The Effect of Se-Methylselenocysteine on Human Transcription Factor E2F1 Expression Levels in HeLa Cells

Introduction

Selenium is a trace element essential to the mammalian diet, (Yin et al. 2004) and has been shown to inhibit the growth of mammalian cells (Sinha and Medina), suggesting a role in cancer treatment. A 2005 study on the effect of selenium treatment on skin cell carcinomas revealed that selenium has an antiproliferative effect on three types of cancers: cancers of the lung, prostate and breast (Clark et al 2005).
Many forms of selenium exist. MSC, a non-methylated organic selenium compound (Sinha and Medina), is a potentially powerful agent in human cancer treatment. MSC has been studied in clinical trials with prostate, lung, and colon carcinoma (Yin et al. 2004) and is suggested to be the most effective selenium treatment for breast cancer (Unni et al. 2005).
E2F is a family of transcription factors that controls transcription of genes required for cell cycle progression (Goodwin and DiMaio 2000), and its inhibition inhibits progression of the cell cycle (Lodish 2003). E2F1, an E2F family member that stimulates transcription, is specifically responsible for activating genes that are required for G1/S-phase transition and cell cycle progression during the S-phase of the cell cycle (Quin et al. 1994, Ginsberg et al. 2002). E2F1 is bound to and controlled by pRB, which represses E2F activity during quiescent periods. In order for replication to occur, a cyclin/cdk complex phosphorylates pRB to inactivate it, allowing E2F to induce transcription (Dick and Dyson 2003). Mutations in pRB and other molecules regulating the pRB/E2F1 interactions are often present in human cancers, resulting in deregulated activity of E2F1 (Ginsberg 2002). Approaches that would enable a decrease the level of E2F1 expression should result in the loss of uncontrolled cell growth in cancerous populations and have an antiproliferative effect on cancerous tumors.
The purpose of this study was to elucidate whether the addition of selenium to cervical cancer cells would have an inhibitory effect on E2F1 expression levels. Cervical cancer, a disease with which about 14,000 women are diagnosed every year (NCCC 2006), is of unknown cause. HeLa cells are a cervical cancer cell line expressing E2F, making them a suitable cell line in which to examine the impact of selenium treatment.

Figures

Figure 1-Figure 1. RT-PCR analysis of E2F1 RNA levels as treated with 0 μmol, 60 μmol and 120 μmol Se-methylselenocysteine. The above gel contains two replicates of the same PCR reaction. Lanes 1-3 show E2F1 and GAPDH (control) as treated with no selenium, 60μmol selenium and 120 μmol selenium respectively. Lanes 4-6 show a duplicate reaction of the same treatments. E2F1 primers (NM_005225.1) were located in exons 3 (492-513), and exon 4 (755-775) and designed to amplify a segment of 284 bases. E2F1 reactions were run for 33 cycles with 10ng RNA. GAPDH primers (NM_002046) were designed to amplify a segment of 228 bases. GAPDH reactions were run for 25 cycles with 5ng RNA.

Figure 2-Figure 2. Sequence alignment of primer product and E2F1. The mRNA product obtained with designed primers was sequenced and aligned with E2F1 (NM_005225.1) with a BLAST search.

Figure 3-Figure 3. Quantitation of RNA expression. RNA expression of E2F1 normalized to GAPDH (control) levels was obtained using Quantity One software from BioRad.

Figure 4-Figure 4. Three-way ANOVA analysis of E2F1 expression levels as compared to GAPDH levels. Statistical analysis to determine whether apparent differences in RNA levels were due to a difference in expression level or were not statistically significant. Since fcalc was less than fstatistic the null hypothesis was not rejected, and no difference in RNA expression could be shown.

To determine whether selenium treatment is effective on cervical cancer, HeLa cells were treated with Se-methylselenocysteine at concentrations of 60 μmol., 120 μmol., and 0 μmol. (control). E2F1 was amplified in an RT-PCR reaction and sequencing confirmed that the amplified product was E2F1. The amount of RNA present was quantified and statistically analyzed with the use of QuantityOne computer software to determine whether E2F1 RNA levels were affected by treatment with selenium. No difference in RNA expression levels was observed. Further studies should be performed to confirm that selenium has no effect on E2F1 in HeLa cells. Testing with a higher dosage of MSC, a longer incubation with selenium, or use of a different selenium compound have the potential to reveal an inhibitory effect of selenium on E2F1 expression in HeLa cells.

Full Paper

Acknowledgments

I would like to thank Dr. Rubin for allowing me to work on this project and guiding me in the process. I would also like to thank Lisa Sarran and Jinsong Qiu for all of their help and advice during the semester.

This document was last modified 05/17/2006.
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