Heterozygous germline BRCA2 mutations predispose to breast, ovarian, pancreatic and other types of cancer. The presence of a pathogenic mutation in patients or their family members warrants close surveillance or prophylactic surgery. Besides clearly pathogenic mutations, variants leading only to a single amino acid substitution are often identified. The influence of such variants on cancer risk is often unknown, making their presence a major clinical problem. When genetic methods are insufficient to classify these variants, functional assays with various cellular models are performed. We developed and applied a new syngeneic model of human cancer cells to test all variants of unknown significance in exon 18 identified by genetic testing of high-risk cancer patients in the Czech Republic, via introduction of constructs containing each of these variants into the wild-type allele of BRCA2-heterozygous DLD1 cells (BRCA2wt/Δex11). We found unaffected DNA repair function of BRCA2 in cell lines BRCA27997G>C/Δex11, BRCA28111C>T/Δex11, BRCA28149G>T/Δex11, BRCA28182G>A/Δex11, and BRCA28182G>T/Δex11, whereas the cell line BRCA28168A>G/Δex11 and the nonsense mutation carrying line BRCA28305G>T/Δex11 did affect protein function. Targeting the BRCA2 wild-type allele with a construct carrying the variant c.7988A> G resulted in incorporation exclusively into the already defective allele in all viable clones, strongly suggesting a detrimental phenotype. Our model thus offers a valuable tool for the functional evaluation of unclassified variants in the BRCA2 gene and provides a stable and distributable cellular resource for further research.

• MeSH
• anamnéza MeSH
• biologické modely * MeSH
• dospělí MeSH
• exony genetika   MeSH
• genetická predispozice k nemoci * MeSH
• genetické testování metody   MeSH
• hodnocení rizik metody   MeSH
• lidé středního věku MeSH
• lidé MeSH
• Markovovy řetězce MeSH
• mutace MeSH
• mutační analýza DNA metody   MeSH
• nádorové buněčné linie MeSH
• nádory diagnóza   genetika   MeSH
• protein BRCA2 genetika   MeSH
• senioři MeSH
• studie proveditelnosti MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH

The tetrahydroisoquinoline trabectedin is a marine compound with approved activity against human soft-tissue sarcoma. It exerts antiproliferative activity mainly by specific binding to the DNA and inducing DNA double-strand breaks (DSB). As homologous recombination repair (HRR)-deficient tumors are more susceptible to trabectedin, hyperthermia-mediated on-demand induction of HRR deficiency represents a novel and promising strategy to boost trabectedin treatment. For the first time, we demonstrate enhancement of trabectedin effectiveness in human sarcoma cell lines by heat and characterize cellular events and molecular mechanisms related to heat-induced effects. Hyperthermic temperatures (41.8 or 43°C) enhanced significantly trabectedin-related clonogenic cell death and G2/M cell cycle arrest followed by cell type-dependent induction of apoptosis or senescence. Heat combination increased accumulation of γH2AX foci as key marker of DSBs. Expression of BRCA2 protein, an integral protein of the HRR machinery, was significantly decreased by heat. Consequently, recruitment of downstream RAD51 to γH2AX-positive repair foci was almost abolished indicating relevant impairment of HRR by heat. Accordingly, enhancement of trabectedin effectiveness was significantly augmented in BRCA2-proficient cells by hyperthermia and alleviated in BRCA2 knockout or siRNA-transfected BRCA2 knockdown cells. In peripheral blood mononuclear cells isolated from sarcoma patients, increased numbers of nuclear γH2AX foci were detected after systemic treatment with trabectedin and hyperthermia of the tumor region. The findings establish BRCA2 degradation by heat as a key factor for a novel treatment strategy that allows targeted chemosensitization to trabectedin and other DNA damaging antitumor drugs by on-demand induction of HRR deficiency.

• MeSH
• antitumorózní látky alkylující farmakologie   MeSH
• apoptóza účinky léků   účinky záření   MeSH
• biologické modely MeSH
• chemorezistence účinky záření   MeSH
• dioxoly farmakologie   MeSH
• histony metabolismus   MeSH
• indukovaná hypertermie * MeSH
• kaspasy metabolismus   MeSH
• kontrolní body buněčného cyklu účinky léků   účinky záření   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• protein BRCA2 metabolismus   MeSH
• proteolýza účinky léků   účinky záření   MeSH
• rekombinační oprava DNA účinky léků   účinky záření   MeSH
• rekombinasa Rad51 metabolismus   MeSH
• sarkom metabolismus   patologie   terapie   MeSH
• tetrahydroisochinoliny farmakologie   MeSH
• transport proteinů MeSH
• vazba proteinů MeSH
• viabilita buněk účinky léků   účinky záření   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH