Together, our gene expression profiling and pathway analysi

These agents do not directly induce DNA breaks, and arrest occurs without the need for Chk1 acti vation. However, supplier ARN-509 Chk1 stabilizes the stalled replication forks and, when inhibited, the replication forks collapse thus producing DNA double strand breaks, Hence, there is a significant difference in the outcome of Chk1 in hibition depending on the type of DNA damage that oc curs, in the latter case, new lethal events occur where no DNA damage existed previously. Consequently, we have found that Chk1 inhibition can induce a far more dra matic sensitization to antimetabolites that induce this rep lication arrest compared to other DNA damaging agents that activate Chk1 through the DNA damage induced checkpoint, Gemcitabine is a deoxynucleoside analogue that is me tabolized to a deoxynucleotide triphosphate, a precursor for incorporation into DNA, and to a deoxynucleotide diphosphate that irreversibly inhibits ribonucleotide re ductase.<br><br> As a consequence, low concentrations of gemci tabine rapidly deplete deoxyribonucleotide pools, inhibit DNA synthesis and induce a long S phase arrest. Here we focus on the combination of gemcitabine with the Chk1 inhibitor MK 8776, We report オーダー AUY922 the efficacy of this combination in cell lines from many different can cers. We also report that the time of addition of MK 8776 can significantly impact the response of tumor cells to gemcitabine both in vitro and in xenograft tumor models. The schedule dependence is critical because of the relatively short half life of MK 8776 in patients plasma, These results have important implications for the design of clinical trials of this combination.<br><br> Methods Materials Gemcitabine was obtained from Eli Lilly, Indianapolis, IN. MK 8776 was pro vided by Merck, Kenilworth, NJ and dissolved in dimethyl sulfoxide, Alisertib 価格 The majority of cell lines are part of the NCI60 panel and were obtained from the Develop mental Therapeutics Program, National Cancer Institute, Bethesda and maintained in RPMI1640 medium plus serum and antibiotics, Other cell lines were obtained from American Type Culture Collection, All lines were used within three months of thawing from frozen stocks. No further reconfirmation of their identity was performed. Cell analysis Cell cycle analysis was performed by flow cytometry as de scribed previously, For cell growth assays, cells were seeded at low density in 96 well plates and then incubated with drugs for various schedules usu ally for 24 h, Following treat ment, cells were washed and grown in fresh media for 6 7 days at 37 C.<br><br> Prior to attaining confluence, cells were washed, lysed, and stained with Hoechst 33258, as previ ously described, Fluorescence was read on a micro plate spectrofluorometer, Results are expressed as the concentration of drug that inhibited growth by 50%, Immunoblotting Cells were harvested and analyzed as previously detailed with the following antibodies, phosphoserine 345 Chk1, phosphoserine 296 Chk1, DNA PK and H2AX, Chk1, phospho 2056 DNA PK, and actin, Immunofluorescence Cells were cultured on glass coverslips, incubated with gemcitabine and or MK 8776, and fixed with 3% para formaldehyde, The cells were then washed 4 × 15 min in PBS T, Slides were then incubated with 200 ng ml anti Rad51 over night, washed in PBS T and incubated with Alexa 555 conjugated goat anti rabbit IgG at 1,1000 dilution for 1 h.