Figure 2

Functional analysis of Akt phosphorylation in human breast cancer cells after treatment with doxorubicin. (a) MCF7 cells were exposed to 1 μM doxorubicin (Doxo) for 24 hours, or irradiated (XRT) with X-rays (5 Gy) and then cultured for a further 24 hours in culture medium supplemented with 10% fetal bovine serum (FBS). MCF7 cells stimulated for 15 min with 10 nM type 1 insulin-like growth factor (IGF-1) were used as a positive control. Untreated (Untx) and the treated MCF7 cells were harvested, lysed and subjected to Western blot analyses with antibodies against Ser473-phosphorylated Akt (p-Akt) and total Akt. (b) MCF7 cells transiently transfected with a His-tagged Akt1 expression construct were left untreated or treated as in (a). Akt1 was immunoprecipitated by using an anti-His tag monoclonal antibody and then assayed for in vitro kinase activities for phosphorylation on Bad and glycogen synthase kinase-3 (GSK3). (c) Cytoplasmic and nuclear fractionations from untreated, doxorubicin-treated and γ-ray-irradiated MCF7 cells were separated as described in Materials and Methods. Equal amounts (40 μg) of cytoplasmic fraction (Cy) and nuclear fraction (Nu) from each sample were subjected to Western blot analysis to determine the distribution of Akt after treatment with doxorubicin or ionizing radiation. β-Actin and poly(ADP-ribose) polymerase (PARP) were used as markers of the cytoplasmic and nuclear fractions, respectively. (d) MCF7 or MDA468 cells were pre-exposed to 10 μM LY294002 for 2 hours before treatment with 1 μM doxorubicin for 24 hours. After treatment the cells were harvested, lysed and subjected to Western blot analyses with antibodies against p-Akt and total Akt.