RESUMEN
Eukaryotic genomes replicate via spatially and temporally regulated origin firing. Cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK) promote origin firing, whereas the S phase checkpoint limits firing to prevent nucleotide and RPA exhaustion. We used chemical genetics to interrogate human DDK with maximum precision, dissect its relationship with the S phase checkpoint, and identify DDK substrates. We show that DDK inhibition (DDKi) leads to graded suppression of origin firing and fork arrest. S phase checkpoint inhibition rescued origin firing in DDKi cells and DDK-depleted Xenopus egg extracts. DDKi also impairs RPA loading, nascent-strand protection, and fork restart. Via quantitative phosphoproteomics, we identify the BRCA1-associated (BRCA1-A) complex subunit MERIT40 and the cohesin accessory subunit PDS5B as DDK effectors in fork protection and restart. Phosphorylation neutralizes autoinhibition mediated by intrinsically disordered regions in both substrates. Our results reveal mechanisms through which DDK controls the duplication of large vertebrate genomes.
Asunto(s)
Replicación del ADN , Origen de Réplica , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Replicación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Puntos de Control de la Fase S del Ciclo Celular , Especificidad por Sustrato , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Xenopus laevisRESUMEN
Crossover recombination is essential for accurate chromosome segregation during meiosis. The MutSγ complex, Msh4-Msh5, facilitates crossing over by binding and stabilizing nascent recombination intermediates. We show that these activities are governed by regulated proteolysis. MutSγ is initially inactive for crossing over due to an N-terminal degron on Msh4 that renders it unstable by directly targeting proteasomal degradation. Activation of MutSγ requires the Dbf4-dependent kinase Cdc7 (DDK), which directly phosphorylates and thereby neutralizes the Msh4 degron. Genetic requirements for Msh4 phosphorylation indicate that DDK targets MutSγ only after it has bound to nascent joint molecules (JMs) in the context of synapsing chromosomes. Overexpression studies confirm that the steady-state level of Msh4, not phosphorylation per se, is the critical determinant for crossing over. At the DNA level, Msh4 phosphorylation enables the formation and crossover-biased resolution of double-Holliday Junction intermediates. Our study establishes regulated protein degradation as a fundamental mechanism underlying meiotic crossing over.
Asunto(s)
Intercambio Genético , Proteínas de Unión al ADN/metabolismo , Meiosis/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Emparejamiento Cromosómico , Proteínas de Unión al ADN/química , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteolisis , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
DNA replication is initiated by assembly of the kinase cell division cycle 7 (CDC7) with its regulatory activation subunit, activator of S-phase kinase (ASK), to activate DNA helicase. However, the mechanism underlying regulation of CDC7-ASK complex is unclear. Here, we show that ADP generated from CDC7-mediated MCM phosphorylation binds to an allosteric region of CDC7, disrupts CDC7-ASK interaction, and inhibits CDC7-ASK activity in a feedback way. EGFR- and ERK-activated casein kinase 2α (CK2α) phosphorylates nuclear phosphoglycerate kinase (PGK) 1 at S256, resulting in interaction of PGK1 with CDC7. CDC7-bound PGK1 converts ADP to ATP, thereby abrogating the inhibitory effect of ADP on CDC7-ASK activity, promoting the recruitment of DNA helicase to replication origins, DNA replication, cell proliferation, and brain tumorigenesis. These findings reveal an instrumental self-regulatory mechanism of CDC7-ASK activity by its kinase reaction product ADP and a nonglycolytic role for PGK1 in abrogating this negative feedback in promoting tumor development.
Asunto(s)
Adenosina Difosfato/metabolismo , Quinasa de la Caseína II/metabolismo , Proteínas de Ciclo Celular/antagonistas & inhibidores , Replicación del ADN , Fosfoglicerato Quinasa/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Animales , Quinasa de la Caseína II/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiología , Línea Celular , Línea Celular Tumoral , ADN Helicasas/genética , ADN Helicasas/metabolismo , Femenino , Xenoinjertos , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Fosfoglicerato Quinasa/genética , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Origen de RéplicaRESUMEN
The evolutionally conserved Cdc7 kinase plays crucial roles in initiation of DNA replication as well as in other chromosomal events. To examine the roles of Cdc7 in brain development, we have generated mice carrying Cdc7 knockout in neural stem cells by using Nestin-Cre. The Cdc7Fl/Fl NestinCre mice were born, but exhibited severe growth retardation and impaired postnatal brain development. These mice exhibited motor dysfunction within 9 days after birth and did not survive for more than 19 days. The cerebral cortical layer formation was impaired, although the cortical cell numbers were not altered in the mutant. In the cerebellum undergoing hypoplasia, granule cells (CGC) decreased in number in Cdc7Fl/F l NestinCre mice compared to the control at E15-18, suggesting that Cdc7 is required for DNA replication and cell proliferation of CGC at mid embryonic stage (before embryonic day 15). On the other hand, the Purkinje cell numbers were not altered but its layer formation was impaired in the mutant. These results indicate differential roles of Cdc7 in DNA replication/cell proliferation in brain. Furthermore, the defects of layer formation suggest a possibility that Cdc7 may play an additional role in cell migration during neural development.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas Serina-Treonina Quinasas , Animales , Ratones , Proteínas de Ciclo Celular/metabolismo , Cerebelo/metabolismo , Replicación del ADN , Nestina/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
IMPORTANCE: The Avibirnavirus infectious bursal disease virus is still an important agent which largely threatens global poultry farming industry economics. VP3 is a multifunctional scaffold structural protein that is involved in virus morphogenesis and the regulation of diverse cellular signaling pathways. However, little is known about the roles of VP3 phosphorylation during the IBDV life cycle. In this study, we determined that IBDV infection induced the upregulation of Cdc7 expression and phosphorylated the VP3 Ser13 site to promote viral replication. Moreover, we confirmed that the negative charge addition of phosphoserine on VP3 at the S13 site was essential for IBDV proliferation. This study provides novel insight into the molecular mechanisms of VP3 phosphorylation-mediated regulation of IBDV replication.
Asunto(s)
Avibirnavirus , Proteínas de Ciclo Celular , Pollos , Virus de la Enfermedad Infecciosa de la Bolsa , Proteínas Serina-Treonina Quinasas , Proteínas Estructurales Virales , Replicación Viral , Animales , Avibirnavirus/química , Avibirnavirus/crecimiento & desarrollo , Avibirnavirus/metabolismo , Infecciones por Birnaviridae/enzimología , Infecciones por Birnaviridae/metabolismo , Infecciones por Birnaviridae/veterinaria , Infecciones por Birnaviridae/virología , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Proteínas de Ciclo Celular/metabolismo , Pollos/virología , Virus de la Enfermedad Infecciosa de la Bolsa/química , Virus de la Enfermedad Infecciosa de la Bolsa/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Estructurales Virales/química , Proteínas Estructurales Virales/metabolismoRESUMEN
Cell division cycle 7 kinase (CDC7) has been found overexpressed in many cancer cell lines being also one of the kinases involved in the nuclear protein TDP-43 phosphorylation in vivo. Thus, inhibitors of CDC7 are emerging drug candidates for the treatment of oncological and neurodegenerative unmet diseases. All the known CDC7 inhibitors are ATP-competitives, lacking of selectivity enough for success in clinical trials. As allosteric sites are less conserved among kinase proteins, discovery of allosteric modulators of CDC7 is a great challenge and opportunity in this field.Using different computational approaches, we have here identified new druggable cavities on the human CDC7 structure and subsequently selective CDC7 inhibitors with allosteric modulation mainly targeting the pockets where the interaction between this kinase and its activator DBF4 takes place.
Asunto(s)
Proteínas Nucleares , Proteínas Serina-Treonina Quinasas , Humanos , Fosforilación , Sitio Alostérico , Línea Celular , Ciclo Celular , Proteínas de Ciclo CelularRESUMEN
In this phase 1 open-label study, we assessed the relative bioavailability of a prototype tablet formulation of TAK-931, a cell division cycle 7 kinase inhibitor, in reference to the current powder-in-capsule (PIC) formulation in patients with advanced solid tumors for whom no effective standard treatment was available. Adult patients were randomized 1:1 in a crossover fashion to receive one dose of TAK-931 80 mg PIC on Day 1 and one dose of TAK-931 80 mg tablet on Day 3 (or the reverse sequence), followed by TAK-931 50 mg PIC once daily (QD) for 12 days starting from Day 5, before a 7-day rest period (Cycle 0). From Cycle 1, all patients received 50 mg PIC QD on Days 1-14 followed by a 7-day rest period. Twenty patients were enrolled. Median Tmax was achieved approximately 2 h post-dose of TAK-931 80 mg for both tablet and PIC. Geometric mean Cmax, AUC exposures, and T1/2z of TAK-931 were similar for both formulations. Geometric mean Cmax, AUClast, and AUCinf ratios were 0.936 (90% confidence interval [CI]: 0.808-1.084), 1.004 (90% CI: 0.899-1.120), and 1.007 (90% CI: 0.903-1.123), respectively, for TAK-931 tablet in reference to PIC. Discontinuation of TAK-931 due to treatment-emergent adverse events (TEAEs) occurred in 1 patient. Four (20%) patients experienced a serious TEAE; none were considered related to TAK-931. Pharmacokinetics and systemic exposure profiles were similar following administration of both formulations, supporting the transition from PIC to tablet in the clinical development of TAK-931. (Trial registration number ClinicalTrials.gov NCT03708211. Registration date October 12, 2018).
Asunto(s)
Neoplasias , Adulto , Humanos , Disponibilidad Biológica , Polvos/uso terapéutico , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Comprimidos/uso terapéutico , Estudios Cruzados , Área Bajo la Curva , Administración Oral , Equivalencia TerapéuticaRESUMEN
BACKGROUND: Hepatocellular carcinoma (HCC) remains one of the most common and lethal malignancies worldwide. Although DBF4-dependent kinase (DDK) complex composed of CDC7 kinase and its regulatory subunit DBF4 has been shown to be overexpressed in primary tumors and promotes tumor development, while its role and prognostic value in HCC remain largely unknown. In the present study, the expression of DBF4 and CDC7 and their relationship with clinical characteristics were comprehensively analyzed. METHODS: The mRNA expression profiles of HCC and the corresponding clinical data of HCC patients were downloaded from TCGA and GEO databases, respectively. The differences in DBF4 and CDC7 expression in tumor tissues and adjacent normal tissues were analyzed. HCC-derived tissue microarray (TMA) was used to evaluate and score the expression of CDC7 by immunohistochemistry (IHC) staining. The Kaplan-Meier method and the Cox regression method were used to analyze the relationship between overall survival and clinical characteristics of the patients. Gene set enrichment analysis (GSEA) was used to analyze the pathway enrichment of DBF4 and CDC7. RESULTS: DBF4 and CDC7 had similar expression patterns in HCC patients. Detailly, compared with adjacent tissues, both mRNA and protein of DBF4 and CDC7 were significantly higher in HCC, and their expression was positively correlated with AJCC_T stage, clinical stage and G stage (grade) of liver cancer patients, and higher DBF4 or CDC7 expression predicted a worse prognosis in HCC patients with shorter overall survival (OS), recurrence-free survival (RFS), disease-specific survival (DSS) and progress-free survival (PFS). Cox regression analysis suggested that both DBF4 and CDC7 were independent risk factors for the prognosis of HCC patients in TCGA dataset. GSEA suggested that both DBF4 and CDC7 were positively correlated with cell cycle and DNA replication. Finally, the prognostic value of CDC7 was furtherly confirmed by TMA-based IHC staining results. CONCLUSIONS: Our study showed that DDK complex was significantly increased in HCC. Both DBF4 and CDC7 may be potential diagnostic and prognostic markers for HCC, and high expression of DDK members predicts a worse prognosis in patients with HCC, which may be associated with high tumor cell proliferation rate.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Carcinoma Hepatocelular/genética , Pronóstico , Neoplasias Hepáticas/genéticaRESUMEN
TDP-43 has been identified as the major component of protein aggregates found in affected neurons in FTLD-TDP and amyotrophic lateral sclerosis (ALS) patients. TDP-43 is hyperphosphorylated, ubiquitinated, and cleaved in the C-terminus. CDC-7 was reported to phosphorylate TDP-43. There are no effective treatments for either FTLD-TDP or ALS, being a pressing need for the search of new therapies. We hypothesized that modulating CDC-7 activity with small molecules that are able to interfere with TDP-43 phosphorylation could be a good therapeutic strategy for these diseases. Here, we have studied the effects of novel brain penetrant, thiopurine-based, CDC-7 inhibitors in TDP-43 homeostasis in immortalized lymphocytes from FTLD-TDP patients, carriers of a loss-of-function GRN mutation, as well as in cells derived from sporadic ALS patients. We found that selective CDC-7 inhibitors, ERP1.14a and ERP1.28a, are able to decrease the enhanced TDP-43 phosphorylation in cells derived from FTLD-TDP and ALS patients and to prevent cytosolic accumulation of TDP-43. Moreover, treatment of FTLD-TDP lymphoblasts with CDC-7 inhibitors leads to recovering the nuclear function of TDP-43-inducing CDK6 repression. We suggest that CDC-7 inhibitors, mainly the heterocyclic compounds here shown, may be considered as promising drug candidates for the ALS/FTD spectrum.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Proteínas de Unión al ADN/metabolismo , Degeneración Lobar Frontotemporal/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Anciano , Células Cultivadas , Proteínas de Unión al ADN/efectos de los fármacos , Femenino , Humanos , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Masculino , Persona de Mediana EdadRESUMEN
The synaptonemal complex (SC) is a proteinaceous macromolecular assembly that forms during meiotic prophase I and mediates adhesion of paired homologous chromosomes along their entire lengths. Although prompt disassembly of the SC during exit from prophase I is a landmark event of meiosis, the underlying mechanism regulating SC destruction has remained elusive. Here, we show that DDK (Dbf4-dependent Cdc7 kinase) is central to SC destruction. Upon exit from prophase I, Dbf4, the regulatory subunit of DDK, directly associates with and is phosphorylated by the Polo-like kinase Cdc5. In parallel, upregulated CDK1 activity also targets Dbf4. An enhanced Dbf4-Cdc5 interaction pronounced phosphorylation of Dbf4 and accelerated SC destruction, while reduced/abolished Dbf4 phosphorylation hampered destruction of SC proteins. SC destruction relieved meiotic inhibition of the ubiquitous recombinase Rad51, suggesting that the mitotic recombination machinery is reactivated following prophase I exit to repair any persisting meiotic DNA double-strand breaks. Taken together, we propose that the concerted action of DDK, Polo-like kinase, and CDK1 promotes efficient SC destruction at the end of prophase I to ensure faithful inheritance of the genome.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Meiosis/fisiología , Proteínas Quinasas/metabolismo , Complejo Sinaptonémico/metabolismo , Fosforilación , Saccharomycetales/metabolismoRESUMEN
MiR-200a acts as a key role in tumor malignant progression. This work purposed to assess the function of miR-200a in Wilm's tumor. Based on bioinformatics analysis, the expression, prognostic value and related pathways of miR-200a and CDC7 (a potential downstream molecule of miR-200a) in Wilm's tumor were analyzed. qRT-PCR was conducted to confirm the miR-200a level in Wilm's tumor cells. The luciferase reporter assay was carried out to verify the binding of miR-200a to 3'-UTR of CDC7. Then, the impacts of miR-200a and CDC7 on cell viability and apoptosis were measured using CCK-8 and flow cytometry assays. Also, western blot was applied to measure the expression of CDC7 as well as Wnt/ß-catenin signaling pathway-related proteins and apoptosis proteins. Herein, we revealed that miR-200a was lowly expressed in Wilm's tumor tissues and cells and the low miR-200a expression is closely bound up with death and poor outcomes. Moreover, miR-200a directly targeted and inhibited CDC7 in Wilm's tumor cells. Biological function experiments illustrated that overexpression of miR-200a reduced the viability and elevated the apoptosis of Wilm's tumor cells, while overexpression of CDC7 reversed the inhibitory impact of miR-200a on cell viability and the promoting impact of miR-200a on cell apoptosis. Besides, we revealed that miR-200a/CDC7 axis can decrease the expression of ß-Catenin, Cyclin D1 and C-Myc as well as the phosphorylation of GSK-3ß, thus inhibiting the Wnt/ß-catenin signaling pathway. Furthermore, blocking the Wnt/ß-catenin signaling pathway caused an increase on cell apoptosis, while overexpression of CDC7 can reverse these impacts. Collectively, miR-200a/CDC7 axis involved in regulating the malignant phenotype of Wilm's tumor through Wnt/ß-catenin signaling pathway, which provides a theoretical basis for targeted molecular therapy of Wilm's tumor.
Asunto(s)
Apoptosis , Proteínas de Ciclo Celular/metabolismo , Neoplasias Renales/metabolismo , MicroARNs/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Neoplásico/metabolismo , Tumor de Wilms/metabolismo , Vía de Señalización Wnt , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Supervivencia Celular/genética , Humanos , Neoplasias Renales/genética , Neoplasias Renales/patología , MicroARNs/genética , Proteínas de Neoplasias/genética , Proteínas Serina-Treonina Quinasas/genética , ARN Neoplásico/genética , Tumor de Wilms/genética , Tumor de Wilms/patologíaRESUMEN
Cdc7 kinase regulates DNA replication. However, its role in DNA repair and recombination is poorly understood. Here we describe a pathway that stabilizes the human Cdc7-ASK (activator of S-phase kinase; also called Dbf4), its regulation, and its function in cellular responses to compromised DNA replication. Stalled DNA replication evoked stabilization of the Cdc7-ASK (Dbf4) complex in a manner dependent on ATR-Chk1-mediated checkpoint signaling and its interplay with the anaphase-promoting complex/cyclosome(Cdh1) (APC/C(Cdh1)) ubiquitin ligase. Mechanistically, Chk1 kinase inactivates APC/C(Cdh1) through degradation of Cdh1 upon replication block, thereby stabilizing APC/C(Cdh1) substrates, including Cdc7-ASK (Dbf4). Furthermore, motif C of ASK (Dbf4) interacts with the N-terminal region of RAD18 ubiquitin ligase, and this interaction is required for chromatin binding of RAD18. Impaired interaction of ASK (Dbf4) with RAD18 disables foci formation by RAD18 and hinders chromatin loading of translesion DNA polymerase η. These findings define a novel mechanism that orchestrates replication checkpoint signaling and ubiquitin-proteasome machinery with the DNA damage bypass pathway to guard against replication collapse under conditions of replication stress.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Daño del ADN , Replicación del ADN , Antígenos CD , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Cadherinas/metabolismo , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Estabilidad de Enzimas , Genes APC/fisiología , Células HEK293 , Células HeLa , Humanos , Unión Proteica , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de SeñalRESUMEN
Combining DNA-damaging drugs with DNA checkpoint inhibitors is an emerging strategy to manage cancer. Checkpoint kinase 1 inhibitors (CHK1is) sensitize most cancer cell lines to DNA-damaging drugs and also elicit single-agent cytotoxicity in 15% of cell lines. Consequently, combination therapy may be effective in a broader patient population. Here, we characterized the molecular mechanism of sensitization to gemcitabine by the CHK1i MK8776. Brief gemcitabine incubation irreversibly inhibited ribonucleotide reductase, depleting dNTPs, resulting in durable S phase arrest. Addition of CHK1i 18 h after gemcitabine elicited cell division cycle 7 (CDC7)- and cyclin-dependent kinase 2 (CDK2)-dependent reactivation of the replicative helicase, but did not reinitiate DNA synthesis due to continued lack of dNTPs. Helicase reactivation generated extensive single-strand (ss)DNA that exceeded the protective capacity of the ssDNA-binding protein, replication protein A. The subsequent cleavage of unprotected ssDNA has been termed replication catastrophe. This mechanism did not occur with concurrent CHK1i plus gemcitabine treatment, providing support for delayed administration of CHK1i in patients. Alternative mechanisms of CHK1i-mediated sensitization to gemcitabine have been proposed, but their role was ruled out; these mechanisms include premature mitosis, inhibition of homologous recombination, and activation of double-strand break repair nuclease (MRE11). In contrast, single-agent activity of CHK1i was MRE11-dependent and was prevented by lower concentrations of a CDK2 inhibitor. Hence, both pathways require CDK2 but appear to depend on different CDK2 substrates. We conclude that a small-molecule inhibitor of CHK1 can elicit at least two distinct, context-dependent mechanisms of cytotoxicity in cancer cells.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Quinasa 2 Dependiente de la Ciclina/metabolismo , Replicación del ADN/efectos de los fármacos , Desoxicitidina/análogos & derivados , Proteínas Serina-Treonina Quinasas/metabolismo , Pirazoles/farmacología , Pirimidinas/farmacología , Puntos de Control de la Fase S del Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Quinasa 2 Dependiente de la Ciclina/genética , ADN de Cadena Simple/biosíntesis , Desoxicitidina/farmacología , Humanos , Células PC-3 , Proteínas Serina-Treonina Quinasas/genética , GemcitabinaRESUMEN
Sequential activation of DNA replication origins is precisely programmed and critical to maintaining genome stability. RecQL4, a member of the conserved RecQ family of helicases, plays an essential role in the initiation of DNA replication in mammalian cells. Here, we showed that RecQL4 protein tethered on the pre-replicative complex (pre-RC) induces early activation of late replicating origins during S phase. Tethering of RecQL4 or its N terminus on pre-RCs via fusion with Orc4 protein resulted in the recruitment of essential initiation factors, such as Mcm10, And-1, Cdc45, and GINS, increasing nascent DNA synthesis in late replicating origins during early S phase. In this origin activation process, tethered RecQL4 was able to recruit Cdc45 even in the absence of cyclin-dependent kinase (CDK) activity, whereas CDK phosphorylation of RecQL4 N terminus was required for interaction with and origin recruitment of And-1 and GINS. In addition, forced activation of replication origins by RecQL4 tethering resulted in increased replication stress and the accumulation of ssDNAs, which can be recovered by transcription inhibition. Collectively, these results suggest that recruitment of RecQL4 to replication origins is an important step for temporal activation of replication origins during S phase. Further, perturbation of replication timing control by unscheduled origin activation significantly induces replication stress, which is mostly caused by transcription-replication conflicts.
Asunto(s)
Replicación del ADN , RecQ Helicasas/metabolismo , Origen de Réplica , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Complejo de Reconocimiento del Origen/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , RecQ Helicasas/genética , Fase S , Activación TranscripcionalRESUMEN
BACKGROUND: Genome-wide association studies have identified the CDC7-TGFBR3 intergenic region on chromosome 1 to be strongly associated with optic disc area size. The mechanism of its function remained unclear until new data on eQTL markers emerged from the Genotype-Tissue Expression project. The target region was found to contain a strong silencer of the distal (800 kb) Transcription Factor (TF) gene GFI1 (Growth Factor Independent Transcription Repressor 1) specifically in neuroendocrine cells (pituitary gland). GFI1 has also been reported to be involved in the development of sensory neurons and hematopoiesis. Therefore, GFI1, being a developmental gene, is likely to affect optic disc area size by altering the expression of the associated genes via long-range interactions. RESULTS: Distribution of haplotypes in the putative enhancer region has been assessed using the data on four continental supergroups generated by the 1000 Genomes Project. The East Asian (EAS) populations were shown to manifest a highly homogenous unimodal haplotype distribution pattern within the region with the major haplotype occurring with the frequency of 0.9. Another European specific haplotype was observed with the frequency of 0.21. The major haplotype appears to be involved in silencing GFI1repressor gene expression, which might be the cause of increased optic disc area characteristic of the EAS populations. The enhancer/eQTL region overlaps AluJo element, which implies that this particular regulatory element is primate-specific and confined to few tissues. CONCLUSION: Population specific distribution of GFI1 enhancer alleles may predispose certain ethnic groups to glaucoma.
Asunto(s)
Elementos de Facilitación Genéticos , Genética de Población , Haplotipos , Disco Óptico/anatomía & histología , Sitios de Carácter Cuantitativo , Pueblo Asiatico/genética , Estudio de Asociación del Genoma Completo , Humanos , Polimorfismo de Nucleótido Simple , Población Blanca/genéticaRESUMEN
In growing cells, DNA replication precedes mitotic cell division to transmit genetic information to the next generation. The slowing or stalling of DNA replication forks at natural or exogenous obstacles causes "replicative stress" that promotes genomic instability and affects cellular fitness. Replicative stress phenotypes can be characterized at the single-molecule level with DNA combing or stretched DNA fibers, but interpreting the results obtained with these approaches is complicated by the fact that the speed of replication forks is connected to the frequency of origin activation. Primary alterations in fork speed trigger secondary responses in origins, and, conversely, primary alterations in the number of active origins induce compensatory changes in fork speed. Here, by employing interventions that temporally restrict either fork speed or origin firing while still allowing interrogation of the other variable, we report a set of experimental conditions to separate cause and effect in any manipulation that affects DNA replication dynamics. Using HeLa cells and chemical inhibition of origin activity (through a CDC7 kinase inhibitor) and of DNA synthesis (via the DNA polymerase inhibitor aphidicolin), we found that primary effects of replicative stress on velocity of replisomes (fork rate) can be readily distinguished from primary effects on origin firing. Identifying the primary cause of replicative stress in each case as demonstrated here may facilitate the design of methods to counteract replication stress in primary cells or to enhance it in cancer cells to increase their susceptibility to therapies that target DNA repair.
Asunto(s)
Afidicolina/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Senescencia Celular/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , ADN/biosíntesis , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Origen de Réplica , Proteínas de Ciclo Celular/metabolismo , Reparación del ADN/efectos de los fármacos , Células HeLa , Humanos , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
BACKGROUND: The effective treatment of triple-negative breast cancer (TNBC) remains a profound clinical challenge. Despite frequent epidermal growth factor receptor (EGFR) overexpression and reliance on downstream signalling pathways in TNBC, resistance to EGFR-tyrosine kinase inhibitors (TKIs) remains endemic. Therefore, the identification of targeted agents, which synergise with current therapeutic options, is paramount. METHODS: Compound-based, high-throughput, proliferation screening was used to profile the response of TNBC cell lines to EGFR-TKIs, western blotting and siRNA transfection being used to examine the effect of inhibitors on EGFR-mediated signal transduction and cellular dependence on such pathways, respectively. A kinase inhibitor combination screen was used to identify compounds that synergised with EGFR-TKIs in TNBC, utilising sulphorhodamine B (SRB) assay as read-out for proliferation. The impact of drug combinations on cell cycle arrest, apoptosis and signal transduction was assessed using flow cytometry, automated live-cell imaging and western blotting, respectively. RNA sequencing was employed to unravel transcriptomic changes elicited by this synergistic combination and to permit identification of the signalling networks most sensitive to co-inhibition. RESULTS: We demonstrate that a dual cdc7/CDK9 inhibitor, PHA-767491, synergises with multiple EGFR-TKIs (lapatinib, erlotinib and gefitinib) to overcome resistance to EGFR-targeted therapy in various TNBC cell lines. Combined inhibition of EGFR and cdc7/CDK9 resulted in reduced cell proliferation, accompanied by induction of apoptosis, G2-M cell cycle arrest, inhibition of DNA replication and abrogation of CDK9-mediated transcriptional elongation, in contrast to mono-inhibition. Moreover, high expression of cdc7 and RNA polymerase II Subunit A (POLR2A), the direct target of CDK9, is significantly correlated with poor metastasis-free survival in a cohort of breast cancer patients. RNA sequencing revealed marked downregulation of pathways governing proliferation, transcription and cell survival in TNBC cells treated with the combination of an EGFR-TKI and a dual cdc7/CDK9 inhibitor. A number of genes enriched in these downregulated pathways are associated with poor metastasis-free survival in TNBC. CONCLUSIONS: Our results highlight that dual inhibition of cdc7 and CDK9 by PHA-767491 is a potential strategy for targeting TNBC resistant to EGFR-TKIs.
Asunto(s)
Antineoplásicos/uso terapéutico , Proteínas de Ciclo Celular/antagonistas & inhibidores , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Resistencia a Antineoplásicos , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Neoplasias de la Mama Triple Negativas/metabolismo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Receptores ErbB/antagonistas & inhibidores , Femenino , Perfilación de la Expresión Génica , Humanos , Terapia Molecular Dirigida , Pronóstico , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/mortalidadRESUMEN
Cancer-associated p53 missense mutants confer gain of function (GOF) and promote tumorigenesis by regulating crucial signaling pathways. However, the role of GOF mutant p53 in regulating DNA replication, a commonly altered pathway in cancer, is less explored. Here, we show that enhanced Cdc7-dependent replication initiation enables mutant p53 to confer oncogenic phenotypes. We demonstrate that mutant p53 cooperates with the oncogenic transcription factor Myb in vivo and transactivates Cdc7 in cancer cells. Moreover, mutant p53 cells exhibit enhanced levels of Dbf4, promoting the activity of Cdc7/Dbf4 complex. Chromatin enrichment of replication initiation factors and subsequent increase in origin firing confirm increased Cdc7-dependent replication initiation in mutant p53 cells. Further, knockdown of CDC7 significantly abrogates mutant p53-driven cancer phenotypes in vitro and in vivo Importantly, high CDC7 expression significantly correlates with p53 mutational status and predicts poor clinical outcome in lung adenocarcinoma patients. Collectively, this study highlights a novel functional interaction between mutant p53 and the DNA replication pathway in cancer cells. We propose that increased Cdc7-dependent replication initiation is a hallmark of p53 gain-of-function mutations.
Asunto(s)
Adenocarcinoma/genética , Proteínas de Ciclo Celular/genética , Replicación del ADN , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , Mutación , Proteínas Serina-Treonina Quinasas/genética , Proteína p53 Supresora de Tumor/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Animales , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Perfilación de la Expresión Génica , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Componente 2 del Complejo de Mantenimiento de Minicromosoma/genética , Componente 2 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Estadificación de Neoplasias , Trasplante de Neoplasias , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-myb/genética , Proteínas Proto-Oncogénicas c-myb/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Análisis de Supervivencia , Activación Transcripcional , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Although Cdc7 protein kinase is important for regulating DNA replication in all eukaryotes and is a target for cancer therapy, it has never been localized in cells. Recently, a novel molecular genomic method used by our laboratory to localize Cdc7 to regions of chromosomes. Originally, mutations in the CDC7 gene were found in the classic cdc mutant collection of Hartwell et al. (Genetics 74:267-286, 1973). The CDC7 gene was found to encode a protein kinase called DDK that has been studied for many years, establishing its precise role in the initiation of DNA replication at origins. Recently, clinical studies are underway with DDK inhibitors against DDK in cancer patients. However, the conundrum is that Cdc7 has never been detected at origins of replication even though many studies have suggested it should be there. We used "Calling Card" system in which DNA binding proteins are localized to the genome via retrotransposon insertion and deep-sequencing methods. We have shown that Cdc7 localizes at many regions of the genome and was enriched at functional origins of replication. These results are consistent with DDK's role in many additional genomic processes including mutagenesis, chromatid cohesion, and meiotic recombination. Thus, the main conclusion from our studies is that Cdc7 kinase is found at many locations in the genome, but is enriched at functional origins of replication. Furthermore, we propose that application of the Calling Card system to other eukaryotes should be useful in identification of functional origins in other eukaryotic cells.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Cromátides/metabolismo , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Genoma Fúngico , Meiosis , Mutagénesis , Recombinación Genética , Origen de Réplica , Saccharomyces cerevisiae/metabolismo , Especificidad por SustratoRESUMEN
CDC7 is a serine/threonine kinase which has an essential role in initiation of DNA proliferation and S phase. It increases the invasion and proliferation in many pathologic lesions. This study aimed to evaluate the expression of CDC7 in the most common odontogenic cysts. We evaluated 17 dentigerous cysts, 18 odontogenic keratocysts (OKC) and 13 radicular cysts immunohistochemically. The mean expression of CDC7 was analyzed using ANOVA and Post-HOC methods. All specimens revealed CDC7 expression. Higher expression of CDC7 in OKC and radicular cyst was shown in comparison to dentigerous cyst (P < 0.001), while radicular cyst and OKC groups showed no difference in CDC7 expression (P = 0.738). The high expression of CDC7 in OKC suggests that this protein could be related to the higher proliferation rate and invasiveness of OKC. On the other hand, the higher CDC7 expression in radicular cyst may simply be related to inflammation as this cyst is neither aggressive nor invasive.