RESUMEN
BACKGROUND: Patients with peritoneal metastases (PMs) originating from colorectal carcinoma (CRC) are curatively treated by cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) with mitomycin C (MMC). We aim to improve patient selection for HIPEC by predicting MMC sensitivity. METHODS: The MMC sensitivity was determined for 12 CRC cell lines and correlated to mRNA expression of 37 genes related to the Fanconi anaemia (FA)-BRCA pathway, ATM-ATR pathway and enzymatic activation of MMC. Functionality of the FA-BRCA pathway in cell lines was assessed using a chromosomal breakage assay and western blot for key protein FANCD2. Bloom syndrome protein (BLM) was further analysed by staining for the corresponding protein with immunohistochemistry (IHC) on both CRC cell lines (n=12) and patient material (n=20). RESULTS: High sensitivity correlated with a low BLM (P=0.01) and BRCA2 (P=0.02) at mRNA expression level. However, FA-BRCA pathway functionality demonstrated no correlation to MMC sensitivity. In cell lines, weak intensity staining of BLM by IHC correlated to high sensitivity (P=0.04) to MMC. Low BLM protein expression was significantly associated with an improved survival in patients after CRS and HIPEC (P=0.04). CONCLUSIONS: Low BLM levels are associated with high MMC sensitivity and an improved survival after HIPEC.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/terapia , Hipertermia Inducida/métodos , Mitomicina/farmacología , Neoplasias Peritoneales/secundario , Neoplasias Peritoneales/terapia , Antibióticos Antineoplásicos/uso terapéutico , Células CACO-2 , Línea Celular Tumoral , Neoplasias Colorrectales/mortalidad , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HCT116 , Células HT29 , Humanos , Mitomicina/uso terapéutico , Neoplasias Peritoneales/mortalidad , RecQ Helicasas/metabolismo , Transducción de Señal/efectos de los fármacos , Análisis de Supervivencia , Investigación Biomédica TraslacionalRESUMEN
DNA interstrand crosslinks (ICLs) are toxic lesions that block the progression of replication and transcription. CtIP is a conserved DNA repair protein that facilitates DNA end resection in the double-strand break (DSB) repair pathway. Here we show that CtIP plays a critical role during initiation of ICL processing in replicating human cells that is distinct from its role in DSB repair. CtIP depletion sensitizes human cells to ICL inducing agents and significantly impairs the accumulation of DNA damage response proteins RPA, ATR, FANCD2, γH2AX, and phosphorylated ATM at sites of laser generated ICLs. In contrast, the appearance of γH2AX and phosphorylated ATM at sites of laser generated double strand breaks (DSBs) is CtIP-independent. We present a model in which CtIP functions early in ICL repair in a BRCA1- and FANCM-dependent manner prior to generation of DSB repair intermediates.
Asunto(s)
Proteínas Portadoras/genética , Reparación del ADN/genética , Replicación del ADN/genética , Proteínas Nucleares/genética , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Roturas del ADN de Doble Cadena/efectos de la radiación , Daño del ADN/efectos de la radiación , Endodesoxirribonucleasas , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Células HEK293 , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Terapia por Luz de Baja Intensidad , Redes y Vías MetabólicasRESUMEN
Cellular senescence is a stress-inducible, naturally irreversible cell cycle arrest, which is likely linked with ageing. Premature ageing of the skin is a prominent side effect of psoralen photoactivation, which is used for the treatment of various skin disorders. Previously, we have shown that DNA interstrand crosslink formation by photoactivated psoralens induces a senescent phenotype in primary fibroblasts that is mediated by Ataxia telangiectasia-mutated and Rad3-related (ATR) kinase. Checkpoint kinase 1 (Chk1) initiates cell cycle checkpoints, and FANCD2 is known to be involved in DNA damage-induced S-phase arrest and crosslink repair. In this study, we examined a role for Chk1 and FANCD2 as downstream effectors of ATR in senescence signalling. We demonstrate that Chk1 and FANCD2 are long-lastingly activated after psoralen photoactivation. Separate and combined reduction in Chk1 and FANCD2 expression by small interfering RNA (siRNA) preceding irradiation partly prevented the initiation of the senescence-like phenotype, whereas siRNA (Chk1 and FANCD2) transfection of senesced fibroblasts released cells from growth arrest. We observed that Chk1 and FANCD2 signal equally and additively for senescence induction, while Chk1 is predominantly responsible for maintaining persistent cell cycle arrest. In conclusion, Chk1 and FANCD2 function downstream of ATR in a non-redundant manner for the establishment and maintenance of psoralen photoactivation-induced senescence.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Envejecimiento de la Piel/fisiología , Proteínas de la Ataxia Telangiectasia Mutada , Células Cultivadas , Senescencia Celular/efectos de los fármacos , Senescencia Celular/fisiología , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Daño del ADN , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/antagonistas & inhibidores , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Furocumarinas/efectos adversos , Humanos , Terapia PUVA/efectos adversos , Proteínas Quinasas/genética , ARN Interferente Pequeño/genética , Transducción de Señal , Envejecimiento de la Piel/efectos de los fármacos , Envejecimiento de la Piel/genéticaRESUMEN
The Fanconi Anemia (FA) DNA damage response pathway is involved in the processing of DNA interstrand crosslinks (ICLs). As such, inhibition of the FA pathway could chemosensitize FA-competent tumor cells to commonly used ICL agents like cisplatin. Moreover, suppression of the FA pathway is synthetic lethal with deficiencies in several other DNA repair pathways, suggesting that FA pathway inhibitors could be used in targeted therapies against specific tumors. To identify such inhibitors, we designed a novel in vitro screening assay utilizing Xenopus egg extracts. Using the DNA-stimulated monoubiquitylation of Xenopus FANCD2 (xFANCD2-L) as readout, a chemical library screen identified DDN (2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone) as a novel and potent FA pathway inhibitor. DDN inhibited xFANCD2-L formation in a dose-dependent manner in both extracts and human cells without disruption of the upstream FA core complex. DDN also inhibited the characteristic subnuclear FANCD2 foci formation following DNA damage. Moreover, DDN displayed a greater synergistic effect with cisplatin in a FA-proficient cancer cell line compared to its FA-deficient isogenic counterpart, suggesting that DDN might be a good lead candidate as cisplatin chemosensitizer in both FA-deficient and FA-competent tumors. This system constitutes the first cell-free screening assay for identifying compounds that inhibit the FA pathway and provides a new biochemical platform for mapping the functions of its various components with specific chemical inhibitors.