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1.
Cell Rep ; 42(2): 112043, 2023 02 28.
Artículo en Inglés | MEDLINE | ID: mdl-36709426

RESUMEN

Cisplatin- and gemcitabine-based chemotherapeutics represent a mainstay of cancer therapy for most solid tumors; however, resistance limits their curative potential. Here, we identify RNA polymerase II-associated factor 1 (PAF1) as a common driver of cisplatin and gemcitabine resistance in human cancers (ovarian, lung, and pancreas). Mechanistically, cisplatin- and gemcitabine-resistant cells show enhanced DNA repair, which is inhibited by PAF1 silencing. We demonstrate an increased interaction of PAF1 with RAD52 in resistant cells. Targeting the PAF1 and RAD52 axis combined with cisplatin or gemcitabine strongly diminishes the survival potential of resistant cells. Overall, this study shows clinical evidence that the expression of PAF1 contributes to chemotherapy resistance and worse clinical outcome for lethal cancers.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Resistencia a Antineoplásicos , Neoplasias Pulmonares , Humanos , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Cisplatino/uso terapéutico , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Gemcitabina/uso terapéutico , Neoplasias Pulmonares/genética , Proteína Recombinante y Reparadora de ADN Rad52 , Factores de Transcripción
2.
PLoS One ; 16(3): e0248941, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33784323

RESUMEN

Synthetic lethality is a successful strategy employed to develop selective chemotherapeutics against cancer cells. Inactivation of RAD52 is synthetically lethal to homologous recombination (HR) deficient cancer cell lines. Replication protein A (RPA) recruits RAD52 to repair sites, and the formation of this protein-protein complex is critical for RAD52 activity. To discover small molecules that inhibit the RPA:RAD52 protein-protein interaction (PPI), we screened chemical libraries with our newly developed Fluorescence-based protein-protein Interaction Assay (FluorIA). Eleven compounds were identified, including FDA-approved drugs (quinacrine, mitoxantrone, and doxorubicin). The FluorIA was used to rank the compounds by their ability to inhibit the RPA:RAD52 PPI and showed mitoxantrone and doxorubicin to be the most effective. Initial studies using the three FDA-approved drugs showed selective killing of BRCA1-mutated breast cancer cells (HCC1937), BRCA2-mutated ovarian cancer cells (PE01), and BRCA1-mutated ovarian cancer cells (UWB1.289). It was noteworthy that selective killing was seen in cells known to be resistant to PARP inhibitors (HCC1937 and UWB1 SYr13). A cell-based double-strand break (DSB) repair assay indicated that mitoxantrone significantly suppressed RAD52-dependent single-strand annealing (SSA) and mitoxantrone treatment disrupted the RPA:RAD52 PPI in cells. Furthermore, mitoxantrone reduced radiation-induced foci-formation of RAD52 with no significant activity against RAD51 foci formation. The results indicate that the RPA:RAD52 PPI could be a therapeutic target for HR-deficient cancers. These data also suggest that RAD52 is one of the targets of mitoxantrone and related compounds.


Asunto(s)
Recombinación Homóloga , Neoplasias/metabolismo , Neoplasias/patología , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Proteína de Replicación A/metabolismo , Apoptosis/efectos de los fármacos , Proteína BRCA1/deficiencia , Proteína BRCA1/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Daño del ADN , Reparación del ADN/efectos de los fármacos , Doxorrubicina/farmacología , Fluorescencia , Ensayos Analíticos de Alto Rendimiento , Recombinación Homóloga/efectos de los fármacos , Humanos , Mitoxantrona/farmacología , Unión Proteica/efectos de los fármacos , Quinacrina/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología
3.
J Nucleic Acids ; 2019: 6357609, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30941207

RESUMEN

Gemcitabine (2', 2'-difluorodeoxycytidine; dFdC) is a deoxycytidine analog and is used primarily against pancreatic cancer. The cytotoxicity of gemcitabine is due to the inhibition of DNA replication. However, a mechanism of removal of the incorporated dFdC is largely unknown. In this report, we discovered that nucleotide excision repair protein XPF-ERCC1 participates in the repair of gemcitabine-induced DNA damage and inactivation of XPF sensitizes cells to gemcitabine. Further analysis identified that XPF-ERCC1 functions together with apurinic/apyrimidinic endonuclease (APE) in the repair of gemcitabine-induced DNA damage. Our results demonstrate the importance of the evaluation of DNA repair activities in gemcitabine treatment.

4.
J Mol Biol ; 413(2): 337-46, 2011 Oct 21.
Artículo en Inglés | MEDLINE | ID: mdl-21875596

RESUMEN

Nucleotide excision repair (NER) is a very important defense system against various types of DNA damage, and it is necessary for maintaining genomic stability. The molecular mechanism of NER has been studied in considerable detail, and it has been shown that proper protein-protein interactions among NER factors are critical for efficient repair. A structure-specific endonuclease, XPF-ERCC1, which makes the 5' incision in NER, was shown to interact with a single-stranded DNA binding protein, RPA. However, the biological significance of this interaction was not studied in detail. We used the yeast two-hybrid assay to determine that XPF interacts with the p70 subunit of RPA. To further examine the role of this XPF-p70 interaction, we isolated a p70-interaction-deficient mutant form of XPF that contains a single amino acid substitution in the N-terminus of XPF by the reverse yeast two-hybrid assay using randomly mutagenized XPF. The biochemical properties of this RPA-interaction-deficient mutant XPF-ERCC1 are very similar to those of wild-type XPF-ERCC1 in vitro. Interestingly, expression of this mutated form of XPF in the XPF-deficient Chinese hamster ovary cell line, UV41, only partially restores NER activity and UV resistance in vivo compared to wild-type XPF. We discovered that the RPA-interaction-deficient XPF is not localized in nuclei and the mislocalization of XPF-ERCC1 prevents the complex from functioning in NER.


Asunto(s)
Daño del ADN/genética , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Proteína de Replicación A/metabolismo , Animales , Fraccionamiento Celular , Células Cultivadas , Cricetinae , Cricetulus , Proteínas de Unión al ADN/genética , Humanos , Mutagénesis , Proteína de Replicación A/genética , Técnicas del Sistema de Dos Híbridos
5.
Biochemistry ; 48(49): 11817-24, 2009 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-19908865

RESUMEN

Human DNA polymerase N (PolN) is an A-family nuclear DNA polymerase whose function is unknown. This study examines the possible role of PolN in DNA repair in human cells treated with PolN-targeted siRNA. HeLa cells with siRNA-mediated knockdown of PolN were more sensitive than control cells to DNA cross-linking agent mitomycin C (MMC) but were not hypersensitive to UV irradiation. The MMC hypersensitivity of PolN knockdown cells was rescued by the overexpression of DNA polymerase-proficient PolN but not by DNA polymerase-deficient PolN. Furthermore, in vitro experiments showed that purified PolN conducts low-efficiency nonmutagenic bypass of a psoralen DNA interstrand cross-link (ICL), whose structure resembles an intermediate in the proposed pathway of ICL repair. These results suggest that PolN might play a role in translesion DNA synthesis during ICL repair in human cells.


Asunto(s)
Daño del ADN , Enzimas Reparadoras del ADN/fisiología , Reparación del ADN , ADN Polimerasa Dirigida por ADN/fisiología , Secuencia de Bases , Núcleo Celular/efectos de los fármacos , Núcleo Celular/enzimología , Núcleo Celular/genética , Reactivos de Enlaces Cruzados/farmacología , Daño del ADN/genética , ADN Helicasas/química , ADN Helicasas/fisiología , Reparación del ADN/efectos de los fármacos , Reparación del ADN/genética , Enzimas Reparadoras del ADN/química , Proteínas de Unión al ADN/genética , ADN Polimerasa Dirigida por ADN/biosíntesis , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/deficiencia , ADN Polimerasa Dirigida por ADN/genética , Células HeLa , Humanos , Mitomicina/farmacología , Datos de Secuencia Molecular , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , ARN Interferente Pequeño/farmacología
6.
J Biol Chem ; 283(3): 1275-1281, 2008 Jan 18.
Artículo en Inglés | MEDLINE | ID: mdl-18006494

RESUMEN

The processing of stalled forks caused by DNA interstrand cross-links (ICLs) has been proposed to be an important step in initiating mammalian ICL repair. To investigate a role of the XPF-ERCC1 complex in this process, we designed a model substrate DNA with a single psoralen ICL at a three-way junction (Y-shaped DNA), which mimics a stalled fork structure. We found that the XPF-ERCC1 complex makes an incision 5' to a psoralen lesion on Y-shaped DNA in a damage-dependent manner. Furthermore, the XPF-ERCC1 complex generates an ICL-specific incision on the 3'-side of an ICL. The ICL-specific 3'-incision, along with the 5'-incision, on the cross-linked Y-shaped DNA resulted in the separation of the two cross-linked strands (the unhooking of the ICL) and the induction of a double strand break near the cross-linked site. These results implicate the XPF-ERCC1 complex in initiating ICL repair by unhooking the ICL, which simultaneously induces a double strand break at a stalled fork.


Asunto(s)
Reactivos de Enlaces Cruzados/farmacología , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Endonucleasas/metabolismo , Ficusina/farmacología , Animales , Células CHO , Cricetinae , Cricetulus , ADN/química , Roturas del ADN de Doble Cadena/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/aislamiento & purificación , Endonucleasas/aislamiento & purificación , Humanos , Modelos Biológicos , Conformación de Ácido Nucleico/efectos de los fármacos , Estructura Terciaria de Proteína , Especificidad por Sustrato/efectos de los fármacos
7.
Cancer Res ; 62(4): 1171-7, 2002 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-11861400

RESUMEN

Using alternative reading frames, the human ARF-INK4a locus encodes two unrelated proteins that both function in tumor suppression. p16(INK4a) maintains the retinoblastoma protein in its growth-suppressive state through inhibition of cyclin D-dependent kinase activity, whereas ARF binds with MDM2 and stabilizes p53. The majority of the activity of ARF to date is ascribed to its ability to activate p53, resulting in a G(1) cell cycle arrest or apoptosis. We show here that ARF colocalizes with DNA replication protein A (RPA32) and that overexpression of ARF reduces the rate of DNA synthesis resulting in accumulation of an S-phase cell population. Impediment of DNA synthesis by ARF can occur and becomes more evident in the absence of p53. Hence, the biological consequence of ARF induction varies dependent on cellular p53 status, inducing predominantly a G(1) arrest or apoptosis in p53-positive cells or causing S-phase retardation when p53 function is comprised.


Asunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina/fisiología , Proteína p53 Supresora de Tumor/fisiología , Proteínas Supresoras de Tumor/fisiología , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , División Celular/fisiología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , ADN de Neoplasias/biosíntesis , Proteínas de Unión al ADN/metabolismo , Humanos , Osteosarcoma/genética , Osteosarcoma/metabolismo , Osteosarcoma/patología , Proteína de Replicación A , Fase S/fisiología , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/biosíntesis , Proteínas Supresoras de Tumor/genética
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