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1.
Anticancer Res ; 43(8): 3717-3726, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37500165

RESUMO

BACKGROUND/AIM: Pyra-Metho-Carnil (PMC) has been identified as a novel candidate compound for treating numerous malignancies; however, its mechanism of action remains unknown. In this study, we conducted RNA-sequencing (RNA-seq) analyses to elucidate the mechanism of PMC against human colorectal cancer cells harboring mutant KRAS (mtKRAS). MATERIALS AND METHODS: RNA-seq analyses of the HKe3-wild-type KRAS and HKe3-mtKRAS spheroids treated with DMSO or PMC for 6 days were performed. RESULTS: RNA-seq data suggested that PMC treatment suppresses the aerobic glycolysis pathway in HKe3-mtKRAS spheroids through the down-regulation of the HIF1 pathway. Indeed, treatment with PMC markedly suppresses the absorption of glucose by spheroids and the secretion of lactate from them. CONCLUSION: PMC suppresses growth of cancer spheroid through down-regulation of cancer-specific glucose metabolism.


Assuntos
Neoplasias Colorretais , Humanos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Linhagem Celular Tumoral , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proliferação de Células , Glicólise
2.
Bio Protoc ; 12(17)2022 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-36213106

RESUMO

In the human cell cycle, complete replication of DNA is a fundamental process for the maintenance of genome integrity. Replication stress interfering with the progression of replication forks causes difficult-to-replicate regions to remain under-replicated until the onset of mitosis. In early mitosis, a homology-directed repair DNA synthesis, called mitotic DNA synthesis (MiDAS), is triggered to complete DNA replication. Here, we present a method to detect MiDAS in human U2OS 40-2-6 cells, in which repetitive lacO sequences integrated into the human chromosome evoke replication stress and concomitant incomplete replication of the lacO array. Immunostaining of BrdU and LacI proteins is applied for visualization of DNA synthesis in early mitosis and the lacO array, respectively. This protocol has been established to easily detect MiDAS at specific loci using only common immunostaining methods and may be optimized for the investigation of other difficult-to-replicate regions marked with site-specific binding proteins.

3.
J Biol Chem ; 298(11): 102528, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36162510

RESUMO

The centromere is an essential chromosomal structure for faithful chromosome segregation during cell division. No protein-coding genes exist at the centromeres, but centromeric DNA is actively transcribed into noncoding RNA (ncRNA). This centromeric transcription and its ncRNA products play important roles in centromere functions. We previously reported that the transcriptional regulator ZFAT (zinc-finger protein with AT hook) plays a pivotal role in ncRNA transcription at the centromere; however, it was unclear how ZFAT involvement was regulated. Here, we show that the death domain-associated protein (DAXX) promotes centromeric localization of ZFAT to regulate ncRNA transcription at the centromere. Coimmunoprecipitation analysis of endogenous proteins clearly reveals that DAXX interacts with ZFAT. In addition, we show that ectopic coexpression of ZFAT with DAXX increases the centromeric levels of both ZFAT and ncRNA, compared with ectopic expression of ZFAT alone. On the other hand, we found that siRNA-mediated depletion of DAXX decreases the centromeric levels of both ZFAT and ncRNA in cells ectopically expressing ZFAT. These results suggest that DAXX promotes the centromeric localization of ZFAT and ZFAT-regulated centromeric ncRNA transcription. Furthermore, we demonstrate that depletion of endogenous DAXX protein is sufficient to cause a decrease in the ncRNA levels at the centromeres of chromosomes 17 and X in which ZFAT regulates the transcription, indicating a physiological significance of DAXX in ZFAT-regulated centromeric ncRNA transcription. Taken together, these results demonstrate that DAXX regulates centromeric ncRNA transcription through ZFAT.


Assuntos
Centrômero , Proteínas Correpressoras , Chaperonas Moleculares , RNA não Traduzido , Fatores de Transcrição , Centrômero/genética , Centrômero/metabolismo , Segregação de Cromossomos , Domínio de Morte , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Dedos de Zinco , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
4.
Anticancer Res ; 42(8): 3993-4001, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35896235

RESUMO

BACKGROUND/AIM: In a screen of compounds to selectively suppress the growth of cancer spheroids, which contained mutant (mt) KRAS, NPD10621 was discovered and associated derivatives were investigated. MATERIALS AND METHODS: Spheroid areas from HCT116-derived HKe3 spheroids expressing wild type (wt) KRAS (HKe3-wtKRAS) and mtKRAS (HKe3-mtKRAS) were treated with 12 NPD10621 derivatives and measured in three-dimensional floating (3DF) cultures. Several cancers were treated with NPD1018 (pyra-metho-carnil: PMC) in 3DF cultures. In a nude mouse assay, 50% cell growth inhibition (GI50) values were determined. RESULTS: From these 12 derivatives, PMC was the most effective inhibitor of HKe3-mtKRAS spheroid growth with the least toxicity. Furthermore, PMC-mediated growth suppression was observed in all tested cancer cell lines, independent of tissue context, driver gene mutations, and drug resistance, suggesting that the PMC target(s) was crucial for cancer growth in a context-independent manner. The GI50 value of PMC in nude mice assay was 7.7 mg/kg and nude mice that were administered 40 mg/kg PMC for 7 days did not show any abnormal blood cell count values. CONCLUSION: PMC is a low-toxicity compound that inhibits the growth of different tumor cell types.


Assuntos
Neoplasias Colorretais , Animais , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/patologia , Camundongos , Camundongos Nus , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Esferoides Celulares/patologia
5.
Nucleic Acids Res ; 49(21): 12234-12251, 2021 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-34761263

RESUMO

Telomeres are intrinsically difficult-to-replicate region of eukaryotic chromosomes. Telomeric repeat binding factor 2 (TRF2) binds to origin recognition complex (ORC) to facilitate the loading of ORC and the replicative helicase MCM complex onto DNA at telomeres. However, the biological significance of the TRF2-ORC interaction for telomere maintenance remains largely elusive. Here, we employed a TRF2 mutant with mutations in two acidic acid residues (E111A and E112A) that inhibited the TRF2-ORC interaction in human cells. The TRF2 mutant was impaired in ORC recruitment to telomeres and showed increased replication stress-associated telomeric DNA damage and telomere instability. Furthermore, overexpression of an ORC1 fragment (amino acids 244-511), which competitively inhibited the TRF2-ORC interaction, increased telomeric DNA damage under replication stress conditions. Taken together, these findings suggest that TRF2-mediated ORC recruitment contributes to the suppression of telomere instability.


Assuntos
Replicação do DNA/genética , Mutação , Complexo de Reconhecimento de Origem/genética , Telômero/genética , Proteína 2 de Ligação a Repetições Teloméricas/genética , Linhagem Celular Tumoral , Dano ao DNA , Regulação da Expressão Gênica , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Microscopia de Fluorescência , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Telômero/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo
6.
Bioengineering (Basel) ; 8(11)2021 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-34821745

RESUMO

The copolyester of 3-hydroxybutyrate (3HB) and 3-hydoxyhexanoate (3HHx), PHBHHx, is one of the most practical kind of bacterial polyhydroxyalkanoates due to its high flexibility and marine biodegradability. PHBHHx is usually produced from vegetable oils or fatty acids through ß-oxidation, whereas biosynthesis from sugars has been achieved by recombinant strains of hydrogen-oxidizing bacterium Cupriavidus necator. This study investigated the biosynthesis of PHBHHx from CO2 as the sole carbon source by engineered C. necator strains. The recombinant strains capable of synthesizing PHBHHx from fructose were cultivated in a flask using complete mineral medium and a substrate gas mixture (H2/O2/CO2 = 8:1:1). The results of GC and 1H NMR analyses indicated that the recombinants of C. necator synthesized PHBHHx from CO2 with high cellular content. When 1.0 g/L (NH4)2SO4 was used as a nitrogen source, the 3HHx composition of PHBHHx in the strain MF01∆B1/pBBP-ccrMeJ4a-emd was 47.7 ± 6.2 mol%. Further investigation demonstrated that the PHA composition can be regulated by using (R)-enoyl-CoA hydratase (PhaJ) with different substrate specificity. The composition of 3HHx in PHBHHx was controlled to about 11 mol%, suitable for practical applications, and high cellular content was kept in the strains transformed with pBPP-ccrMeJAc-emd harboring short-chain-length-specific PhaJ.

7.
J Biol Chem ; 297(4): 101213, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34547289

RESUMO

The centromere is a chromosomal locus that is essential for the accurate segregation of chromosomes during cell division. Transcription of noncoding RNA (ncRNA) at the centromere plays a crucial role in centromere function. The zinc-finger transcriptional regulator ZFAT binds to a specific 8-bp DNA sequence at the centromere, named the ZFAT box, to control ncRNA transcription. However, the precise molecular mechanisms by which ZFAT localizes to the centromere remain elusive. Here we show that the centromeric protein CENP-B is required for the centromeric localization of ZFAT to regulate ncRNA transcription. The ectopic expression of CENP-B induces the accumulation of both endogenous and ectopically expressed ZFAT protein at the centromere in human cells, suggesting that the centromeric localization of ZFAT requires the presence of CENP-B. Coimmunoprecipitation analysis reveals that ZFAT interacts with the acidic domain of CENP-B, and depletion of endogenous CENP-B reduces the centromeric levels of ZFAT protein, further supporting that CENP-B is required for the centromeric localization of ZFAT. In addition, knockdown of CENP-B significantly decreased the expression levels of ncRNA at the centromere where ZFAT regulates the transcription, suggesting that CENP-B is involved in the ZFAT-regulated centromeric ncRNA transcription. Thus, we concluded that CENP-B contributes to the establishment of the centromeric localization of ZFAT to regulate ncRNA transcription.


Assuntos
Proteína B de Centrômero/metabolismo , Centrômero/metabolismo , RNA não Traduzido/biossíntese , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Centrômero/genética , Proteína B de Centrômero/genética , Células HEK293 , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , RNA não Traduzido/genética , Fatores de Transcrição/genética
8.
Cell Mol Life Sci ; 78(21-22): 6763-6773, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34463774

RESUMO

During duplication of the genome, eukaryotic cells may experience various exogenous and endogenous replication stresses that impede progression of DNA replication along chromosomes. Chemical alterations in template DNA, imbalances of deoxynucleotide pools, repetitive sequences, tight DNA-protein complexes, and conflict with transcription can negatively affect the replication machineries. If not properly resolved, stalled replication forks can cause chromosome breaks leading to genomic instability and tumor development. Replication stress is enhanced in cancer cells due, for example, to the loss of DNA repair genes or replication-transcription conflict caused by activation of oncogenic pathways. To prevent these serious consequences, cells are equipped with diverse mechanisms that enhance the resilience of replication machineries to replication stresses. This review describes DNA damage responses activated at stressed replication forks and summarizes current knowledge on the pathways that promote faithful chromosome replication and protect chromosome integrity, including ATR-dependent replication checkpoint signaling, DNA cross-link repair, and SLX4-mediated responses to tight DNA-protein complexes that act as barriers. This review also focuses on the relevance of replication stress responses to selective cancer chemotherapies.


Assuntos
Dano ao DNA/genética , Replicação do DNA/genética , DNA/genética , Animais , Cromossomos/genética , Reparo do DNA/genética , Humanos , Proteínas/genética
9.
J Cell Biol ; 220(1)2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33347546

RESUMO

The DNA damage response (DDR) has a critical role in the maintenance of genomic integrity during chromosome replication. However, responses to replication stress evoked by tight DNA-protein complexes have not been fully elucidated. Here, we used bacterial LacI protein binding to lacO arrays to make site-specific replication fork barriers on the human chromosome. These barriers induced the accumulation of single-stranded DNA (ssDNA) and various DDR proteins at the lacO site. SLX4-XPF functioned as an upstream factor for the accumulation of DDR proteins, and consequently, ATR and FANCD2 were interdependently recruited. Moreover, LacI binding in S phase caused underreplication and abnormal mitotic segregation of the lacO arrays. Finally, we show that the SLX4-ATR axis represses the anaphase abnormality induced by LacI binding. Our results outline a long-term process by which human cells manage nucleoprotein obstacles ahead of the replication fork to prevent chromosomal instability.


Assuntos
Dano ao DNA , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Recombinases/metabolismo , Estresse Fisiológico , Anáfase , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Segregação de Cromossomos , Cromossomos Humanos/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Humanos , Modelos Biológicos , Ligação Proteica , Fase S
10.
J Biochem ; 167(1): 15-24, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31545368

RESUMO

Glutamate-rich WD40 repeat containing 1 (GRWD1) functions as a histone chaperone to promote loading of the MCM replication helicase at replication origins. GRWD1 is overexpressed in several cancer cell lines, and GRWD1 overexpression confers tumorigenic potential in human cells. However, less is known concerning its oncogenic activity. Our previous analysis showed that GRWD1 negatively regulates the tumour suppressor p53 via the RPL11-MDM2-p53 and RPL23-MDM2-p53 axes. Here, we demonstrate that GRWD1 directly interacts with p53 via the p53 DNA-binding domain. Upon DNA damage, GRWD1 downregulation resulted in increased p21 expression. Conversely, GRWD1 co-expression suppressed several p53-regulated promoters. GRWD1 interacted with the p21 and MDM2 promoters, and these interactions required p53. By using the Human Cancer Genome Atlas database, we found that GRWD1 expression levels are inversely correlated with the expression levels of some p53-target genes. Interestingly, high GRWD1 expression in combination with low expression levels of some p53-target genes was significantly correlated with poor prognosis in skin melanoma patients with wild-type p53. Taken together, our findings suggest a novel oncogenic function of GRWD1 as a transcriptional regulator of p53 and that GRWD1 might be an attractive therapeutic target and prognostic marker in cancer therapy.


Assuntos
Proteínas de Transporte/metabolismo , Ativação Transcricional , Proteína Supressora de Tumor p53/genética , Células HCT116 , Humanos
11.
Sci Rep ; 9(1): 16825, 2019 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-31727981

RESUMO

We previously reported the identification of a novel antimitotic agent with carbazole and benzohydrazide structures: N'-[(9-ethyl-9H-carbazol-3-yl)methylene]-2-iodobenzohydrazide (code number NP-10). However, the mechanism(s) underlying the cancer cell-selective inhibition of mitotic progression by NP-10 remains unclear. Here, we identified NP-10-interacting proteins by affinity purification from HeLa cell lysates using NP-10-immobilized beads followed by mass spectrometry. The results showed that several mitosis-associated factors specifically bind to active NP-10, but not to an inactive NP-10 derivative. Among them, NUP155 and importin ß may be involved in NP-10-mediated mitotic arrest. Because NP-10 did not show antitumor activity in vivo in a previous study, we synthesized 19 NP-10 derivatives to identify more effective NP-10-related compounds. HMI83-2, an NP-10-related compound with a Cl moiety, inhibited HCT116 cell tumor formation in nude mice without significant loss of body weight, suggesting that HMI83-2 is a promising lead compound for the development of novel antimitotic agents.


Assuntos
Antineoplásicos/administração & dosagem , Neoplasias do Colo/tratamento farmacológico , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Polietilenoglicóis/administração & dosagem , beta Carioferinas/metabolismo , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Células HeLa , Humanos , Camundongos , Camundongos Nus , Polietilenoglicóis/síntese química , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Cancer Sci ; 110(3): 1044-1053, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30648820

RESUMO

MCM8 and MCM9 are paralogues of the MCM2-7 eukaryotic DNA replication helicase proteins and play a crucial role in a homologous recombination-mediated repair process to resolve replication stress by fork stalling. Thus, deficiency of MCM8-9 sensitizes cells to replication stress caused, for example, by platinum compounds that induce interstrand cross-links. It is suggested that cancer cells undergo more replication stress than normal cells due to hyperstimulation of growth. Therefore, it is possible that inhibiting MCM8-9 selectively hypersensitizes cancer cells to platinum compounds and poly(ADP-ribose) polymerase inhibitors, both of which hamper replication fork progression. Here, we inhibited MCM8-9 in transformed and nontransformed cells and examined their sensitivity to cisplatin and olaparib. We found that knockout of MCM9 or knockdown of MCM8 selectively hypersensitized transformed cells to cisplatin and olaparib. In agreement with reported findings, RAS- and human papilloma virus type 16 E7-mediated transformation of human fibroblasts increased replication stress, as indicated by induction of multiple DNA damage responses (including formation of Rad51 foci). Such replication stress induced by oncogenes was further increased by knockdown of MCM8, providing a rationale for cancer-specific hypersensitization to cisplatin and olaparib. Finally, we showed that knocking out MCM9 increased the sensitivity of HCT116 xenograft tumors to cisplatin. Taken together, the data suggest that conceptual MCM8-9 inhibitors will be powerful cancer-specific chemosensitizers for platinum compounds and poly(ADP-ribose) polymerase inhibitors, thereby opening new avenues to the design of novel cancer chemotherapeutic strategies.


Assuntos
Cisplatino/farmacologia , Proteínas de Manutenção de Minicromossomo/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Ftalazinas/farmacologia , Piperazinas/farmacologia , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Feminino , Células HCT116 , Recombinação Homóloga/efeitos dos fármacos , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/metabolismo , Compostos Organoplatínicos/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Reparo de DNA por Recombinação/efeitos dos fármacos
13.
J Cell Sci ; 131(15)2018 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-29991511

RESUMO

Glutamate-rich WD40 repeat-containing 1 (GRWD1) is a Cdt1-binding protein that promotes mini-chromosome maintenance (MCM) loading through its histone chaperone activity. GRWD1 acts as a tumor-promoting factor by downregulating p53 (also known as TP53) via the RPL11-MDM2-p53 axis. Here, we identified GRWD1-interacting proteins using a proteomics approach and showed that GRWD1 interacts with various proteins involved in transcription, translation, DNA replication and repair, chromatin organization, and ubiquitin-mediated proteolysis. We focused on the ribosomal protein ribosomal protein L23 (RPL23), which positively regulates nucleolar stress responses through MDM2 binding and inhibition, thereby functioning as a tumor suppressor. Overexpression of GRWD1 decreased RPL23 protein levels and stability; this effect was restored upon treatment with the proteasome inhibitor MG132. EDD (also known as UBR5), an E3 ubiquitin ligase that interacts with GRWD1, also downregulated RPL23, and the decrease was further enhanced by co-expression of GRWD1. Conversely, siRNA-mediated GRWD1 knockdown upregulated RPL23. Co-expression of GRWD1 and EDD promoted RPL23 ubiquitylation. These data suggest that GRWD1 acts together with EDD to negatively regulate RPL23 via the ubiquitin-proteasome system. GRWD1 expression reversed the RPL23-mediated inhibition of anchorage-independent growth in cancer cells. Our data suggest that GRWD1-induced RPL23 proteolysis plays a role in downregulation of p53 and tumorigenesis.


Assuntos
Proteínas de Transporte/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Ribossômicas/metabolismo , Células HEK293 , Humanos , Leupeptinas/farmacologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos
14.
Nucleic Acids Res ; 46(13): 6683-6696, 2018 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-29893900

RESUMO

In metazoan cells, only a limited number of mini chromosome maintenance (MCM) complexes are fired during S phase, while the majority remain dormant. Several methods have been used to map replication origins, but such methods cannot identify dormant origins. Herein, we determined MCM7-binding sites in human cells using ChIP-Seq, classified them into firing and dormant origins using origin data and analysed their association with various chromatin signatures. Firing origins, but not dormant origins, were well correlated with open chromatin regions and were enriched upstream of transcription start sites (TSSs) of transcribed genes. Aggregation plots of MCM7 signals revealed minimal difference in the efficacy of MCM loading between firing and dormant origins. We also analysed common fragile sites (CFSs) and found a low density of origins at these sites. Nevertheless, firing origins were enriched upstream of the TSSs. Based on the results, we propose a model in which excessive MCMs are actively loaded in a genome-wide manner, irrespective of chromatin status, but only a fraction are passively fired in chromatin areas with an accessible open structure, such as regions upstream of TSSs of transcribed genes. This plasticity in the specification of replication origins may minimize collisions between replication and transcription.


Assuntos
Origem de Replicação , Composição de Bases , Sítios de Ligação , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Sítios Frágeis do Cromossomo , DNA/química , Genoma Humano , Células HeLa , Humanos , Componente 7 do Complexo de Manutenção de Minicromossomo/metabolismo , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição
15.
Genes (Basel) ; 8(4)2017 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-28350373

RESUMO

Telomeres are essential chromosomal regions that prevent critical shortening of linear chromosomes and genomic instability in eukaryotic cells. The bulk of telomeric DNA is replicated by semi-conservative DNA replication in the same way as the rest of the genome. However, recent findings revealed that replication of telomeric repeats is a potential cause of chromosomal instability, because DNA replication through telomeres is challenged by the repetitive telomeric sequences and specific structures that hamper the replication fork. In this review, we summarize current understanding of the mechanisms by which telomeres are faithfully and safely replicated in mammalian cells. Various telomere-associated proteins ensure efficient telomere replication at different steps, such as licensing of replication origins, passage of replication forks, proper fork restart after replication stress, and dissolution of post-replicative structures. In particular, shelterin proteins have central roles in the control of telomere replication. Through physical interactions, accessory proteins are recruited to maintain telomere integrity during DNA replication. Dormant replication origins and/or homology-directed repair may rescue inappropriate fork stalling or collapse that can cause defects in telomere structure and functions.

16.
Biochim Biophys Acta Mol Cell Res ; 1864(1): 191-201, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27836746

RESUMO

Telomeres are specialized chromatin structures that prevent the degradation and instability of the ends of linear chromosomes. While telomerase maintains long stretches of the telomeric repeat, the majority of telomeric DNA is duplicated by conventional DNA replication. A fundamental step in eukaryotic DNA replication involves chromatin binding of the origin recognition complex (ORC). In human cells, telomeric repeat binding factor 2 (TRF2) is thought to play a role in the recruitment of ORC onto telomeres. To better understand the mechanism of TRF2-mediated ORC recruitment, we utilized a lacO-LacI protein tethering system in U2OS cells and found that ectopically targeted TRF2, but not TRF1, can recruit ORC onto the lacO array. We further found that the TRF homology (TRFH) dimerization domain of TRF2, but not its mutant defective in dimerization, is sufficient for ORC and minichromosome maintenance (MCM) recruitment. Mutations impairing the dimerization also compromised ORC recruitment by full-length TRF2. Similar results were obtained using immunoprecipitation and GST pull-down assays. Together, these results suggest that dimerized TRF2 recruits ORC and stimulates pre-replication complex (pre-RC) formation at telomeres through the TRFH domain.


Assuntos
Cromatina/química , Proteínas de Manutenção de Minicromossomo/metabolismo , Complexo de Reconhecimento de Origem/metabolismo , Telômero/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Linhagem Celular Tumoral , Cromatina/metabolismo , Replicação do DNA , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Repressores Lac/genética , Repressores Lac/metabolismo , Proteínas de Manutenção de Minicromossomo/genética , Mutação , Complexo de Reconhecimento de Origem/genética , Osteoblastos/citologia , Osteoblastos/metabolismo , Domínios Proteicos , Multimerização Proteica , Transdução de Sinais , Telômero/ultraestrutura , Proteína 1 de Ligação a Repetições Teloméricas/genética , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/química , Proteína 2 de Ligação a Repetições Teloméricas/genética
17.
EMBO Rep ; 18(1): 123-137, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27856536

RESUMO

The ribosomal protein L11 (RPL11) binds and inhibits the MDM2 ubiquitin ligase, thereby promoting p53 stability. Thus, RPL11 acts as a tumor suppressor. Here, we show that GRWD1 (glutamate-rich WD40 repeat containing 1) physically and functionally interacts with RPL11. GRWD1 is localized to nucleoli and is released into the nucleoplasm upon nucleolar stress. Silencing of GRWD1 increases p53 induction by nucleolar stress, whereas overexpression of GRWD1 reduces p53 induction. Furthermore, GRWD1 overexpression competitively inhibits the RPL11-MDM2 interaction and alleviates RPL11-mediated suppression of MDM2 ubiquitin ligase activity toward p53. These effects are mediated by the N-terminal region of GRWD1, including the acidic domain. Finally, we show that GRWD1 overexpression in combination with HPV16 E7 and activated KRAS confers anchorage-independent growth and tumorigenic capacity on normal human fibroblasts. Consistent with this, GRWD1 overexpression is associated with poor prognosis in cancer patients. Taken together, our results suggest that GRWD1 is a novel negative regulator of p53 and a potential oncogene.


Assuntos
Proteínas de Transporte/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Ribossômicas/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Animais , Proteínas de Transporte/química , Linhagem Celular Tumoral , Transformação Celular Viral , Modelos Animais de Doenças , Feminino , Expressão Gênica , Inativação Gênica , Genes ras , Xenoenxertos , Humanos , Camundongos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/mortalidade , Neoplasias/patologia , Prognóstico , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Estresse Fisiológico , Proteína Supressora de Tumor p53/metabolismo
18.
Biochim Biophys Acta ; 1863(11): 2739-2748, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27552915

RESUMO

GRWD1 was previously identified as a novel Cdt1-binding protein that possesses histone-binding and nucleosome assembly activities and promotes MCM loading, probably by maintaining chromatin openness at replication origins. However, the molecular mechanisms underlying these activities remain unknown. We prepared reconstituted mononucleosomes from recombinant histones and a DNA fragment containing a nucleosome positioning sequence, and investigated the effects of GRWD1 on them. GRWD1 could disassemble these preformed mononucleosomes in vitro in an ATP-independent manner. Thus, our data suggest that GRWD1 facilitates removal of H2A-H2B dimers from nucleosomes, resulting in formation of hexasomes. The activity was compromised by deletion of the acidic domain, which is required for efficient histone binding. In contrast, nucleosome assembly activity of GRWD1 was not affected by deletion of the acidic domain. In HeLa cells, the acidic domain of GRWD1 was necessary to maintain chromatin openness and promote MCM loading at replication origins. Taken together, our results suggest that GRWD1 promotes chromatin fluidity by influencing nucleosome structures, e.g., by transient eviction of H2A-H2B, and thereby promotes efficient MCM loading at replication origins.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Montagem e Desmontagem da Cromatina , Replicação do DNA , DNA/metabolismo , Nucleossomos/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , DNA/química , DNA/genética , Células HeLa , Histonas/metabolismo , Humanos , Proteínas de Manutenção de Minicromossomo/metabolismo , Conformação de Ácido Nucleico , Nucleossomos/química , Nucleossomos/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , Origem de Replicação , Transdução de Sinais , Relação Estrutura-Atividade , Transfecção
19.
Biochim Biophys Acta ; 1850(9): 1676-84, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25960391

RESUMO

BACKGROUND: The mitotic spindles are among the most successful targets of anti-cancer chemotherapy, and they still hold promise as targets for novel drugs. The anti-mitotic drugs in current clinical use, including taxanes, epothilones, vinca alkaloids, and halichondrins, are all microtubule-targeting agents. Although these drugs are effective for cancer chemotherapy, they have some critical problems; e.g., neurotoxicity caused by damage to neuronal microtubules, as well as innate or acquired drug resistance. To overcome these problems, a great deal of effort has been expended on development of novel anti-mitotics. METHODS: We identified novel microtubule-targeting agents with carbazole and benzohydrazide structures: N'-[(9-ethyl-9H-carbazol-3-yl)methylene]-2-methylbenzohydrazide (code number HND-007) and its related compounds. We investigated their activities against cancer cells using various methods including cell growth assay, immunofluorescence analysis, cell cycle analysis, tubulin polymerization assay, and tumor inhibition assay in nude mice. RESULTS: HND-007 inhibits tubulin polymerization in vitro and blocks microtubule formation and centrosome separation in cancer cells. Consequently, it suppresses the growth of various cancer cell lines, with IC50 values in the range 1.3-4.6µM. In addition, HND-007 can inhibit the growth of taxane-resistant cancer cells that overexpress P-glycoprotein. Finally, HND-007 can inhibit HeLa cell tumor growth in nude mice. CONCLUSIONS AND GENERAL SIGNIFICANCE: Taken together, these findings suggest that HND-007 is a promising lead compound for development of novel anti-mitotic, anti-microtubule chemotherapeutic agents.


Assuntos
Antimitóticos/farmacologia , Antineoplásicos/farmacologia , Carbazóis/farmacologia , Microtúbulos/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Moduladores de Tubulina/farmacologia
20.
Nucleic Acids Res ; 43(12): 5898-911, 2015 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-25990725

RESUMO

Efficient pre-replication complex (pre-RC) formation on chromatin templates is crucial for the maintenance of genome integrity. However, the regulation of chromatin dynamics during this process has remained elusive. We found that a conserved protein, GRWD1 (glutamate-rich WD40 repeat containing 1), binds to two representative replication origins specifically during G1 phase in a CDC6- and Cdt1-dependent manner, and that depletion of GRWD1 reduces loading of MCM but not CDC6 and Cdt1. Furthermore, chromatin immunoprecipitation coupled with high-throughput sequencing (Seq) revealed significant genome-wide co-localization of GRWD1 with CDC6. We found that GRWD1 has histone-binding activity. To investigate the effect of GRWD1 on chromatin architecture, we used formaldehyde-assisted isolation of regulatory elements (FAIRE)-seq or FAIRE-quantitative PCR analyses, and the results suggest that GRWD1 regulates chromatin openness at specific chromatin locations. Taken together, these findings suggest that GRWD1 may be a novel histone-binding protein that regulates chromatin dynamics and MCM loading at replication origins.


Assuntos
Proteínas de Transporte/metabolismo , Cromatina/metabolismo , Histonas/metabolismo , Proteínas de Manutenção de Minicromossomo/metabolismo , Origem de Replicação , Proteínas de Transporte/análise , Proteínas de Transporte/fisiologia , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/química , Cromatina/química , Replicação do DNA , Células HEK293 , Células HeLa , Humanos , Lamina Tipo B/genética , Componente 4 do Complexo de Manutenção de Minicromossomo/genética , Proteínas Nucleares/metabolismo
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