Our results provide a rationale for further development of new generations of analog drugs with improved specificity and decreased toxicity, as well as pre clinical testing in appropriate animal models. Further evaluation of these combinations in cisplatin resistant tumors may lead to the selleck chemical development of efficient cancer treatments. Materials and methods Cell lines and culture conditions HeLa, U2OS, TOV 21 G and GFPu 1 cells were purchased from the American Type Culture Collections. 2008 and FANCF corrected 2008 FANCF ovarian cancer cells, TOV 21 G and FANCF corrected TOV 21 G FANCF ovarian cancer cells were described previously. FANCD2 deficient fibroblast line, PD20 corrected with wild type FANCD2 and enhanced green fluorescent protein FANCD2 were described previously.
U2OS DR GFP cells were a gift from Drs. Maria Jasin and Koji Nakanishi. Cell lines were grown in DMEM supplemented with 10% fetal calf serum. Gamma irradiation was delivered using a JL Shepherd Mark Inhibitors,Modulators,Libraries I Inhibitors,Modulators,Libraries Cesium Irradiator. The present research has been approved by the Institutional Review Board Committee at the Fred Hutchinson Cancer Research Center. Chemicals The chemical libraries, Commercial Diversity Set 1, Chembridge DiverSet Library and NINDS II library were used to identify inhibitors of the FA pathway. For subsequent studies, chemicals were purchased from Biomol 13 HODE EMD biochemicals 3, H 9, K 252c, MG132, nifedipine, propidium iodide, puromycin, roscovitine, SB218078, spermine NONOate, TPEN, trichos tatin A, wortmannin Cayman Chemical, Chembridge Corporation, Fisher, Millen ium Pharmaceutical, MP, Sigma, Tocris, VWR.
Screen for small molecules that inhibit the FA pathway The PD20 EGFP FANCD2 clone 7 was used in the screen. The cell based screening of ICCB bioactives and Commercial Diversity Set 1 was done at the Institute for Chemistry and Cell Biology and a partial result was previously reported. The cell based screening of Chembridge DiverSet Inhibitors,Modulators,Libraries Library and NINDS II Inhibitors,Modulators,Libraries library was done at Fred Hutchinson Cancer Research Center. For this screening, duplicate 96 well plates were seeded with PD20 EGFP FANCD2 clone 7 cells. Chemical compounds from the library were added, five compounds per well, at a single concentration of 7. 5 umol/L. After a 12 hour incubation, cells were irradiated and fixed for EGFP microscopy 12 hours later.
Photomicrographs Inhibitors,Modulators,Libraries were obtained for each well and wells with significant reduction in percentage of EGFP FANCD2 foci positive cells were identified by visual inspection. The five compounds of each well identified with reduced EGFP FANCD2 foci were then individually tested. Immunofluorescence microscopy was performed as described previously. Antibodies against BRCA1, H2AX, FANCD2 and RAD51 were used. Species specific fluores cein isothiocyanate or Cy3 conjugated second ary antibodies diluted 17-DMAG clinical trial in blocking buffer were incubated for 1 hour at room temperature.
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