.
Asunto(s)
Adaptación Biológica/genética , Telómero/genética , Cromosomas Fúngicos , Reparación del ADN , Duplicación de Gen , Genes del Tipo Sexual de los Hongos/genética , Mutación con Pérdida de Función , Mutación , Recombinación Genética , Telómero/metabolismo , Levaduras/genéticaRESUMEN
Ribosome inactivating proteins (RIPs) are among the most toxic agents known. More than a dozen clinical trials against refractory cancers have been initiated using modified RIPs with impressive results. However, dose-limiting toxicity due to vascular leak syndrome limits success of the therapy. We have previously reported some tight-binding transition state analogues of Saporin L3 that mimic small oligonucleotide substrates in which the susceptible adenosine has been replaced by a 9-deazaadenyl hydroxypyrrolidinol derivative. They provide the first step in the development of rescue agents to prevent Saporin L3 toxicity on non-targeted cells. Here we report the synthesis, using solution phase chemistry, of these and a larger group of transition state analogues. They were tested for inhibition against Saporin L3 giving Ki values as low as 3.3 nM and indicating the structural requirements for inhibition.
Asunto(s)
Materiales Biomiméticos/química , Materiales Biomiméticos/farmacología , Oligonucleótidos/química , Oligonucleótidos/farmacología , Proteínas Inactivadoras de Ribosomas Tipo 1/química , Secuencia de Bases , Modelos Moleculares , Oligonucleótidos/genética , Conformación Proteica , ARN/genética , ARN/metabolismo , Proteínas Inactivadoras de Ribosomas Tipo 1/metabolismo , Proteínas Inactivadoras de Ribosomas Tipo 1/toxicidad , SaporinasRESUMEN
In response to chromosomal double-strand breaks (DSBs), eukaryotic cells activate the DNA damage checkpoint, which is orchestrated by the PI3 kinase-like protein kinases ATR and ATM (Mec1 and Tel1 in budding yeast). Following DSB formation, Mec1 and Tel1 phosphorylate histone H2A on serine 129 (known as γ-H2AX). We used caffeine to inhibit the checkpoint kinases after DSB induction. We show that prolonged phosphorylation of H2A-S129 does not require continuous Mec1 and Tel1 activity. Unexpectedly, caffeine treatment impaired homologous recombination by inhibiting 5' to 3' end resection, independent of Mec1 and Tel1 inhibition. Caffeine treatment led to the rapid loss, by proteasomal degradation, of both Sae2, a nuclease that plays a role in early steps of resection, and Dna2, a nuclease that facilitates one of two extensive resection pathways. Sae2's instability is evident in the absence of DNA damage. A similar loss is seen when protein synthesis is inhibited by cycloheximide. Caffeine treatment had similar effects on irradiated HeLa cells, blocking the formation of RPA and Rad51 foci that depend on 5' to 3' resection of broken chromosome ends. Our findings provide insight toward the use of caffeine as a DNA damage-sensitizing agent in cancer cells.
Asunto(s)
Cafeína/farmacología , Roturas del ADN de Doble Cadena , ADN Helicasas/metabolismo , Reparación del ADN/efectos de los fármacos , Endonucleasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Células HeLa , Histonas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Recombinasa Rad51/metabolismo , Proteína de Replicación A/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidoresRESUMEN
Efficient repair of chromosomal double-strand breaks (DSBs) by homologous recombination relies on the formation of a Rad51 recombinase filament that forms on single-stranded DNA (ssDNA) created at DSB ends. This filament facilitates the search for a homologous donor sequence and promotes strand invasion. Recently caffeine treatment has been shown to prevent gene targeting in mammalian cells by increasing non-productive Rad51 interactions between the DSB and random regions of the genome. Here we show that caffeine treatment prevents gene conversion in yeast, independently of its inhibition of the Mec1(ATR)/Tel1(ATM)-dependent DNA damage response or caffeine's inhibition of 5' to 3' resection of DSB ends. Caffeine treatment results in a dosage-dependent eviction of Rad51 from ssDNA. Gene conversion is impaired even at low concentrations of caffeine, where there is no discernible dismantling of the Rad51 filament. Loss of the Rad51 filament integrity is independent of Srs2's Rad51 filament dismantling activity or Rad51's ATPase activity and does not depend on non-specific Rad51 binding to undamaged double-stranded DNA. Caffeine treatment had similar effects on irradiated HeLa cells, promoting loss of previously assembled Rad51 foci. We conclude that caffeine treatment can disrupt gene conversion by disrupting Rad51 filaments.
Asunto(s)
Cafeína/farmacología , ADN de Cadena Simple/metabolismo , Conversión Génica/efectos de los fármacos , Recombinasa Rad51/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidoresRESUMEN
The RAD54 family DNA translocases have several biochemical activities. One activity, demonstrated previously for the budding yeast translocases, is ATPase-dependent disruption of RAD51-dsDNA binding. This activity is thought to promote dissociation of RAD51 from heteroduplex DNA following strand exchange during homologous recombination. In addition, previous experiments in budding yeast have shown that the same activity of Rad54 removes Rad51 from undamaged sites on chromosomes; mutants lacking Rad54 accumulate nonrepair-associated complexes that can block growth and lead to chromosome loss. Here, we show that human RAD54 also promotes the dissociation of RAD51 from dsDNA and not ssDNA. We also show that translocase depletion in tumor cell lines leads to the accumulation of RAD51 on chromosomes, forming complexes that are not associated with markers of DNA damage. We further show that combined depletion of RAD54L and RAD54B and/or artificial induction of RAD51 overexpression blocks replication and promotes chromosome segregation defects. These results support a model in which RAD54L and RAD54B counteract genome-destabilizing effects of direct binding of RAD51 to dsDNA in human tumor cells. Thus, in addition to having genome-stabilizing DNA repair activity, human RAD51 has genome-destabilizing activity when expressed at high levels, as is the case in many human tumors.
Asunto(s)
ADN Helicasas/metabolismo , Reparación del ADN , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Recombinasa Rad51/metabolismo , Línea Celular Tumoral , Proliferación Celular , ADN Helicasas/antagonistas & inhibidores , ADN Helicasas/genética , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Proteínas de Unión al ADN , Humanos , Células MCF-7 , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mutágenos/metabolismo , Neoplasias/patología , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , ARN Interferente Pequeño/genética , Recombinasa Rad51/genética , Proteína de Replicación A/genética , Proteína de Replicación A/metabolismoRESUMEN
A levulinoyl ester-containing cellulose polymer is introduced as a waterborne coating. Incorporation of the biomass-derived levulinic acid proceeds via an unexpected intermediate and provides the unusual feature of a cellulose derivative that is readily chemically modified. The levulinoyl-cellulose ester could be chemically manipulated, allowing it to be dispersed to generate a waterborne hydrocolloid latex. This was capable of film-formation at room temperature, and was formulated for use as a coating of high-renewable content.
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Celulosa/química , Agua/química , Biomasa , Ésteres , Industrias , Cetonas/química , Ácidos Levulínicos/químicaRESUMEN
RAD51 is the central protein that catalyzes DNA repair via homologous recombination, a process that ensures genomic stability. RAD51 protein is commonly expressed at high levels in cancer cells relative to their noncancerous precursors. High levels of RAD51 expression can lead to the formation of genotoxic RAD51 protein complexes on undamaged chromatin. We developed a therapeutic approach that exploits this potentially toxic feature of malignancy, using compounds that stimulate the DNA-binding activity of RAD51 to promote cancer cell death. A panel of immortalized cell lines was challenged with the RAD51-stimulatory compound RS-1. Resistance to RS-1 tended to occur in cells with higher levels of RAD54L and RAD54B, which are Swi2/Snf2-related translocases known to dissociate RAD51 filaments from dsDNA. In PC3 prostate cancer cells, RS-1-induced lethality was accompanied by the formation of microscopically visible RAD51 nuclear protein foci occurring in the absence of any DNA-damaging treatment. Treatment with RS-1 promoted significant antitumor responses in a mouse model, providing proof-of-principle for this novel therapeutic strategy.
Asunto(s)
Benzamidas/farmacología , ADN Helicasas/genética , Neoplasias/genética , Proteínas Nucleares/genética , Recombinasa Rad51/genética , Sulfonamidas/farmacología , Animales , Línea Celular Tumoral , Supervivencia Celular , Cromatina/metabolismo , ADN Helicasas/biosíntesis , Reparación del ADN/efectos de los fármacos , Reparación del ADN/genética , Replicación del ADN/efectos de los fármacos , Replicación del ADN/genética , Proteínas de Unión al ADN , Células HEK293 , Recombinación Homóloga/genética , Humanos , Células MCF-7 , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias/tratamiento farmacológico , Proteínas Nucleares/biosíntesis , Unión Proteica , Interferencia de ARN , Recombinasa Rad51/biosíntesisRESUMEN
SNM1B/Apollo is a DNA nuclease that has important functions in telomere maintenance and repair of DNA interstrand crosslinks (ICLs) within the Fanconi anemia (FA) pathway. SNM1B is required for efficient localization of key repair proteins, such as the FA protein, FANCD2, to sites of ICL damage and functions epistatically to FANCD2 in cellular survival to ICLs and homology-directed repair. The FA pathway is also activated in response to replication fork stalling. Here, we sought to determine the importance of SNM1B in cellular responses to stalled forks in the absence of a blocking lesion, such as ICLs. We found that depletion of SNM1B results in hypersensitivity to aphidicolin, a DNA polymerase inhibitor that causes replication stress. We observed that the SNM1B nuclease is required for efficient localization of the DNA repair proteins, FANCD2 and BRCA1, to subnuclear foci upon aphidicolin treatment, thereby indicating SNM1B facilitates direct repair of stalled forks. Consistent with a role for SNM1B subsequent to recognition of the lesion, we found that SNM1B is dispensable for upstream events, including activation of ATR-dependent signaling and localization of RPA, γH2AX and the MRE11/RAD50/NBS1 complex to aphidicolin-induced foci. We determined that a major consequence of SNM1B depletion is a marked increase in spontaneous and aphidicolin-induced chromosomal gaps and breaks, including breakage at common fragile sites. Thus, this study provides evidence that SNM1B functions in resolving replication stress and preventing accumulation of genomic damage.
Asunto(s)
Sitios Frágiles del Cromosoma , Enzimas Reparadoras del ADN/metabolismo , Replicación del ADN , Inestabilidad Genómica , Proteínas Nucleares/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Afidicolina/farmacología , Proteína BRCA1/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Cromatina/metabolismo , Daño del ADN , Reparación del ADN , Enzimas Reparadoras del ADN/química , Enzimas Reparadoras del ADN/genética , Exodesoxirribonucleasas , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Expresión Génica , Histonas/metabolismo , Humanos , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Proteína de Replicación A/metabolismo , Transducción de Señal/efectos de los fármacos , UbiquitinaciónRESUMEN
The pathogenic protozoa responsible for malaria lack enzymes for the de novo synthesis of purines and rely on purine salvage from the host. In Plasmodium falciparum (Pf), hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) converts hypoxanthine to inosine monophosphate and is essential for purine salvage making the enzyme an anti-malarial drug target. We have synthesized a number of simple acyclic aza-C-nucleosides and shown that some are potent inhibitors of Pf HGXPRT while showing excellent selectivity for the Pf versus the human enzyme.
Asunto(s)
Antimaláricos/química , Inhibidores Enzimáticos/química , Nucleósidos/química , Pentosiltransferasa/antagonistas & inhibidores , Plasmodium falciparum/enzimología , Antimaláricos/síntesis química , Antimaláricos/farmacología , Compuestos Aza/química , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Humanos , Cinética , Nucleósidos/síntesis química , Nucleósidos/farmacología , Pentosiltransferasa/metabolismo , Plasmodium falciparum/efectos de los fármacos , Unión ProteicaRESUMEN
Several acyclic hydroxy-methylthio-amines with 3-5 carbon atoms were prepared and coupled via a methylene link to 9-deazaadenine. The products were tested for inhibition against human MTAP and Escherichia coli and Neisseria meningitidis MTANs and gave K(i) values as low as 0.23 nM. These results were compared to those obtained with 1st and 2nd generation inhibitors (1S)-1-(9-deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-5-methylthio-D-ribitol (MT-Immucillin-A, 3) and (3R,4S)-1-[9-deazaadenin-9-yl)methyl]3-hydroxy-4-methylthiomethylpyrrolidine (MT-DADMe-Immucillin-A, 4). The best inhibitors were found to exhibit binding affinities of approximately 2- to 4-fold those of 3 but were significantly weaker than 4. Cleavage of the 2,3 carbon-carbon bond in MT-Immucillin-A (3) gave an acyclic product (79) with a 21,500 fold loss of activity against E. coli MTAN. In another case, N-methylation of a side chain secondary amine resulted in a 250-fold loss of activity against the same enzyme [(±)-65 vs (±)-68]. The inhibition results were also contrasted with those acyclic derivatives previously prepared as inhibitors for a related enzyme, purine nucleoside phosphorylase (PNP), where some inhibitors in the latter case were found to be more potent than their cyclic counterparts.