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4% of the cancer sam ples, whereas the remaining 105 samples displayed weak

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 4% of the cancer sam ples, whereas the remaining 105 samples displayed weak Empty 4% of the cancer sam ples, whereas the remaining 105 samples displayed weak

Mensagem  kai123 Seg Nov 09, 2015 10:14 pm

To identify robust DDR readouts which could be leveraged to recognize cells with functionally defective or impaired HRR pathway activity, オーダー 17-AAG the cellular response to PARPi AZD2281 was examined using an expanded panel of DDR readouts and cell lines with known mutations in HRR machinery, including BRCA2. BRCA1, and BRCA1lines. Cells were treated with PARPi temozolomide for 48 72 h and analyzed for the induction of 6 DDR proteins in all cells, in CyclinA2 and in CyclinA2 cell subsets within the same cell line. This choice of conditions was based on previous experiments in cell lines demonstrating that PARPi AZD2281 temozolomide selectively in duced DSBs in CyclinA2 cells while etoposide induces DSBs in both CyclinA2 and CyclinA2 cells. PARPi induced DDR responses from all cells, CyclinA2.<br><br> and CyclinA2 subsets were examined in the 17-DMAG 臨床試験 HRR mutant and proficient cell lines in the context of their HRR status. Elevated levels of PARPi induced p ATM, p BRCA1, p RPA2, and p DNA PKcs were detected in the BRCA2 cell line in CyclinA2 cells at both 48 h and 72 h. BRCA1vs BRCA1cell lines were not distinguished by readouts for PARPi TMZ induced p21, p ATM, p BRCA1, p RPA2, and p DNA PKcs in any population analyzed. Although some BRCA1samples demonstrated a trend of higher PARPi induced p21 levels vs. BRCA1samples, the difference was not significant. The most sensitive readout for HRR pathway function was p H2AX. Analysis of CyclinA2 cells consistently demonstrated highest induced p H2AX levels in the homozygous BRCA2 cell line, intermediate p H2AX levels in heterozygous BRCA1cell lines, and lowest p H2AX levels in BRCA1cell lines.<br><br> Im portantly, gating on CyclinA2 cells distinguished BRCA1from BRCA1lines. p H2AX 価格 A66 levels were significantly higher in BRCA1lines com pared to BRCA1lines in CyclinA2 cells but not in CyclinA2 cells or in the parent population at all time points and treatment conditions tested. These data suggest that analysis of DDR readouts spe cifically in CyclinA2 cells enables more accurate quan tification of HRR proficiency, possibly by reducing the confounding effects of different pro liferation rates across samples. To further examine CyclinA2 gating as a method to con trol for proliferation rate, the above PARPi induced DDR data from BRCA2.<br><br> BRCA1, and BRCA1 cell lines were evaluated for the percentage of CyclinA2 cells as a measure of proliferation for each cell line and used to compute correlations between proliferation and PARPi temozolomide induced DDR readouts measured in three populationsa CyclinA2 cells only, b CyclinA2 cells only or b all healthy cells. While DDR readouts measured in all live cells or CyclinA2 cells demonstrated higher correlations with proliferation, DDR readouts from CyclinA2 sub sets were less correlated with proliferation. These data confirm that analysis of DDR readouts specifically in CyclinA2 cells helps normalize for cell proliferation rate, enabling more accurate quantifi cation of HRR proficiency. In addition, examination of the percentages of live, healthy cells with or without PARPi treatment demonstrated no significant differences in apop tosis between BRCA1and BRCA1samples, which is consistent with published literature, and suggests that DDR pathway measurements may be more sensitive than apoptosis measurements in identifying altered DNA repair phenotypes.

kai123

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