Transduction of E2F-1 Tat fusion proteins into primary invasive ductal breast carcinoma cell lines and subsequent effects on gene transcription

Abstract

Scope and Method of Study: Breast carcinomas harbor specific genetic alterations that contribute to their immortalized state, including overexpression of telomerase and p53 tumor suppressor gene mutations. E2F-1 is a known transcriptional repressor of the catalytic subunit of telomerase, hTERT. In p53 mutated cells, E2F-1 possesses the ability to transactivate the p73 tumor suppressor pathway and p53-homologues involved in cell cycle arrest such as p21WAF1/CIP1. We investigated an alternative method of introducing the E2F-1 transcription factor via Tat-mediated protein transduction to effectively target and reversibly impact gene expression in breast cancer cell lines. Using affinity chromatography, the E2F-1 Tat fusion proteins were isolated and purified via FPLC and dialysis. Real-time RT-qPCR was utilized to assess the effects of E2F-1 Tat fusion protein treatment on gene expression of breast carcinomas. We investigated repression of hTERT and induction of p73 and p21WAF1/CIP1 genes in HCC1937 and HCC1599 primary invasive ductal breast cancer cell lines. Following cell treatment with E2F-1 Tat protein therapy, apoptotic activity was monitored via TUNEL assays.

Findings and Conclusions: The E2F-1 Tat fusion proteins effectively transduced greater than 95% of cancer cells. Protein therapy with E2F-1/TatHA repressed expression of hTERT by 3.5-fold in HCC1937 cells and by 4.0-fold in HCC1599 cells versus control proteins. Treatment of HCC1937 and HCC1599 carcinoma cells with E2F-1 Tat fusion proteins resulted in greater than 2-fold induction of p73 gene expression. Upregulation of the p53 responsive gene, p21WAF1/CIP1, was observed in HCC1937 and HCC1599 cells following treatment with E2F-1 Tat proteins. A 3.3-fold induction of p21WAF1/CIP1 was observed in HCC1937 cells versus a smaller induction of 1.4-fold in HCC1599 cells, which was attributed to the presence of a BRCA1 mutation in HCC1599 cells. Following 24 hours of treatment with E2F-1 Tat fusion proteins, apoptotic activity was detected in approximately 10% of breast cancer cells versus control proteins. Overall, the E2F-1 Tat protein therapy proved to be a moderately effective repressor of hTERT and activator of both p73 and p21WAF1/CIP1 resulting in detectable apoptotic activity. This suggests a potential application of E2F-1 Tat protein therapy in cancer therapeutics to modulate gene expression in breast carcinomas.