Hit rates did not systematically differ between and or with [17, 19]. for treatment response towards ATR- or CHK1-inhibitors that are currently tested in clinical trials. and deficiency [16C19] as well as and oncogenic overexpression [20, 21]. The aim of this study was to identify synthetically lethal interactions between and certain DNA-repair genes, applying a siRNA library of all major DNA-repair genes in a well-characterized genetic knock-in model of DLD1 colorectal malignancy (CRC) cells [14, 22, 23] harboring the hypomorphic were further characterized. RESULTS siRNA library screening to identify synthetic lethal interactions between ATR and DNA-repair genes in DLD1 cells To identify potential synthetically lethal interactions between and certain DNA-repair genes, we compared the effects of siRNA-mediated knockdown of single genes on the proliferation rate of DLD1 cancer cells harboring the knock-in Seckel mutation [23], using a focused siRNA library directed against 288 DNA repair genes each targeted by three different siRNAs. Prior to screening, deficiency of cells was verified on the protein level by demonstration of ATR protein suppression below the detection limit of our assay (Figure ?(Figure1A)1A) and functionally through confirmation of hypersensitivity towards the DNA interstrand-crosslinking (ICL) agent mitomycin C (MMC) (Figure ?(Figure1B)1B) [24, 25]. The experimental screening design is schematically outlined in Figure ?Figure1C1C and Figure ?Figure1D.1D. In short, parental and cells were transfected simultaneously using a previously established siRNA library. At 120 h post transfection, proliferation differences between genotype-dependent and genotype-independent proliferation inhibition, respectively, according to the criteria described in Rabbit polyclonal to ZFHX3 the Material&Methods section. Taken together, each candidate gene was validated based on the average growth inhibition ratio of four independent experiments. The top six gene targets displaying selective (9-fold growth inhibition ratio with an average relative survival of 5% of cells) and therefore chosen for further in-depth characterization. Open in a separate window Figure 1 Experimental design and screening process of the siRNA library screeningA. ATR protein synthesis was assessed in parental and cells by immunoblotting. -ACTIN served as loading control. B. MMC sensitivity of parental and genotype-dependent DNA-repair gene targets cells. The mean growth inhibition ratio and SEM were determined from four individual growth inhibition ratio values that each represent triplicates from three different oligonucleotides targeting one particular gene, as described in Material&Methods. cells) (Table ?(Table2).2). Notably, siRNA-mediated knockdown of and caused a virtually complete loss of proliferation, extending the known essential functions of these genes also to DLD1 colorectal cancer cells [26, 27]. Table 2 Identified genotype-independent DNA-repair gene targets cells. The mean growth inhibition ratio and SEM were determined from four individual growth inhibition ratio values that each represent triplicates from three different oligonucleotides Carboxypeptidase G2 (CPG2) Inhibitor targeting one particular Carboxypeptidase G2 (CPG2) Inhibitor gene. **The average relative survival of parental and ATRs/s cells, respectively, was calculated by the mean of four individual growth inhibition values for each cell line from three different oligonucleotides targeting one particular gene, as described in Material&Methods. Validation of synthetic lethality of with in cells To validate the synthetic lethal relationship of with cells. The detrimental effects of knockdown selectively on cells were time-dependent, as shown by a proliferation inhibition of at least 50%, starting at 96 h and further peaking at 120 h post transfection, as compared to mock- and untreated cells (Figure ?(Figure2A).2A). Efficient siRNA-mediated knockdown at 96 h post transfection was confirmed on the protein level in parental and cells (Figure ?(Figure2B).2B). Similarly, the effects of knockdown on cells were dose-dependent, as shown at 120 h post transfection by a proliferation inhibition of at least 70% at concentrations ranging from 2.5 nM to 40 nM (Figure ?(Figure2C).2C). Expectedly, cells upon treatment at higher and likely toxic siRNA concentrations starting from 80 nM. Importantly, clonally selected heterozygous cells also remained unaffected by knockdown in DLD1 cancer cellsA. Proliferation inhibition over time of siRNA-mediated knockdown (10 nM) was assessed in cells. B. Efficient siRNA-mediated POLD1 protein depletion was confirmed at 96 h after treatment in parental and cells. siGAL served as transfection control, -ACTIN as loading control. C. concentration-dependent proliferation inhibition was assessed at 120 h after treatment in parental and cells. D+E. Effects on proliferation of ATR- and CHK1-inhibitors (D) or common chemotherapeutics (E), respectively, were assessed at 120 h after treatment in control-, mock- or knockdown for each line (Figure ?(Figure3A),3A), the cells were treated with NU6027, VE-822 or UCN-01, respectively. As compared to control cells, POLD1 depletion sensitized RKO cells towards NU6027 (IC50 ratio 3) and.Consistent with the increased sub-G1 fraction of genotype-dependent effects of POLD1 depletion on cell cycle profile and apoptosisCell cycle and apoptosis analyses were performed upon siRNA-mediated knockdown at 10 nM in parental and cells. currently tested in clinical trials. and deficiency [16C19] as well as and oncogenic overexpression [20, 21]. The aim of this study was to identify synthetically lethal Carboxypeptidase G2 (CPG2) Inhibitor interactions between and certain DNA-repair genes, applying a siRNA library of all major DNA-repair genes in a well-characterized genetic knock-in model of DLD1 colorectal cancer (CRC) cells [14, 22, 23] harboring the hypomorphic were further characterized. RESULTS siRNA library screening to identify synthetic lethal interactions between ATR and DNA-repair genes in DLD1 cells To identify potential synthetically lethal interactions between and certain DNA-repair genes, we compared the effects of siRNA-mediated knockdown of single genes on the proliferation rate of DLD1 cancer cells harboring the knock-in Seckel mutation [23], using a focused siRNA library directed against 288 DNA repair genes each targeted by three different siRNAs. Prior to screening, deficiency of cells was verified on the protein level by demonstration of ATR protein suppression below the detection limit of our assay (Figure ?(Figure1A)1A) and functionally through confirmation of hypersensitivity towards the DNA interstrand-crosslinking (ICL) agent mitomycin C (MMC) (Figure ?(Figure1B)1B) [24, 25]. The experimental screening design is schematically outlined in Figure ?Figure1C1C and Figure ?Figure1D.1D. In short, parental and cells were transfected simultaneously using a previously established siRNA library. At 120 h post transfection, proliferation differences between genotype-dependent and genotype-independent proliferation inhibition, respectively, according to the criteria described in the Material&Methods section. Taken together, each candidate gene was validated based on the average growth inhibition ratio of four independent experiments. The top six gene targets displaying selective (9-fold growth inhibition ratio with an average relative survival of 5% of cells) and therefore chosen for further in-depth characterization. Open in a separate window Figure 1 Experimental design and screening process of the siRNA library screeningA. ATR protein synthesis was assessed in parental and cells by immunoblotting. -ACTIN served as loading control. B. MMC sensitivity of parental and genotype-dependent DNA-repair gene targets cells. The mean growth inhibition ratio and SEM were determined from four individual growth inhibition ratio values that each represent triplicates from three different oligonucleotides targeting one particular gene, as described in Material&Methods. cells) (Table ?(Table2).2). Notably, siRNA-mediated knockdown of and caused a virtually complete loss of proliferation, extending the known essential functions of these genes also to DLD1 colorectal cancer cells [26, 27]. Table 2 Identified genotype-independent DNA-repair gene targets cells. The mean growth inhibition ratio and SEM were determined from four individual growth inhibition ratio values that each represent triplicates from three different oligonucleotides targeting one particular gene. **The average relative survival of parental and ATRs/s cells, respectively, was calculated by the mean of four individual growth inhibition values for each cell line from three different oligonucleotides targeting one particular gene, as described in Material&Methods. Validation of synthetic lethality of with in cells To validate the synthetic lethal relationship of with cells. The detrimental effects of knockdown selectively on cells were time-dependent, as shown by a proliferation inhibition of at least 50%, starting at 96 h and further peaking at 120 h post transfection, as compared to mock- and untreated cells (Figure ?(Figure2A).2A). Efficient siRNA-mediated knockdown at 96 h post transfection was confirmed on the protein level in parental and cells (Figure ?(Figure2B).2B). Similarly, the effects of knockdown on cells were dose-dependent, as shown at 120 h post transfection by a proliferation inhibition of at least 70% at concentrations ranging from 2.5 nM to 40 nM (Figure ?(Figure2C).2C). Expectedly, cells upon treatment at higher and likely toxic siRNA concentrations starting from 80 nM. Importantly, clonally selected heterozygous cells also remained unaffected by knockdown in DLD1 cancer cellsA. Proliferation inhibition over time of siRNA-mediated knockdown (10 nM) was assessed in cells. B. Efficient siRNA-mediated POLD1 protein depletion was confirmed at 96 h after treatment in parental and cells. siGAL served as transfection control, -ACTIN as loading control. C. concentration-dependent proliferation inhibition was assessed at 120 h after treatment in parental and cells. D+E. Effects on proliferation of ATR- and CHK1-inhibitors (D) or common chemotherapeutics (E), respectively, were assessed at 120 h after treatment in control-, mock- or knockdown for each line (Figure ?(Figure3A),3A), the cells were treated with NU6027, VE-822 or UCN-01, respectively. As compared to control cells, POLD1 depletion sensitized RKO cells towards NU6027 (IC50 ratio 3) and VE-822 (IC50 ratio 2) (Figure ?(Figure3B,3B, upper panel), SW480 cells towards NU6027 (IC50 ratio 2) and UCN-01 (IC50 ratio.