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    2019-09-23


    To search for additional HR gene mutations, DNA from HR-deficient cases that lacked deleterious BRCA1 or BRCA2 mutations was isolated from FFPE slides by laser capture microdissection and assayed for muta-tions in genes involved in DNA repair (Table S1) by BROCA-HR DNA se-quencing as previously described [22]. Mutations were considered deleterious if they were truncating or were missense mutations with evidence of functional compromise. Sanger sequencing was used to confirm deleterious mutations.
    2.3. Methylation analysis
    As previously reported [5,23], DNA was bisulfite converted (EZ Methylation Direct kit, Zymo Research, Irvine, CA) and evaluated with methylation sensitive PCR for BRCA1 and RAD51C. In vitro methylated DNA and known unmethylated DNA (Zymo Research #D5014) were used as positive and negative controls, respectively, for bisulfite conver-sion and validation of amplicon size. Water (H2O) was substituted for DNA to rule out cross-contamination of samples.
    The identity of all cell lines was confirmed by short tandem repeat analysis in the Mayo Clinic Cytogenetics Core. To assess the impact of 53BP1 on PARP inhibitor sensitivity, BRCA1-mutant COV362 (−)-Apomorphine ([24], kind gift from Robert van Waardenburg, University of Alabama Birmingham) were utilized. To provide positive and negative controls for IHC, the following cell lines were utilized: OVCAR8 (kind gift from Dominic Scudiero, NCI Frederick) as well as OVCAR8 cells transiently transfected with siRNA targeting the RAD51, KU80, KU70, XRCC4, or 53BP1 mRNAs; parental and PARP1−/− HCT116 cells ([25], a kind gift from Eric Hendrickson, University of Minnesota); or parental MO59J cells (lacking DNA-PKCS) and MO59K cells expressing DNA-PKCS ([26], kind gift from Jann Sarkaria, Mayo Clinic, Rochester, MN). HR-proficient OV90 ([24], kind gift from Robert van Waardenburg, Univer-sity of Alabama Birmingham) and HR-deficient, BRCA2-mutant PE01 cells [11,23] served as positive and negative controls, respectively, for the plasmid reactivation assays.
    To generate 53BP1 knockout cells, the oligonucleotides (5′-TTGATC TCACTTGTGATTCG-3′) guiding to human 53BP1 2023–2042 (accession number: AF078776.1) were synthesized, annealed, and cloned into the BsmBI site of lentiCRISPR-v2 plasmid (Addgene, Cambridge, MA). 53BP1 targeting virus and empty vector were packaged by transfecting HEK293T cells with the packaging vector psPAX3, envelope vector pMD2.G, and lentiCRISPR-v2-53BP1 2023–2042 or empty vector using Lipofectamine 2000 (ThermoFisher, Waltham, MA). Two days after viral transduction, COV362 cells were selected with 3 μg/mL puromycin. Pooled cells that repopulated the cultures after puromycin selection were utilized for the assays described below. 53BP1 knockout was veri-fied by immunoblotting.
    The following siRNA constructs were purchased from Dharmacon (Lafayette, CO): RAD51 (3M-003530-04, SMARTpool Human), KU80 (J-010491-07, ON-TARGETplus siRNA), and XRCC4 (5′-AUAUGUUGG UGAACUGAGATT-3′) [27]; or from Ambion (Austin, TX): KU70 (s5457, 5′-GACAUAUCCUUGUUCUACA-3′), 53BP1 (s14313, 5′-GAAGGACGGAG UACUAAUA-3′), and Negative Control No. 1 (cat. No. 4390884). Oligo-nucleotides were resuspended according to the suppliers' instructions. Cells suspended in medium A were subjected to electroporation using a BTX830 square wave electroporator (Harvard Apparatus, Holliston, MA, USA) delivering two 10-ms pulses at 280 V. After a 48-h incubation, 90% of the cells were fixed and embedded to serve as immunohisto-chemistry (IHC) controls. Whole cell lysates were prepared from the re-maining cells to confirm knockdown of siRNA targets by immunoblotting.
    2.5. Clonogenic assays
    Aliquots containing 500 COV362 EV or COV362 53BP1−/− cells in medium A containing 3 μg/mL puromycin, were plated in 35 mm plates, allowed to adhere for 14–18 h, and treated with varying concentrations of veliparib (added from 1000× stocks in DMSO). Cells were incubated for 10 days to allow colony formation. PE01 and OV90 cells were assayed similarly except that 400 and 600 cells, respectively, were plated and cells were grown in their usual growth media. Survival was calculated as the ratio of colonies per well treated with drug compared with diluent.