Abstract
BackgroundTelomerase expression is detectable in 81–95% of breast carcinomas and may serve as a therapeutic target. The objective of this study was to investigate repression of telomerase activity in primary ductal breast cancer cells through transcriptional regulation of the catalytic subunit hTERT. We hypothesized that inhibition of telomerase expression could be achieved via Tat mediated protein transduction of the repressor protein E2F-1.MethodsProtein purification techniques were refined to yield biologically active Tat fusion proteins (TFPs) capable of transducing the breast cancer cell lines HCC1937 and HCC1599. Cell lines were treated with wildtype E2F-1 (E2F-1/TatHA), mutant E2F-1 (E132/TatHA) and a control Tat peptide (TatHA) for 24 hours. Total RNA was isolated from treated cells, reverse transcribed and fold changes in gene expression for hTERT determined via real-time RT-qPCR.ResultsSignificant repression of the catalytic subunit of telomerase (hTERT) was present in both HCC1937 and HCC1599 cells following treatment with E2F-1/TatHA. In HCC1937 cells, hTERT was repressed 3.5-fold by E2F-1/TatHA in comparison to E132/TatHA (p < 0.0012) and the TatHA peptide controls (p < 0.0024). In HCC1599 cells, hTERT was also repressed with E2F-1/TatHA treatment by 4.0-fold when compared to the E132/TatHA control (p < 0.0001). A slightly lower hTERT repression of 3.3-fold was observed with E2F-1/TatHA in the HCC1599 cells when compared to the TatHA control (p < 0.0001).ConclusionThese results suggest that transduction of E2F-1/TatHA fusion proteins in vitro is an effective repressor of hTERT expression in the primary ductal breast cancer cell lines HCC1937 and HCC1599.
Highlights
Telomerase expression is detectable in 81–95% of breast carcinomas and may serve as a therapeutic target
The biologically active E2F-1/TatHA, E132/TatHA and TatHA proteins effectively transduced greater than 95% of cell lines tested as detected via immunocytochemistry
In HCC1599 cells, hTERT was repressed with E2F-1/TatHA treatment by 4.0-fold when compared to the E132/TatHA control
Summary
Telomerase expression is detectable in 81–95% of breast carcinomas and may serve as a therapeutic target. The objective of this study was to investigate repression of telomerase activity in primary ductal breast cancer cells through transcriptional regulation of the catalytic subunit hTERT. Telomerase activity is detectable in 80–90% of malignancies and is absent in most normal somatic cells [1]. (2000) reported that 95% of advanced stage breast cancers express telomerase [3]. Due to the majority of tumor cells expressing telomerase, this protein is being evaluated as a tumor marker for breast cancer and other solid malignancies [4]. Telomerase includes an RNA component (hTR) that serves as the template for telomeric DNA and a protein catalytic subunit (hTERT) with reverse transcriptase activity [5]. The activity of telomerase is regulated by the transcription of hTERT, and the cloning and characterization of the hTERT-promoter has permitted examination of elements controlling transcriptional activation and repression of hTERT [6]
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