Because BRCA1 also has ubiquitin ligase activity toward tubulin, ERa and phosphorylated Tipifarnib myeloid Akt, and because we observed a pronounced increase in intracellular EGFR upon BRCA1 suppression, we tested whether BRCA1 suppression affects EGFR stability after blockade of protein biosynthesis with cycloheximide. Interestingly, BRCA1 suppression increased the half life of the EGFR protein from less than 30 minutes to over 75 minutes. Thus, there appear to be at least two mechanisms that result in an increase in EGFR protein levels upon BRCA1 suppression, transcriptional regula tion and protein stabilization. ALDH1 positive cells show an increase in EGFR expression Using immunofluorescence imaging, we noted heteroge neity with regard to EGFR expression in both control MECs as well as in MECs after BRCA1 inhibition.

An increased expression of EGFR in basal cells was previously observed in murine MECs and hMECs, and a drift toward high EGFR expression was seen in cell line models of basaloid breast cancer, which led us to examine whether Inhibitors,Modulators,Libraries the EGFR levels differed between stem and non stem cells as defined by the expression of ALDH1. We found that mean numbers of EGFR were higher in the ALDH1 positive fractions of MECs than in the ALDH1 negative Inhibitors,Modulators,Libraries fractions. Con sistently, ALDH1 positive MECs showed an increased binding of Rh labeled EGF when compared to the ALDH1 negative fraction. Given these differences in cell sur face expressed EGFR, we compared the kinetics of EGF binding and internalization between ALDH1 positive and ALDH1 negative MECs.

For the binding assay, cells were incubated with increasing concentrations Inhibitors,Modulators,Libraries of Rh labeled EGF, and binding was analyzed using flow cyto metry. Inhibitors,Modulators,Libraries Scatchard analysis of Rh EGF binding at 4 C showed that both the ALDH1 positive and ALDH1 negative population bound EGF with similar affinity. For binding and internalization, cells were preincubated with Rh EGF at 4 C to allow for binding, followed by removal of unbound Rh EGF incubated for the indicated times and concentrations with Rh labeled EGF at 37 C, and then washed with either PBS or an acidified buffer as described previously, followed by ALDH1 stain ing. While the Inhibitors,Modulators,Libraries PBS wash removes only unbound Rh EGF, the acidified buffer removes both receptor bound and receptor unbound EGF, that is, fluorescence after the acidic wash is representative of internalized EGF.

We found that EGF binding was biphasic, both in ALDH1 positive and ALDH1 negative cells, with an initial saturation of EGF binding sites after Ganetespib OSA 5 minutes, followed by a second, slower phase of binding and inter nalization. Internalization was complete after 30 minutes at 37 C. In summary, binding and internalization kinetics were similar in ALDH1 positive and ALDH1 negative MECs, while the total number of circulating EGF receptors was increased in the ALDH1 positive fraction.