RESUMEN
As p53-binding protein 1 (53BP1) localizes to the sites of DNA double-strand breaks and rapidly forms nuclear foci (NF), and its presence may be an indicator of endogenous genomic instability (GIN). We previously showed that 53BP1 NF in cervical cells increase with neoplastic progression, indicating the significance of 53BP1 expression for the estimation of malignant potential during cervical carcinogenesis. This study aimed to further elucidate the impact of 53BP1 expression as a biomarker for cervical squamous intraepithelial lesion (SIL). A total of 81 tissue samples, including 17 of normal cervical epithelium, 22 of cervical intraepithelial neoplasia (CIN) 1, 21 of CIN2, and 21 of CIN3, from patients positive for high-risk human papillomavirus (HR-HPV) were used for double-label immunofluorescence of 53BP1 and Ki-67/p16INK4a expression and HR-HPV in situ hybridization. We analyzed associations between 53BP1 expression type with parameters such as CIN grade, HR-HPV infection status, p16INK4a expression, and CIN prognosis. Expression type of 53BP1 was significantly associated with histological grade of CIN and HR-HPV in situ hybridization signal pattern (P < .0001). There was a significant correlation between 53BP1 and p16INK4a expression levels (r = .73, P < .0001). However, there was no association between 53BP1 expression type and CIN prognosis. We propose that 53BP1 expression type is a valuable biomarker for SIL, which can help estimate the grade and GIN of cervical lesions reflecting replication stress caused by the integration of HR-HPV to the host genome.
Asunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina/biosíntesis , Infecciones por Papillomavirus/metabolismo , Lesiones Intraepiteliales Escamosas/metabolismo , Lesiones Intraepiteliales Escamosas/virología , Proteína 1 de Unión al Supresor Tumoral P53/biosíntesis , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/virología , Adulto , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Femenino , Humanos , Papillomaviridae/genética , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/patología , Infecciones por Papillomavirus/virología , Lesiones Intraepiteliales Escamosas/genética , Lesiones Intraepiteliales Escamosas/patología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/genética , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patologíaRESUMEN
BACKGROUND: Abnormal DNA damage response (DDR) leads to genomic instability and carcinogenesis. P53-binding protein 1 (53 BP1), a DDR molecule, is known to accumulate at the sites of DNA double-strand breaks. The aim of this study was to analyze the expression pattern of 53 BP1-nuclear foci (NF) in esophageal neoplasms in order to visualize the state of DDR in esophageal carcinogenesis and to clarify its significance in the molecular pathology of the disease. METHODS: A total of 61 lesions from 22 surgically resected samples of esophageal cancer, including histologically normal squamous epithelium, low-grade intraepithelial neoplasia (LG-IN), high-grade intraepithelial neoplasia (HG-IN), carcinoma in situ (CIS), and invasive squamous cell carcinoma (SCC), were included in the study. 53 BP1 and Ki-67 expression were analyzed by double-labeled immunofluorescence. RESULTS: The number of discrete 53 BP1-NF increased as the tumor progressed from normal epithelium through LG-IN, HG-IN, CIS, and SCC. 53 BP1-NF larger than 1⯵m in diameter (large foci), indicating intensive DDR, also showed a stepwise increase during the progression of carcinogenesis. Of note, large foci of 53 BP1 were found in significantly higher numbers in HG-IN than in LG-IN. Furthermore, localization of 53 BP1-NF in Ki-67-positive cells, indicating the abnormal timing of DDR, also increased with malignancy progression. CONCLUSIONS: 53 BP1-NF accumulation increases during cancer progression from LG-IN to HG-IN to CIS to SCC. Detection of 53 BP1-NF by immunofluorescence, especially large foci, is a feasible method of estimating DNA instability and the malignant potential of esophageal intraepithelial neoplasia.
Asunto(s)
Biomarcadores de Tumor/análisis , Carcinoma in Situ/patología , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/patología , Proteína 1 de Unión al Supresor Tumoral P53/biosíntesis , Anciano , Carcinogénesis/metabolismo , Daño del ADN , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Estudios RetrospectivosRESUMEN
OBJECTIVE: Poly(ADP-ribose) polymerase (PARP) inhibitors have shown substantial activity in homologous recombination- (HR-) deficient ovarian cancer and are undergoing testing in other HR-deficient tumors. For reasons that are incompletely understood, not all patients with HR-deficient cancers respond to these agents. Preclinical studies have demonstrated that changes in alternative DNA repair pathways affect PARP inhibitor (PARPi) sensitivity in ovarian cancer models. This has not previously been assessed in the clinical setting. METHODS: Clonogenic and plasmid-based HR repair assays were performed to compare BRCA1-mutant COV362 ovarian cancer cells with or without 53BP1 gene deletion. Archival biopsies from ovarian cancer patients in the phase I, open-label clinical trial of PARPi ABT-767 were stained for PARP1, RAD51, 53BP1 and multiple components of the nonhomologous end-joining (NHEJ) DNA repair pathway. Modified histochemistry- (H-) scores were determined for each repair protein in each sample. HRD score was determined from tumor DNA. RESULTS: 53BP1 deletion increased HR in BRCA1-mutant COV362 cells and decreased PARPi sensitivity in vitro. In 36 women with relapsed ovarian cancer, responses to the PARPi ABT-767 were observed exclusively in cancers with HR deficiency. In this subset, 7 of 18 patients (39%) had objective responses. The actual HRD score did not further correlate with change from baseline tumor volume (râ¯=â¯0.050; pâ¯=â¯0.87). However, in the HR-deficient subset, decreased 53BP1 H-score was associated with decreased antitumor efficacy of ABT-767 (râ¯=â¯-0.69, pâ¯=â¯0.004). CONCLUSION: Differences in complementary repair pathways, particularly 53BP1, correlate with PARPi response of HR-deficient ovarian cancers.
Asunto(s)
Benzamidas/administración & dosificación , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/administración & dosificación , Sulfonamidas/administración & dosificación , Proteína 1 de Unión al Supresor Tumoral P53/genética , Línea Celular Tumoral , Reparación del ADN , Resistencia a Antineoplásicos , Femenino , Genes BRCA1 , Genes BRCA2 , Recombinación Homóloga , Humanos , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Poli(ADP-Ribosa) Polimerasa-1/biosíntesis , Poli(ADP-Ribosa) Polimerasa-1/genética , Proteína 1 de Unión al Supresor Tumoral P53/biosíntesis , Proteína 1 de Unión al Supresor Tumoral P53/deficienciaRESUMEN
PURPOSE: Loss of P53 binding protein 1 (53BP1) is considered a poor prognostic factor for colorectal cancer. However, its effect on chemosensitivity of colorectal cancer to 5-fluorouracil (5-FU) remains elusive. This study aimed to examine the association of 53BP1 expression with chemosensitivity of colorectal cancer cells to 5-FU. METHODS: Immunohistochemistry was performed on 30 metastatic colorectal cancer samples to assess the associations of 53BP1 levels with clinical therapeutic effects. In vitro, IC50 values for 5-FU and 53BP1 levels were determined by MTT assay and Western blot in 5 colorectal cancer cell lines. Then, 53BP1 was silenced in HCT116 and HT29 cells, and cell proliferation, apoptosis and cell cycle distribution were evaluated. Relative protein levels of ATM-CHK2-P53 pathway effectors and Bcl-2 family members were measured by Western blot. Finally, the effects of 53BP1 knockdown on tumor growth and 5-FU chemoresistance were investigated in vivo. RESULTS: 53BP1 expression was closely related to time to progression (TTP) after first-line chemotherapy. Namely, 53BP1 downregulation resulted in reduced TTP. In addition, 53BP1 silencing increased proliferation, inhibited apoptosis and induced S phase arrest in HCT116 and HT29 cells after 5-FU treatment. Moreover, 53BP1 knockdown also reduced the protein levels of ATM-CHK2-P53 apoptotic pathway effectors, caspase9 and caspase3, while increasing Bcl-2 expression. In vivo, 53BP1 silencing accelerated tumor proliferation in nude mice and enhanced resistance to 5-FU. CONCLUSIONS: These findings confirmed that 53BP1 loss might be a negative factor for chemotherapy efficacy, promoting cell proliferation and inhibiting apoptosis by suppressing ATM-CHK2-P53 signaling, and finally inducing 5-FU resistance.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/genética , Quinasa de Punto de Control 2/genética , Neoplasias Colorrectales/tratamiento farmacológico , Proteína p53 Supresora de Tumor/genética , Proteína 1 de Unión al Supresor Tumoral P53/genética , Animales , Proliferación Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Daño del ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Fluorouracilo/administración & dosificación , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Transducción de Señal/efectos de los fármacos , Proteína 1 de Unión al Supresor Tumoral P53/antagonistas & inhibidores , Proteína 1 de Unión al Supresor Tumoral P53/biosíntesisRESUMEN
BACKGROUND: The ability to image DNA repair in cancer cells after irradiation, as well as its inhibition by potential therapeutic agents, is important for the further development of effective cancer therapy. 53BP1 is a DNA repair protein that is overexpressed and forms foci when double-stranded DNA breaks occur in DNA. MATERIALS AND METHODS: The re-localization of green fluorescent protein (GFP) fused to the chromatin-binding domain of 53BP1 to form foci was imaged after UVC irradiation of breast and pancreatic cancer cells expressing 53BP1-GFP using confocal microscopy. RESULTS: During live-cell imaging, 53BP1-GFP focus formation was observed within 10 minutes after UVC irradiation. Most 53BP1 foci resolved by 100 minutes. To block UVC-induced double-strand break repair in cancer cells, poly(ADP-ribose) polymerase (PARP) was targeted with ABT-888 (veliparib). PARP inhibition markedly enhanced UVC-irradiation-induced persistence of 53BP1-foci, even beyond 100 minutes after UVC irradiation, and reduced proliferation of breast and pancreatic cancer cells. CONCLUSION: Confocal microscopy of 53BP1-GFP is a powerful method for imaging UVC-induced DNA damage and repair, as well as inhibition of repair.
Asunto(s)
Neoplasias Pancreáticas/diagnóstico por imagen , Neoplasias Pancreáticas/terapia , Poli(ADP-Ribosa) Polimerasa-1/genética , Proteína 1 de Unión al Supresor Tumoral P53/genética , Bencimidazoles/administración & dosificación , Línea Celular Tumoral , Roturas del ADN de Doble Cadena/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de la radiación , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de los fármacos , Reparación del ADN/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Proteínas Fluorescentes Verdes/química , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Fotoquimioterapia , Poli(ADP-Ribosa) Polimerasa-1/biosíntesis , Inhibidores de Poli(ADP-Ribosa) Polimerasas/administración & dosificación , Proteína 1 de Unión al Supresor Tumoral P53/biosíntesis , Rayos UltravioletaRESUMEN
H2AX is expressed at very low levels in quiescent normal cells in vivo and in vitro. Such cells repair DNA double-strand breaks (DSBs) induced by γ-irradiation through a transient stabilization of H2AX. However, the resultant cells accumulate small numbers of irreparable (or persistent) DSBs via an unknown mechanism. We found that quiescent cells that had repaired DSBs directly induced by γ-rays were prone to accumulate DSBs during the subsequent DNA replication. Unlike directly induced DSBs, secondary DSBs were not efficiently repaired, although Rad51 and 53BP1 were recruited to these sites. H2AX was dramatically stabilized in response to DSBs directly caused by γ-rays, enabling γH2AX foci formation and DSB repair, whereas H2AX was barely stabilized in response to secondary DSBs, in which γH2AX foci were small and DSBs were not efficiently repaired. Our results show a pathway that leads to the persistent DSB formation after γ-irradiation.
Asunto(s)
Roturas del ADN de Doble Cadena/efectos de la radiación , Replicación del ADN/genética , Histonas/genética , Recombinasa Rad51/genética , Proteína 1 de Unión al Supresor Tumoral P53/genética , Células 3T3 , Animales , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de la radiación , Replicación del ADN/efectos de la radiación , Fibroblastos , Rayos gamma , Regulación de la Expresión Génica/efectos de la radiación , Histonas/biosíntesis , Humanos , Ratones , Recombinasa Rad51/biosíntesis , Proteína 1 de Unión al Supresor Tumoral P53/biosíntesisRESUMEN
Oncogenic Ras expression is associated with activation of the DNA damage response (DDR) pathway, as evidenced by elevated DNA damage, primarily DNA double-strand breaks (DSBs), and activation of DNA damage checkpoints, which in primary human cells leads to entry into senescence. DDR activation is viewed as a physiological barrier against uncontrolled proliferation in oncogenic Ras-expressing cells, and arises in response to genotoxic stress due to the production of reactive oxygen species that damage DNA and to hyper-replication stress. Although oncogene-induced senescence (OIS) is considered a tumor suppressor mechanism, the accumulation of DNA damage in senescent cells is thought to cause genomic instability, eventually allowing secondary hits in the genome that promote tumorigenesis. To date, the molecular mechanisms behind DNA repair defects during OIS remain poorly understood. Here, we show that oncogenic Ras expression in human primary cells results in the downregulation of BRCA1 and 53BP1, two key factors in DNA DSB repair by homologous recombination and non-homologous end joining, respectively. As a consequence, Ras-induced senescent cells are hindered in their ability to recruit BRCA1 and 53BP1 to DNA damage sites. Whereas BRCA1 is downregulated at transcripts levels, 53BP1 loss is caused by activation of cathepsin L-mediated degradation of 53BP1 protein. Moreover, we discovered a marked downregulation of vitamin D receptor (VDR) during OIS, and a role for the vitamin D/VDR axis regulating the levels of these DNA repair factors during OIS. This study reveals a new functional relationship between the oncogene Ras, the vitamin D/VDR axis and the expression of DNA repair factors, in the context of OIS. The observed deficiencies in DNA repair factors in senescent cells could contribute to the genomic instability that allows senescence bypass and tumorigenesis.