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1.
Nat Microbiol ; 6(7): 842-851, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34083769

RESUMO

The emergent fungal pathogen Candida auris exhibits high resistance to antifungal drugs and environmental stresses, impeding treatment and decontamination1-3. The fungal factors mediating this stress tolerance are largely unknown. In the present study, we performed piggyBac, transposon-mediated, genome-wide mutagenesis and genetic screening in C. auris, and identified a mutant that grew constitutively in the filamentous form. Mapping the transposon insertion site revealed the disruption of a long non-coding RNA, named DINOR for DNA damage-inducible non-coding RNA. Deletion of DINOR caused DNA damage and an upregulation of genes involved in morphogenesis, DNA damage and DNA replication. The DNA checkpoint kinase Rad53 was hyperphosphorylated in dinorΔ mutants, and deletion of RAD53 abolished DNA damage-induced filamentation. DNA-alkylating agents, which cause similar filamentous growth, induced DINOR expression, suggesting a role for DINOR in maintaining genome integrity. Upregulation of DINOR also occurred during exposure to the antifungal drugs caspofungin and amphotericin B, macrophages, H2O2 and sodium dodecylsulfate, indicating that DINOR orchestrates multiple stress responses. Consistently, dinorΔ mutants displayed increased sensitivity to these stresses and were attenuated for virulence in mice. Moreover, genome-wide genetic interaction studies revealed links between the function of DINOR and TOR signalling, an evolutionarily conserved pathway that regulates the stress response. Identification of the mechanism(s) by which DINOR regulates stress responses in C. auris may provide future opportunities for the development of therapeutics.


Assuntos
Candida/patogenicidade , RNA Fúngico/metabolismo , RNA Longo não Codificante/metabolismo , Fatores de Virulência/metabolismo , Animais , Antifúngicos/farmacologia , Candida/genética , Candida/crescimento & desenvolvimento , Candidíase/microbiologia , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Dano ao DNA , Replicação do DNA , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação da Expressão Gênica , Peróxido de Hidrogênio/farmacologia , Camundongos , Morfogênese , Mutação , Fosforilação , RNA Fúngico/genética , RNA Longo não Codificante/genética , Virulência , Fatores de Virulência/genética
2.
FASEB J ; 35(6): e21680, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34042225

RESUMO

Hepatitis B virus (HBV) is a human hepatotropic pathogen causing hepatocellular carcinoma. We recently obtained HBV-susceptible immortalized human hepatocyte NKNT-3 by exogenously expressing NTCP and its derived cell clones, #28.3.8 and #28.3.25.13 exhibiting different levels of HBV susceptibility. In the present study, we showed that HBV infection activated the ATM-Chk2 signaling pathway in #28.3.25.13 cells but not in #28.3.8 cells. Both the cell culture supernatant and extracellular vesicles (EVs) derived from HBV-infected #28.3.25.13 cells also activated the ATM-Chk2 signaling pathway in naïve #28.3.25.13 cells. Interestingly, EVs derived from HBV-infected #28.3.25.13 cells included higher level of mitochondrial DNA (mtDNA) than those from HBV-infected #28.3.8 cells. Based on our results, we propose the novel model that EVs mediate the activation of ATM-Chk2 signaling pathway by the intercellular transfer of mtDNA in HBV-infected human hepatocyte.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , DNA Mitocondrial/genética , Vesículas Extracelulares/metabolismo , Hepatite B/patologia , Hepatócitos/patologia , Replicação Viral , Proteínas Mutadas de Ataxia Telangiectasia/genética , Quinase do Ponto de Checagem 2/genética , DNA Mitocondrial/metabolismo , Células Hep G2 , Hepatite B/genética , Hepatite B/metabolismo , Hepatite B/microbiologia , Vírus da Hepatite B/fisiologia , Hepatócitos/metabolismo , Hepatócitos/microbiologia , Humanos
3.
Zhongguo Fei Ai Za Zhi ; 24(4): 265-270, 2021 Apr 20.
Artigo em Chinês | MEDLINE | ID: mdl-33910274

RESUMO

Lung cancer is the most commonly diagnosed cancer and the leading cause of cancer death. Although great progress has been made in chemotherapy, radiotherapy and targeted therapy, the emergence of acquired drug resistance hinders the efficacy of clinical treatment. Studies have shown that tumor is a class of diseases with damaged cell cycle regulation mechanism, in which checkpoint kinase (Chk) plays a core role, Chk1 and Chk2 are very important protein kinases in the checkpoint. In recent years, it has been found that the regulation of Chk1 and Chk2 plays an important role in the clinical treatment and drug resistance mechanism of lung cancer. This article reviews the mechanism of cell cycle checkpoint kinase and drug resistance of lung cancer, and expounds the effective therapeutic targets and methods of lung cancer.
.


Assuntos
Quinase 1 do Ponto de Checagem/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/enzimologia , Animais , Antineoplásicos/administração & dosagem , Pontos de Checagem do Ciclo Celular , Quinase 1 do Ponto de Checagem/genética , Quinase do Ponto de Checagem 2/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/fisiopatologia
4.
Acta Biochim Biophys Sin (Shanghai) ; 53(6): 726-738, 2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-33913495

RESUMO

The cellular response to DNA damage is crucial for maintaining the integrity and stability of molecular structure. To maintain genome stability, DNA-damaged cells should be arrested so that mutations can be repaired before replication. Although several key components required for this arrest have been discovered, the majority of the pathways are still unclear. Through a number of assays, including cell viability, colony formation, and apotheosis assay, we found that AKR1B10 protected cells from UVC-induced DNA damage. Surprisingly, UVC-induced γH2AX foci and DNA double-strand breaks in the AKR1B10-overexpressing cells were ∼4-5 folds lower than those in the control group. The expression levels of AKR1B10, p53, chk1, chk2, nuclear factor (NF)-κB, and p65 showed dynamic changes in response to UVC irradiation. Our results suggested that AKR1B10 is involved in the pathway of cell cycle checkpoint and NF-κB in DNA damage. Taken together, our results suggest that AKR1B10 is involved in the repair of the DNA double-strand break, which provides a new insight into the role of AKR1B10 in DNA damage repair and indicates a new trail in tumorigenesis and cancer drug resistance.


Assuntos
Aldo-Ceto Redutases/metabolismo , Neoplasias da Mama/metabolismo , Dano ao DNA/efeitos da radiação , Transdução de Sinais/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Aldo-Ceto Redutases/genética , Apoptose/efeitos da radiação , Neoplasias da Mama/patologia , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Quinase 1 do Ponto de Checagem/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA/genética , Feminino , Vetores Genéticos/genética , Histonas/metabolismo , Humanos , Células MCF-7 , NF-kappa B/metabolismo , Transfecção , Proteína Supressora de Tumor p53/metabolismo
5.
J Photochem Photobiol B ; 219: 112188, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33901880

RESUMO

Far-infrared (FIR) irradiation is reported to inhibit cell proliferation in various types of cancer cells; the underlying mechanism, however, remains unclear. We explored the molecular mechanisms using MDA-MB-231 human breast cancer cells. FIR irradiation significantly inhibited cell proliferation and colony formation compared to hyperthermal stimulus, with no alteration in cell viability. No increase in DNA fragmentation or phosphorylation of DNA damage kinases including ataxia-telangiectasia mutated kinase, ataxia telangiectasia and Rad3-related kinase, and DNA-dependent protein kinase indicated no DNA damage. FIR irradiation increased the phosphorylation of checkpoint kinase 2 (Chk2) at Thr68 (p-Chk2-Thr68) but not that of checkpoint kinase 1 at Ser345. Increased nuclear p-Chk2-Thr68 and Ca2+/CaM accumulations were found in FIR-irradiated cells, as observed in confocal microscopic analyses and cell fractionation assays. In silico analysis predicted that Chk2 possesses a Ca2+/calmodulin (CaM) binding motif ahead of its kinase domain. Indeed, Chk2 physically interacted with CaM in the presence of Ca2+, with their binding markedly pronounced in FIR-irradiated cells. Pre-treatment with a Ca2+ chelator significantly reversed FIR irradiation-increased p-Chk2-Thr68 expression. In addition, a CaM antagonist or small interfering RNA-mediated knockdown of the CaM gene expression significantly attenuated FIR irradiation-increased p-Chk2-Thr68 expression. Finally, pre-treatment with a potent Chk2 inhibitor significantly reversed both FIR irradiation-stimulated p-Chk2-Thr68 expression and irradiation-repressed cell proliferation. In conclusion, our results demonstrate that FIR irradiation inhibited breast cancer cell proliferation, independently of DNA damage, by activating the Ca2+/CaM/Chk2 signaling pathway in the nucleus. These results demonstrate a novel Chk2 activation mechanism that functions irrespective of DNA damage.


Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , Proliferação de Células/efeitos da radiação , Quinase do Ponto de Checagem 2/metabolismo , Dano ao DNA/efeitos da radiação , Raios Infravermelhos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos da radiação , Quinase do Ponto de Checagem 2/antagonistas & inibidores , Quinase do Ponto de Checagem 2/genética , Feminino , Humanos , Fosforilação/efeitos da radiação , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Regulação para Cima/efeitos da radiação
6.
J Clin Invest ; 131(8)2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33690219

RESUMO

Although cancer cells are frequently faced with a nutrient- and oxygen-poor microenvironment, elevated hexosamine-biosynthesis pathway (HBP) activity and protein O-GlcNAcylation (a nutrient sensor) contribute to rapid growth of tumor and are emerging hallmarks of cancer. Inhibiting O-GlcNAcylation could be a promising anticancer strategy. The gluconeogenic enzyme phosphoenolpyruvate carboxykinase 1 (PCK1) is downregulated in hepatocellular carcinoma (HCC). However, little is known about the potential role of PCK1 in enhanced HBP activity and HCC carcinogenesis under glucose-limited conditions. In this study, PCK1 knockout markedly enhanced the global O-GlcNAcylation levels under low-glucose conditions. Mechanistically, metabolic reprogramming in PCK1-loss hepatoma cells led to oxaloacetate accumulation and increased de novo uridine triphosphate synthesis contributing to uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) biosynthesis. Meanwhile, deletion of PCK1 also resulted in AMPK-GFAT1 axis inactivation, promoting UDP-GlcNAc synthesis for elevated O-GlcNAcylation. Notably, lower expression of PCK1 promoted CHK2 threonine 378 O-GlcNAcylation, counteracting its stability and dimer formation, increasing CHK2-dependent Rb phosphorylation and HCC cell proliferation. Moreover, aminooxyacetic acid hemihydrochloride and 6-diazo-5-oxo-L-norleucine blocked HBP-mediated O-GlcNAcylation and suppressed tumor progression in liver-specific Pck1-knockout mice. We reveal a link between PCK1 depletion and hyper-O-GlcNAcylation that underlies HCC oncogenesis and suggest therapeutic targets for HCC that act by inhibiting O-GlcNAcylation.


Assuntos
Carcinoma Hepatocelular , Quinase do Ponto de Checagem 2/metabolismo , Gluconeogênese/efeitos dos fármacos , Glucose/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Neoplasias Hepáticas , Fosfoenolpiruvato Carboxiquinase (GTP)/deficiência , Acilação/efeitos dos fármacos , Acilação/genética , Animais , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Quinase do Ponto de Checagem 2/genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Nus , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo
7.
PLoS One ; 16(3): e0248468, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33724994

RESUMO

Diterpenoids jolkinolide A and B, were first isolated from Euphorbia fischeriana. In our previous research, 19-(Benzyloxy)-19-oxojolkinolide B (19-BJB), a derivative of jolkinolides, was synthesized as a novel ent -abietane diterpene diepoxide. In this study, 19-BJB showed strong in vitro activity against bladder cancer cell lines. DNA damage which was observed through the interaction of 19-BJB with nucleotide chains and affected DNA repair resulted in the activation of checkpoint kinase 1 (Chk1) and checkpoint kinase 2 (Chk2) in bladder cancer cell lines. In vivo testing in nude mice also proved that 19-BJB revealed a potential inhibitory effect on tumor growth. Additionally, the 3D-QSAR models of jolkinolides were established. Briefly, we proved that 19-BJB could potentially be used as a drug to inhibit the growth of bladder tumor.


Assuntos
Abietanos/farmacologia , Dano ao DNA/efeitos dos fármacos , Diterpenos/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Abietanos/uso terapêutico , Animais , Antineoplásicos Fitogênicos , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Reparo do DNA , Diterpenos/uso terapêutico , Medicamentos de Ervas Chinesas/uso terapêutico , Euphorbia/química , Humanos , Masculino , Camundongos , Simulação de Acoplamento Molecular , Neoplasias da Bexiga Urinária/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Cancer Res ; 81(11): 2861-2873, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33762357

RESUMO

Defective mitosis with chromosome missegregation can have a dramatic effect on genome integrity by causing DNA damage, activation of the DNA damage response (DDR), and chromosomal instability. Although this is an energy-dependent process, mechanisms linking DDR to cellular metabolism are unknown. Here we show that checkpoint kinase 2 (CHK2), a central effector of DDR, regulates cellular energy production by affecting glycolysis and mitochondrial functions. Patients with hepatocellular carcinoma (HCC) had increased CHK2 mRNA in blood, which was associated with elevated tricarboxylic acid cycle (TCA) metabolites. CHK2 controlled expression of succinate dehydrogenase (SDH) and intervened with mitochondrial functions. DNA damage and CHK2 promoted SDH activity marked by increased succinate oxidation through the TCA cycle; this was confirmed in a transgenic model of HCC with elevated DNA damage. Mitochondrial analysis identified CHK2-controlled expression of SDH as key in sustaining reactive oxygen species production. Cells with DNA damage and elevated CHK2 relied significantly on glycolysis for ATP production due to dysfunctional mitochondria, which was abolished by CHK2 knockdown. This represents a vulnerability created by the DNA damage response that could be exploited for development of new therapies. SIGNIFICANCE: This study uncovers a link between a central effector of DNA damage response, CHK2, and cellular metabolism, revealing potential therapeutic strategies for targeting hepatocellular carcinoma.


Assuntos
Carcinoma Hepatocelular/patologia , Quinase do Ponto de Checagem 2/metabolismo , Dano ao DNA , Glicólise , Neoplasias Hepáticas/patologia , Metaboloma , Transcriptoma , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Quinase do Ponto de Checagem 2/genética , Ciclo do Ácido Cítrico , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Mitose , Espécies Reativas de Oxigênio/metabolismo , Succinatos/metabolismo
9.
BMC Biol ; 19(1): 35, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33607997

RESUMO

BACKGROUND: The G1 checkpoint is a critical regulator of genomic stability in untransformed cells, preventing cell cycle progression after DNA damage. DNA double-strand breaks (DSBs) recruit and activate ATM, a kinase which in turn activates the CHK2 kinase to establish G1 arrest. While the onset of G1 arrest is well understood, the specific role that ATM and CHK2 play in regulating G1 checkpoint maintenance remains poorly characterized. RESULTS: Here we examine the impact of ATM and CHK2 activities on G1 checkpoint maintenance in untransformed cells after DNA damage caused by DSBs. We show that ATM becomes dispensable for G1 checkpoint maintenance as early as 1 h after DSB induction. In contrast, CHK2 kinase activity is necessary to maintain the G1 arrest, independently of ATM, ATR, and DNA-PKcs, implying that the G1 arrest is maintained in a lesion-independent manner. Sustained CHK2 activity is achieved through auto-activation and its acute inhibition enables cells to abrogate the G1-checkpoint and enter into S-phase. Accordingly, we show that CHK2 activity is lost in cells that recover from the G1 arrest, pointing to the involvement of a phosphatase with fast turnover. CONCLUSION: Our data indicate that G1 checkpoint maintenance relies on CHK2 and that its negative regulation is crucial for G1 checkpoint recovery after DSB induction.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Quinase do Ponto de Checagem 2/genética , Dano ao DNA , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular , Quinase do Ponto de Checagem 2/metabolismo , Humanos
10.
PLoS Genet ; 17(2): e1009391, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33600490

RESUMO

During DNA replication newly synthesized histones are incorporated into the chromatin of the replicating sister chromatids. In the yeast Saccharomyces cerevisiae new histone H3 molecules are acetylated at lysine 56. This modification is carefully regulated during the cell cycle, and any disruption of this process is a source of genomic instability. Here we show that the protein kinase Dun1 is necessary in order to maintain viability in the absence of the histone deacetylases Hst3 and Hst4, which remove the acetyl moiety from histone H3. This lethality is not due to the well-characterized role of Dun1 in upregulating dNTPs, but rather because Dun1 is needed in order to counteract the checkpoint kinase Rad53 (human CHK2) that represses the activity of late firing origins. Deletion of CTF18, encoding the large subunit of an alternative RFC-like complex (RLC), but not of components of the Elg1 or Rad24 RLCs, is enough to overcome the dependency of cells with hyper-acetylated histones on Dun1. We show that the detrimental function of Ctf18 depends on its interaction with the leading strand polymerase, Polε. Our results thus show that the main problem of cells with hyper-acetylated histones is the regulation of their temporal and replication programs, and uncover novel functions for the Dun1 protein kinase and the Ctf18 clamp loader.


Assuntos
Proteínas de Ciclo Celular/genética , Histona Desacetilases/genética , Histonas/genética , Mutação , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Acetilação , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisina/genética , Lisina/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Temperatura
11.
J Zhejiang Univ Sci B ; 22(1): 63-72, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33448188

RESUMO

DNA is the hereditary material in humans and almost all other organisms. It is essential for maintaining accurate transmission of genetic information. In the life cycle, DNA replication, cell division, or genome damage, including that caused by endogenous and exogenous agents, may cause DNA aberrations. Of all forms of DNA damage, DNA double-strand breaks (DSBs) are the most serious. If the repair function is defective, DNA damage may cause gene mutation, genome instability, and cell chromosome loss, which in turn can even lead to tumorigenesis. DNA damage can be repaired through multiple mechanisms. Homologous recombination (HR) and non-homologous end joining (NHEJ) are the two main repair mechanisms for DNA DSBs. Increasing amounts of evidence reveal that protein modifications play an essential role in DNA damage repair. Protein deubiquitination is a vital post-translational modification which removes ubiquitin molecules or polyubiquitinated chains from substrates in order to reverse the ubiquitination reaction. This review discusses the role of deubiquitinating enzymes (DUBs) in repairing DNA DSBs. Exploring the molecular mechanisms of DUB regulation in DSB repair will provide new insights to combat human diseases and develop novel therapeutic approaches.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , Enzimas Desubiquitinantes/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quinase 1 do Ponto de Checagem/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Reparo do DNA por Junção de Extremidades/fisiologia , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Recombinação Homóloga , Humanos , Ubiquitina-Proteína Ligases/metabolismo
12.
J Cell Biol ; 220(2)2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33404608

RESUMO

Cancer cells develop strong genetic dependencies, enabling survival under oncogenic stress. MYC is a key oncogene activated across most cancers, and identifying associated synthetic lethality or sickness can provide important clues about its activity and potential therapeutic strategies. On the basis of previously conducted genome-wide screenings in MCF10A cells expressing MYC fused to an estrogen receptor fragment, we identified UVSSA, a gene involved in transcription-coupled repair, whose knockdown or knockout decreased cell viability when combined with MYC expression. Synthetic sick interactions between MYC expression and UVSSA down-regulation correlated with ATM/CHK2 activation, suggesting increased genome instability. We show that the synthetic sick interaction is diminished by attenuating RNA polymerase II (RNAPII) activity; yet, it is independent of UV-induced damage repair, suggesting that UVSSA has a critical function in regulating RNAPII in the absence of exogenous DNA damage. Supporting this hypothesis, RNAPII ChIP-seq revealed that MYC-dependent increases in RNAPII promoter occupancy are reduced or abrogated by UVSSA knockdown, suggesting that UVSSA influences RNAPII dynamics during MYC-dependent transcription. Taken together, our data show that the UVSSA complex has a significant function in supporting MYC-dependent RNAPII dynamics and maintaining cell survival during MYC addiction. While the role of UVSSA in regulating RNAPII has been documented thus far only in the context of UV-induced DNA damage repair, we propose that its activity is also required to cope with transcriptional changes induced by oncogene activation.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Estresse Fisiológico/genética , Transcrição Genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Quinase do Ponto de Checagem 2/metabolismo , Dano ao DNA , Reparo do DNA , Regulação para Baixo , Humanos , Modelos Biológicos , Fenótipo , Ligação Proteica , Estruturas R-Loop/genética , RNA Polimerase II/metabolismo , Mutações Sintéticas Letais/genética , Sítio de Iniciação de Transcrição
13.
DNA Repair (Amst) ; 98: 103047, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33454524

RESUMO

Our genome bears tens of thousands of harms and devastations per day; In this regard, numerous sophisticated and complicated mechanisms are embedded by our cells in furtherance of remitting an unchanged and stable genome to their next generation. These mechanisms, that are collectively called DDR, have the duty of detecting the lesions and repairing them. it's necessary for the viability of any living cell that sustain the integrity and stability of its genetic content and this highlights the role of mediators that transduce the signals of DNA damage to the cell cycle in order to prevent the replication of a defective DNA. In this paper, we review the signaling pathways that lie between these processes and define how different ingredients of DDR are also able to affect the checkpoint signaling.


Assuntos
Pontos de Checagem do Ciclo Celular , Quebras de DNA , Reparo do DNA , Transdução de Sinais , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quinase 1 do Ponto de Checagem/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , DNA/metabolismo , Eucariotos/genética , Eucariotos/metabolismo , Humanos
14.
Int J Sports Med ; 42(3): 283-290, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32947637

RESUMO

Aging muscle is prone to sarcopenia and its associated telomere shortening and increased oxidative stress. Telomeres are protected by a shelterin protein complex, proteins expressed in response to DNA damage. Aerobic exercise training has shown to positively modulate these proteins while aging, but the effects of resistance training are less clear. This investigation was to examine the role of dynamic and isometric RT on markers of senescence and muscle apoptosis: checkpoint kinase 2, 53 kDa protein, shelterin telomere repeat binding 1 and 2, DNA repair, telomere length and redox state in the quadriceps muscle. Fifteen 49-week-old male rats were divided into three groups: control, dynamic resistance training, and isometric resistance training. Dynamic and isometric groups completed five sessions per week during 16 weeks at low to moderate intensity (20-70% maximal load). Only dynamic group decreased expression of 53 kDa protein, proteins from shelterin complex, oxidative stress, and improved antioxidant defense. There was no difference among groups regarding telomere length. In conclusion, dynamic resistance training was more effective than isometric in reducing markers of aging and muscle apoptosis in elderly rats. This modality should be considered as valuable tool do counteract the deleterious effects of aging.


Assuntos
Envelhecimento/fisiologia , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Treinamento de Força/métodos , Animais , Apoptose , Biomarcadores/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Reparo do DNA , Genes p53 , Contração Isométrica , Masculino , Músculo Esquelético/citologia , Oxirredução , Estresse Oxidativo , Condicionamento Físico Animal , Ratos Wistar , Encurtamento do Telômero , Proteínas de Ligação a Telômeros/fisiologia
15.
J Cell Biol ; 220(2)2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33355621

RESUMO

During cell division, in response to chromatin bridges, the chromosomal passenger complex (CPC) delays abscission to prevent chromosome breakage or tetraploidization. Here, we show that inhibition of ATM or Chk2 kinases impairs CPC localization to the midbody center, accelerates midbody resolution in normally segregating cells, and correlates with premature abscission and chromatin breakage in cytokinesis with trapped chromatin. In cultured human cells, ATM activates Chk2 at late midbodies. In turn, Chk2 phosphorylates human INCENP-Ser91 to promote INCENP binding to Mklp2 kinesin and CPC localization to the midbody center through Mklp2 association with Cep55. Expression of truncated Mklp2 that does not bind to Cep55 or nonphosphorylatable INCENP-Ser91A impairs CPC midbody localization and accelerates abscission. In contrast, expression of phosphomimetic INCENP-Ser91D or a chimeric INCENP protein that is targeted to the midbody center rescues the abscission delay in Chk2-deficient or ATM-deficient cells. Furthermore, the Mre11-Rad50-Nbs1 complex is required for ATM activation at the midbody in cytokinesis with chromatin bridges. These results identify an ATM-Chk2-INCENP pathway that imposes the abscission checkpoint by regulating CPC midbody localization.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Pontos de Checagem do Ciclo Celular , Quinase do Ponto de Checagem 2/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Citocinese , Transdução de Sinais , Aurora Quinase B/metabolismo , Proliferação de Células , Quinase do Ponto de Checagem 2/antagonistas & inibidores , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Cinesina/metabolismo , Proteína Homóloga a MRE11/metabolismo , Complexos Multiproteicos/metabolismo , Mutação/genética , Fosforilação
16.
mBio ; 11(6)2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33323516

RESUMO

DNA damage checkpoints are key guardians of genome integrity. Eukaryotic cells respond to DNA damage by triggering extensive phosphorylation of Rad53/CHK2 effector kinase, whereupon activated Rad53/CHK2 mediates further aspects of checkpoint activation, including cell cycle arrest and transcriptional changes. Budding yeast Candida glabrata, closely related to model eukaryote Saccharomyces cerevisiae, is an opportunistic pathogen characterized by high genetic diversity and rapid emergence of drug-resistant mutants. However, the mechanisms underlying this genetic variability are unclear. We used Western blotting and mass spectrometry to show that, unlike S. cerevisiae, C. glabrata cells exposed to DNA damage did not induce C. glabrata Rad53 (CgRad53) phosphorylation. Furthermore, flow cytometry analysis showed that, unlike S. cerevisiae, C. glabrata cells did not accumulate in S phase upon DNA damage. Consistent with these observations, time-lapse microscopy showed C. glabrata cells continuing to divide in the presence of DNA damage, resulting in mitotic errors and cell death. Finally, transcriptome sequencing (RNAseq) analysis revealed transcriptional rewiring of the DNA damage response in C. glabrata and identified several key protectors of genome stability upregulated by DNA damage in S. cerevisiae but downregulated in C. glabrata, including proliferating cell nuclear antigen (PCNA). Together, our results reveal a noncanonical fungal DNA damage response in C. glabrata, which may contribute to rapidly generating genetic change and drug resistance.IMPORTANCE In order to preserve genome integrity, all cells must mount appropriate responses to DNA damage, including slowing down or arresting the cell cycle to give the cells time to repair the damage and changing gene expression, for example to induce genes involved in DNA repair. The Rad53 protein kinase is a conserved central mediator of these responses in eukaryotic cells, and its extensive phosphorylation upon DNA damage is necessary for its activation and subsequent activity. Interestingly, here we show that in the opportunistic fungal pathogen Candida glabrata, Rad53 phosphorylation is not induced by DNA damage, nor do these cells arrest in S phase under these conditions, in contrast to the closely related yeast Saccharomyces cerevisiae Instead, C. glabrata cells continue to divide in the presence of DNA damage, resulting in significant cell lethality. Finally, we show that a number of genes involved in DNA repair are strongly induced by DNA damage in S. cerevisiae but repressed in C. glabrata Together, these findings shed new light on mechanisms regulating genome stability in fungal pathogens.


Assuntos
Pontos de Checagem do Ciclo Celular , Dano ao DNA , Saccharomycetales/citologia , Saccharomycetales/genética , Divisão Celular , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Humanos , Micoses/microbiologia , Fosforilação , Fase S , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/enzimologia
17.
Cells ; 9(12)2020 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-33322746

RESUMO

Germline alterations in many genes coding for proteins regulating DNA repair and DNA damage response (DDR) to DNA double-strand breaks (DDSB) have been recognized as pathogenic factors in hereditary cancer predisposition. The ATM-CHEK2-p53 axis has been documented as a backbone for DDR and hypothesized as a barrier against cancer initiation. However, although CHK2 kinase coded by the CHEK2 gene expedites the DDR signal, its function in activation of p53-dependent cell cycle arrest is dispensable. CHEK2 mutations rank among the most frequent germline alterations revealed by germline genetic testing for various hereditary cancer predispositions, but their interpretation is not trivial. From the perspective of interpretation of germline CHEK2 variants, we review the current knowledge related to the structure of the CHEK2 gene, the function of CHK2 kinase, and the clinical significance of CHEK2 germline mutations in patients with hereditary breast, prostate, kidney, thyroid, and colon cancers.


Assuntos
Quinase do Ponto de Checagem 2/genética , Predisposição Genética para Doença , Mutação em Linhagem Germinativa/genética , Neoplasias/enzimologia , Neoplasias/genética , Animais , Quinase do Ponto de Checagem 2/química , Quinase do Ponto de Checagem 2/metabolismo , Humanos , Taxa de Mutação , Especificidade por Substrato
18.
PLoS Genet ; 16(11): e1009067, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33206637

RESUMO

Mammalian oogonia proliferate without completing cytokinesis, forming cysts. Within these, oocytes differentiate and initiate meiosis, promoting double-strand break (DSBs) formation, which are repaired by homologous recombination (HR) causing the pairing and synapsis of the homologs. Errors in these processes activate checkpoint mechanisms, leading to apoptosis. At the end of prophase I, in contrast with what is observed in spermatocytes, oocytes accumulate unrepaired DSBs. Simultaneously to the cyst breakdown, there is a massive oocyte death, which has been proposed to be necessary to enable the individualization of the oocytes to form follicles. Based upon all the above-mentioned information, we hypothesize that the apparently inefficient HR occurring in the oocytes may be a requirement to first eliminate most of the oocytes and enable cyst breakdown and follicle formation. To test this idea, we compared perinatal ovaries from control and mutant mice for the effector kinase of the DNA Damage Response (DDR), CHK2. We found that CHK2 is required to eliminate ~50% of the fetal oocyte population. Nevertheless, the number of oocytes and follicles found in Chk2-mutant ovaries three days after birth was equivalent to that of the controls. These data revealed the existence of another mechanism capable of eliminating oocytes. In vitro inhibition of CHK1 rescued the oocyte number in Chk2-/- mice, implying that CHK1 regulates postnatal oocyte death. Moreover, we found that CHK1 and CHK2 functions are required for the timely breakdown of the cyst and to form follicles. Thus, we uncovered a novel CHK1 function in regulating the oocyte population in mice. Based upon these data, we propose that the CHK1- and CHK2-dependent DDR controls the number of oocytes and is required to properly break down oocyte cysts and form follicles in mammals.


Assuntos
Dano ao DNA/genética , Oogônios/metabolismo , Folículo Ovariano/metabolismo , Animais , Apoptose/fisiologia , Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Cistos/metabolismo , Dano ao DNA/fisiologia , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Feminino , Meiose/fisiologia , Prófase Meiótica I/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Oócitos/metabolismo , Oócitos/fisiologia , Oogônios/fisiologia , Folículo Ovariano/fisiologia , Ovário/metabolismo , Progesterona/metabolismo
19.
J Clin Invest ; 130(11): 5951-5966, 2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-33016929

RESUMO

ARID1A, a component of the chromatin-remodeling complex SWI/SNF, is one of the most frequently mutated genes in human cancer. We sought to develop rational combination therapy to potentiate the efficacy of immune checkpoint blockade in ARID1A-deficient tumors. In a proteomic analysis of a data set from The Cancer Genomic Atlas, we found enhanced expression of Chk2, a DNA damage checkpoint kinase, in ARID1A-mutated/deficient tumors. Surprisingly, we found that ARID1A targets the nonchromatin substrate Chk2 for ubiquitination. Loss of ARID1A increased the Chk2 level through modulating autoubiquitination of the E3-ligase RNF8 and thereby reducing RNF8-mediated Chk2 degradation. Inhibition of the ATM/Chk2 DNA damage checkpoint axis led to replication stress and accumulation of cytosolic DNA, which subsequently activated the DNA sensor STING-mediated innate immune response in ARID1A-deficient tumors. As expected, tumors with mutation or low expression of both ARID1A and ATM/Chk2 exhibited increased tumor-infiltrating lymphocytes and were associated with longer patient survival. Notably, an ATM inhibitor selectively potentiated the efficacy of immune checkpoint blockade in ARID1A-depleted tumors but not in WT tumors. Together, these results suggest that ARID1A's targeting of the nonchromatin substrate Chk2 for ubiquitination makes it possible to selectively modulate cancer cell-intrinsic innate immunity to enhance the antitumor activity of immune checkpoint blockade.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias , Neoplasias , Nucleotidiltransferases/metabolismo , Transdução de Sinais , Fatores de Transcrição/deficiência , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Quinase do Ponto de Checagem 2/genética , Proteínas de Ligação a DNA/metabolismo , Células HCT116 , Humanos , Proteínas de Membrana/genética , Camundongos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Nucleotidiltransferases/genética , Proteólise , Fatores de Transcrição/metabolismo
20.
Appl. cancer res ; 40: 1-6, Oct. 19, 2020. ilus, tab
Artigo em Inglês | LILACS, Inca | ID: biblio-1282611

RESUMO

Background: Oral squamous cell carcinoma (OSCC) is the most frequently occurring malignant tumor of the head and neck region. Chk2 (Checkpoint kinase 2) is considered a tumor suppressor gene that acts on the cellular response to DNA damage. However, the role of Chk2 in OSCC prognosis is not yet fully understood. The objective of this study was to evaluate Chk2 immunoexpression in OSCC and to elucidate the association between its expression and clinicopathological parameters of prognostic importance, including overall survival, disease-free survival, and metastasis-free survival. Methods: Chk2 expression was analyzed in 101 samples from patients with OSCC using immunohistochemistry. We stratified the patients into high expression (> 66% of cells positive for Chk2) and low expression (< 66%) groups. Results: Chk2 showed high expression in 57.43% of OSCC. In our study, the expression of Chk2 did not correlate with any of the prognostic parameters evaluated. There was no difference between overall survival, metastasis-free survival, and disease-free survival according to Chk2 expression. Conclusion: Despite the great importance of Chk2 in the development of different types of cancer, our findings do not favor Chk2 as a prognostic marker in oral squamous cell carcinoma.


Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Neoplasias Bucais/metabolismo , Imuno-Histoquímica , Carcinoma de Células Escamosas/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Prognóstico , Análise de Sobrevida
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