In contrast, B RafV600E did not affect sensitivity to wards conventional chemotherapeutic compounds such as selleck chemical mitomycin C, oxaliplatin, paclitaxel, etoposide, or 5 fluorouracil Inhibitors,Modulators,Libraries in our model. Also, no sensitivity was ob served towards the therapeutic EGFR antibody cetuxi mab, although the EGF receptor was similarly expressed in all RKO derived cell clones. Dissecting the effect of se lective B Raf inhibition, neither vemurafenib nor RAF265 induced proliferation differences among wild type and mutant clones. In contrast, dabrafenib exhibited an obvious BRAF status dependent inhibitory effect on cell prolifera tion. Together with the highly robust molecular effects of dabrafenib on phospho Erk and phospho Mek induction, this possibly indicates a high specificity of the compound.
On the other hand, off target effects could also have con tributed, since all small molecule kinase inhibitors are multi kinase inhibitors to some extent. Kinomic ap proaches to obtain detailed inhibition profiles appear as a promising tool for future studies to reveal the key differen tial modes of action between Inhibitors,Modulators,Libraries the utilized Inhibitors,Modulators,Libraries compounds. Methods Tissue culture Cell culture reagents and antibiotics were purchased from PAA Laboratories. HEK293 and RKO were purchased from ATCC and validated by DNA profil ing at the German Biological Resource Center. Additional RKO clones har boring deleted BRAF alleles were kindly provided by B. Vogelstein HEK293, RKO, and derivative cell clones were maintained at 37 C in a water saturated atmosphere containing 5% CO2 in high glucose DMEM supplemented with 100 units mL penicillin, 100 mg L streptomycin, and 10% FBS if not indicated differently.
Somatic cell gene targeting For somatic cell gene targeting, Inhibitors,Modulators,Libraries the AAV Helper Free System was used. The chromosomally stable target cell line RKO shows slight aneuploidy leading to triplication at the BRAF locus. In order to target two BRAF alleles serially, two AAV targeting constructs were cloned containing either hygromycin or neomycin resist ance. The resistance cassette was flanked by sequences homologous to regions flanking BRAF exon 15. These homology arms were amplified by PCR using primer LHA FW NotI respectively. Preparation of AAV particles was done according to Kohli et al. After a limiting dilution of RKO cells, the single clone RKO E1 was infected with AAV containing the hyg re sistance gene and seeded in a limiting dilution.
After three weeks of incubation with 2. 0 g L hygromycin B, single colonies were screened with two primer pairs LHA upstream FW From a clone of the desired BRAF genotype the knockout cell line RBOW was established. RBOW cells were infected with AAV particles Inhibitors,Modulators,Libraries mediating neomy cin resistance, diluted selleck catalog and incubated with 4. 5 g L G418 sulphate. For PCR screening of the single colonies, LHA upstream FW was combined with Western blotting Western blot samples were prepared with phospho protein lysis buffer.