824

Mutations in the p53 tumor suppressor gene are present in over 60% of breast carcinomas. In cancer cells harboring a p53 mutation, overexpression of a p53 homologue (the p73 tumor suppressor) induces apoptosis. Upregulation of p73 and subsequent cell death have been achieved via transfection of the E2F-1 transcription factor gene. Here we report an alternative, reversible methodology to induce p73 expression in breast carcinoma cells utilizing protein transduction with an E2F-1 Tat fusion protein (TFP) to target and kill breast cancer cells. We have produced recombinant E2F-1/TatHA (HA= hemagglutinin) fusion proteins in BLR(DE3)pLysS cells followed by purification with FPLC using affinity chromatography. Transduction efficiency of the recombinant E2F-1 TFP was verified via immunocytochemistry. Greater than 95% of treated cells demonstrated high levels of E2F-1/TatHA accumulation within the perinuclear, nuclear and nucleolar cell compartments. Biological activity of the E2F-1 TFP was determined by monitoring induction of p73 expression using real-time qRT-PCR, and apoptosis was assessed via brightfield cell morphology and TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling) assay. Two p53 deficient breast cancer cell lines, CRL-2331 and CRL-2336, were exposed to 2 μM E2F-1 TFP or control TatHA protein for 24 hours in the presence and absence of 200 μM chloroquine. Total RNA was isolated from cells, reverse transcribed, and cDNA pools were analyzed via real-time qPCR using p73 and β-actin primers. The 2-ΔΔCTmethod was used to compare relative p73 expression between treatment groups. In both cancer cell lines, greater than 2-fold induction of p73 was observed in E2F-1 TFP treated cells as compared to TatHA treated controls. Following E2F-1 TFP treatment for 24 hours, significant apoptotic activity was evident in the breast cancer cells. The greatest overall apoptotic effect was observed in E2F-1 TFP treated cells in the presence of the lysosmotropic agent chloroquine. In conclusion, the E2F-1/TatHA protein effectively transduced into CRL-2336 and CRL-2331 cells, activated transcription of p73, and induced apoptosis. This study supports the hypothesis that transduced E2F-1/TatHA fusion protein activates the p73 tumor suppressor pathway in p53-mutated breast carcinoma cell lines.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]