RESUMO
DNA double-strand break (DSB) is one of the most serious forms of damage induced by ionizing irradiation and is mainly repaired by the non-homologous end joining (NHEJ) repair. Immunohistochemical analysis of proteins involved in NHEJ, such as XRCC4 (X-ray repair cross-complementing protein 4), Ku86 and DNA-PKcs (DNA-dependent protein kinase, catalytic subunits), may be useful for predicting tumor radiosensitivity. We examined 92 patients with esophageal squamous cell carcinoma (ECSS) who were treated by radiotherapy between 1999 and 2008. Immunohistochemical examination of tumor tissue for Ki-67 and DSB-related proteins, including XRCC4, Ku86, and DNA-PKcs, was performed using pretreatment biopsy specimens. Low expression of XRCC4 was detected in 31 of 92 examined samples (33.7 %). The 5-year overall survival (OS) rate was 67.7 % in the low expression group and 31.0 % in the high expression group (P = 0.00). Multivariate analysis confirmed that advanced T-stage (HR 3.24, P = 0.01), radiation dose less than 66 Gy (HR 2.23, P = 0.02), absence of systemic chemotherapy (HR 2.59, P = 0.05), and high expression of XRCC4 (HR 12.0, P = 0.02) were independent prognostic factors for predicting poor OS. Other DSB-related proteins and Ki-67 were not predictive factors. XRCC4 expression might have an influence on results of radiotherapy for patients with ESCC.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/radioterapia , Idoso , Idoso de 80 Anos ou mais , Proteína Quinase Ativada por DNA/metabolismo , Feminino , Humanos , Autoantígeno Ku/metabolismo , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Estadiamento de Neoplasias , Modelos de Riscos Proporcionais , Resultado do TratamentoRESUMO
DNA double-strand breaks (DSB) are severe damages induced by ionizing radiation. Non-homologous end joining (NHEJ) is a major mechanism for repairing DSB. Immunohistochemical analysis of proteins involved in NHEJ, such as Ku86 and XRCC4 (X-ray repair cross-complementing protein 4) may be useful for predicting tumor radiosensitivity. We examined the relationship between expression of DSB-related proteins in biopsy specimens of uterine cervical cancer and the pathological effect of 40 Gy of preoperative radiotherapy. 119 patients with uterine cervical cancer were treated between 2000 and 2011. Pathological effects of preoperative radiotherapy were classified by examining hysterectomy specimens. Patients with complete response (pCR) had a significantly better overall 5-year survival rate than those without pCR (96.3 vs. 76.9 %, P = 0.02). The pCR rate was significantly higher in patients with low Ku86 and XRCC4 expression than in other patients (47.4 vs. 21.3 %, P = 0.04). Logistic regression analysis also demonstrated that low Ku86 and XRCC4 expression was a significant predictor of pCR (P = 0.03). Patients with high Ku86 and XRCC4 expression had a significantly lower 5-year metastasis-free rate than others (79.3 vs. 93.5 %, P = 0.02). Proteins involved with NHEJ might have an influence on results of radiotherapy for uterine cervical cancer.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Autoantígeno Ku/metabolismo , Cuidados Pré-Operatórios , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/cirurgia , Adulto , Idoso , Biópsia , Contagem de Células , Reparo do DNA por Junção de Extremidades/genética , Intervalo Livre de Doença , Feminino , Humanos , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Resultado do Tratamento , Neoplasias do Colo do Útero/patologiaRESUMO
Cancer metastasis is the major cause of death from cancer (Massague and Obenauf, 2016; Steeg, 2016). The extensive genetic heterogeneity and cellular plasticity of metastatic tumors set a prime barrier for the current cancer treatment protocols (Boumahdi and de Sauvage, 2020). In addition, acquired therapy resistance has become an insurmountable obstacle that abolishes the beneficial effects of numerous anti-cancer regimens (De Angelis et al., 2019; Boumahdi and de Sauvage, 2020). Here we report that deficiency of Ku leads to the exploitation of host cells in human cancer cell line models. We found that, upon conditional deletion of XRCC6 that codes for Ku70, HCT116 human colorectal cancer cells gain a parasitic lifestyle that is characterized by the continuous cycle of host cell exploitation. We also found that DAOY cells, a human medulloblastoma cell line, innately lack nuclear Ku70/Ku86 proteins and utilize the host-cell invasion/exit mechanism for maintenance of their survival, similarly to the Ku70 conditionally-null HCT116 cells. Our study demonstrates that a functional loss of Ku protein promotes an adaptive, opportunistic switch to a parasitic lifestyle in human cancer cells, providing evidence for a previously unknown mechanism of cell survival in response to severe genomic stress. We anticipate that our study will bring a new perspective for understanding the mechanisms of cancer cell evolution, leading to a shift in the current concepts of cancer therapy protocols directed to the prevention of cancer metastasis and therapy resistance.
RESUMO
Drug resistance and recurrence are significant contributors to the poor prognosis of serous ovarian cancer (SOC). Radiotherapy is primarily used for the treatment of cancer recurrence; however, it is rarely applied in cases of SOC. Ku86, also known as XRCC5(X-ray repair cross complementing 5), has rarely been reported in the radiotherapy of SOC. Therefore, this study aimed to investigate the role of Ku86 in the development of SOC and in radiotherapy sensitivity induced by X-ray. In vitro experiments and database analysis showed significantly elevated expression of Ku86 in SOC. Further, after down-regulating Ku86 using RNAi technology, cell proliferation was inhibited. Further, the cell cycle was blocked in the G2 phase, and G2/G1 was increased since X-ray irradiation led to an increase in γ-H2AX. Down-regulation of Ku86 in the case of X-ray irradiation will promote the above biological effects, with more γ-H2AX and higher G2/G1. Here, we further deduce that Ku86 promotes the X-ray induced effect is most likely to activate the ATR pathway to block the cell cycle while inhibiting the NHEJ pathway to inhibit DNA damage response(DDR). Together these ï¬ndings suggest that the down-regulation of Ku86 can improve X-ray-induced radiotherapy by affecting ATR and NHEJ pathways, and provide a new strategy for the treatment of ovarian cancer.
Assuntos
Carcinoma Epitelial do Ovário/radioterapia , Autoantígeno Ku/efeitos da radiação , Feminino , Humanos , Raios XRESUMO
OBJECTIVE: The aim of this study was to observe the effect of Ku86 on cellular senescence and apoptosis induced by various doses of ionizing radiation in human umbilical vein endothelial cells (HUVECs). METHODS: Senescence-associated ß-galactosidase activity was detected to evaluate cell senescence. Apoptosis was determined by flow cytometry and a caspase enzyme determination kit. p16Ink4a, Sirt1, superoxide dismutase 2 (SOD2), xanthine oxidase (XOD), and Bcl-2 protein expression levels were measured by western blotting. RESULTS: Low doses of ionizing radiation induced cellular senescence and apoptosis in a dose-dependent manner. The Ku86 protein was negatively correlated with ionization intensity. After transfection of Ku86 with a vector (pcDNA 3.1), or interference with siRNA (si-Ku86), apoptosis/senescence and related protein expression were observed. Western blot results revealed that this induction of senescence was associated with activated Sirt1 and SOD2, and downregulation of p16Ink4a and XOD in 0.2 Gy ionizing radiation. The expression levels of apoptosis-associated proteins, such as Bcl-2, cleaved caspase-3, caspase-8, and caspase-9, were significantly altered in both the presence and absence of Ku86 with ionizing radiation (0.2 Gy). CONCLUSIONS: Our study revealed that Ku86 overexpression inhibits HUVEC apoptosis and senescence induced by low doses of ionizing radiation.
Assuntos
Apoptose/fisiologia , Senescência Celular/fisiologia , Células Endoteliais da Veia Umbilical Humana/patologia , Autoantígeno Ku/metabolismo , Radiação Ionizante , Apoptose/efeitos da radiação , Senescência Celular/efeitos da radiação , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos da radiação , HumanosRESUMO
A novel role for HSF1 as an inhibitor of non-homologous end joining (NHEJ) repair activity was identified. HSF1 interacted directly with both of the N-terminal sequences of the Ku70 and Ku86 proteins, which inhibited the endogenous heterodimeric interaction between Ku70 and Ku86. The blocking of the Ku70 and Ku86 interaction by HSF1 induced defective NHEJ repair activity and ultimately activated genomic instability after ionizing radiation (IR), which was similar to effects seen in Ku70 or Ku80 knockout cells. The binding activity between HSF1 and Ku70 or Ku86 was dependent on DNA damage response such as IR exposure, but not on the heat shock mediated transcriptional activation of HSF1. Moreover, the posttranslational modification such as phosphorylation, acetylation and sumoylation of HSF1 did not alter the binding activities of HSF1-Ku70 or HSF1-Ku86. Furthermore, the defect in DNA repair activity by HSF1 was observed regardless of p53 status. Rat mammary tumors derived using dimethylbenz(a)anthracence revealed that high levels of HSF1 expression which correlate with aggressive malignancy, interfered with the binding of Ku70-Ku80. This data suggests that HSF1 interacts with both Ku70 and Ku86 to induce defective NHEJ repair activity and genomic instability, which in turn suggests a novel mechanism of HSF1-mediated cellular carcinogenesis.
Assuntos
Antígenos Nucleares/metabolismo , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Antígenos Nucleares/genética , Linhagem Celular Tumoral , Células Cultivadas , Dano ao DNA , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos/citologia , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos da radiação , Células HEK293 , Fatores de Transcrição de Choque Térmico , Humanos , Immunoblotting , Imuno-Histoquímica , Autoantígeno Ku , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/metabolismo , Camundongos Knockout , Radiação Ionizante , Ratos Sprague-Dawley , Fatores de Transcrição/genéticaRESUMO
The Ku heterodimer, composed of Ku70 and Ku80, is the initiating factor of the nonhomologous end joining (NHEJ) double-strand break (DSB) repair pathway. Ku is also thought to impede the homologous recombination (HR) repair pathway via inhibition of DNA end resection. Using the cell-free Xenopus laevis egg extract system, we had previously discovered that Ku80 becomes polyubiquitylated upon binding to DSBs, leading to its removal from DNA and subsequent proteasomal degradation. Here we show that the Skp1-Cul1-F box (SCF) E3 ubiquitin ligase complex is required for Ku80 ubiquitylation and removal from DNA. A screen for DSB-binding F box proteins revealed that the F box protein Fbxl12 was recruited to DNA in a DSB- and Ku-sensitive manner. Immunodepletion of Fbxl12 prevented Cul1 and Skp1 binding to DSBs and Ku80 ubiquitylation, indicating that Fbxl12 is the F box protein responsible for Ku80 substrate recognition. Unlike typical F box proteins, the F box of Fbxl12 was essential for binding to both Skp1 and its substrate Ku80. Besides Fbxl12, six other chromatin-binding F box proteins were identified in our screen of a subset of Xenopus F box proteins: ß-TrCP, Fbh1, Fbxl19, Fbxo24, Fbxo28 and Kdm2b. Our study unveils a novel function for the SCF ubiquitin ligase in regulating the dynamic interaction between DNA repair machineries and DSBs.
Assuntos
Antígenos Nucleares/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Xenopus/genética , Xenopus laevis/genética , Animais , Antígenos Nucleares/metabolismo , Sistema Livre de Células , Proteínas Culina/genética , Proteínas Culina/metabolismo , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Regulação da Expressão Gênica , Autoantígeno Ku , Dados de Sequência Molecular , Ligação Proteica , Multimerização Proteica , Proteínas Quinases Associadas a Fase S/genética , Proteínas Quinases Associadas a Fase S/metabolismo , Alinhamento de Sequência , Transdução de Sinais , Ubiquitinação , Proteínas de Xenopus/metabolismo , Xenopus laevis/crescimento & desenvolvimento , Xenopus laevis/metabolismo , Zigoto/químicaRESUMO
NaCl induces DNA breaks, thus leading to cellular senescence. Here we showed that Ku86 deficiency accelerated the high NaCl-induced cellular senescence. We find that 1) high NaCl induces rapid cellular senescence in Ku86 deficient(xrs5) cells, 2) Ku86 deficiency shortens lifespan of C. elegans in high NaCl, and 3) cellular senescence is greatly accelerated in renal inner medullas of Ku86 (-/-) mice. Further, although water balance is known to be compromised in old mice, this occurs at much earlier age in Ku86(-/-) mice. When subjected to mild water restriction, 3 month old Ku86(-/-), but not Ku86(+/+),mice rapidly become dehydrated as evidenced by decrease in body weight, increased production of antidiuretic hormone,increased urine osmolality and decreased urine volume. The deficiency in water balance does not occur in Ku86(+/+)mice until they are much older (14 months). We conclude that Ku86 deficiency accelerates high NaCl(-) induced cellular senescence,particularly in the renal medulla where NaCl normally is high.