EGCG, a component of green tea, was investigated for its effects on epigenetic regulation of the RNA polymerase III subunit Brf2 in lung cancer cells. Treatment of lung cancer cells with EGCG or green tea for 24 hours showed no change in methylation of the Brf2 promoter region or significant change in cell proliferation, respectively. Restriction digestion and PCR analysis indicated EGCG did not alter methylation of the Brf2 promoter. Preliminary data also showed green tea treatment did not significantly affect cancer cell growth over 24 hours.

1. Epigenetic Regulation of the RNA Polymerase III
Subunit Brf2 by the Green Tea Component EGCG in
Lung Cancer Cells
By Gina Viavattene

2. Abstract
RNA polymerase III (RNA pol III) is responsible for the transcription of many structural
RNA molecules involved in RNA translation, assisting in the regulation of cell growth. Initiation
of RNA pol III transcription depends on accurate recruitment by the transcription factor TFIIIB.
In mammals, there are two known forms of TFIIIB. The first is made up of the TBP, Bdp1, and
Brf1 subunits, and is required for initiation of RNA pol III transcription from gene internal
promoters. The second form is comprised of the TBP, Bdp1, and Brf2 subunits, and is required
for initiation of RNA pol III transcription from gene external promoters. The TFIIIB subunit Brf2
has recently been demonstrated to behave as an oncogene, highlighting the importance of
strict regulation of TFIIIB expression and RNA pol III activity. Epigallocatechin gallate (EGCG), a
major component found in green tea, has been demonstrated to inhibit the growth of cancer
cells. I considered the idea that EGCG is acting to inflict epigenetic changes upon the Brf2
promoter region, specifically demethylation, and have so far determined that treatment of lung
cancer cells with EGCG does not change the methylation status of the Brf2 promoter in vitro.
Additionally, since EGCG is found in green tea, I began treating lung cancer cells with green tea
and have so far noticed no significant change in cell proliferation after treatment for 24 hours.
Introduction
Cancer is a serious health problem affecting millions of people worldwide. Among each
of its various forms, lung cancer is the deadliest, with a death toll greater than that of all other
forms of cancer combined [1]. The most dominant characteristic of cancer is its uncontrolled
and elevated levels of cell proliferation. RNA polymerase III (RNA pol III) is responsible for
transcribing several structural RNA molecules, making it key regulator of cell growth in humans.
Cancer cells have been shown to exhibit high levels of RNA pol III transcription in comparison to
non-cancerous cells, confirming the belief that regulation of RNA pol III transcription must be
compromised in some way.
Proper initiation of RNA pol III transcription depends upon the function of the
transcription factor TFIIIB, which is responsible for recruiting RNA pol III to its promoters.
Mammalian TFIIIB exists in two forms: that which is required to initiate transcription from gene
internal promoters, containing the TBP, Bdp1, and Brf1 subunits, and that which is required for
initiating transcription from gene external promoters, containing the TBP, Bdp1, and Brf2
subunits [3]. The fact that RNA pol III transcription is shown to be elevated in cancers makes
TFIIIB a target in cancer research. Recent studies have demonstrated that Brf2, the gene
encoding the Brf2 subunit in TFIIIB recruiting RNA pol III to gene external promoters, behaves as
an oncogene in lung squamous cell carcinoma [2]. Additionally, studies have shown that
transcription by TFIIIB containing the Brf2 subunit is regulated by tumor suppressors and
oncogenes, and that Brf2 has the potential to be used as a biomarker in cancer patients [3].
Each of these studies confirms the notion that Brf2 is a strong potential target in cancer
therapy.
Epigenetics is the study of mechanisms that make functionally relevant changes to a
genome without altering the nucleotide sequence, such as acetylation and methylation.
Methylated DNA is unable to be transcribed, and proper regulation of this epigenetic process
by DNA methyltransferases (DNMTs) is required for normal behavior of transcription machinery
like RNA pol III and its associated transcription factors.

3. Epigallocatechin gallate (EGCG), a polyphenol, has been shown to possess
chemopreventative properties, as it is able to inhibit cancer cell growth [4]. It has been already
been demonstrated that EGCG is capable of inhibiting RNA pol III transcription in cervical
carcinoma cells [5]. It has also been shown to decrease DNMT activity, reviving tumor
suppressor function in breast cancer cells, highlighting its role in epigenetic regulation [6]. Thus,
throughout this past semester, my studies have focused on determining the methylation status
of the Brf2 promoter in A549 male lung adenocarcinoma cells, following treatment with EGCG
for 24 hours, in order to determine if EGCG has an effect on oncogenes like Brf2. Through
restriction digestion of the DNA isolated from these A549 cells, I have demonstrated that EGCG
treatment seems to have no effect on the methylation status of the Brf2 promoter.
Additionally, since EGCG is found in green tea, I began looking into the effects of treating my
A549 cells with gunpowder green tea. It makes sense to believe that since EGCG has been
demonstrated to slow the growth of cancer cells, the EGCG in brewed green tea may lead to
the same effect. So far I see no significant change in A549 cell proliferation following 24 hour
treatment with the green tea, though my research in this area is new.
Materials and Methods
Cell Culture and Growth Assays
A549 cells were obtained from the American Type Culture Collection (Rockville, MD). Cells were
cultured in RPMI supplemented with FBS (5% v/v), nonessential amino acids (100 mM), L-
glutamine (5 mM), streptomycin (100 μg/ml), and penicillin (100 units/ml); all
fromBioWhittaker, Walkersville, MD. Cells were grown at 37°C in a humidified atmosphere of
95% air and 5% CO2. To measure cell proliferation in A549 cells following Green Tea/EGCG
treatment, the TheCelltiter-Glo® Luminescent Cell Viability Assay kit (Promega) was used
following the manufacturer's instruction and the luminescence was read on a Sirius single tube
luminometer (Berthold).
Methylation Analysis of the Brf2 promoter
The Brf2 promoter was analyzed using MethPrimer to identify potential CpG islands and
NEBcutterV2.0 for potential restriction enzyme sites sensitive to changes in methylation status.
Genomic DNA was isolated from 0 μM and 20 μM EGCG treated A549 cancer cells using DNeasy
Blood & Tissue isolation kit (Qiagen) using the manufacturers protocol. DNA concentration was
quantified by spectrophotometry. For the Brf2 promoter methylation analysis, genomic DNA
isolated from untreated and treated A549 cells were digested with the BseYI restriction enzyme
(New England Biolabs). Restriction digestions were then analyzed by semi-quantitative PCR
using primers spanning the Brf2 promoter region as noted in Figure 1, as well as GAPDH primers
as a control. PCR products were visualized on a 1% agarose gel. Hyperladder II (Bioline) was
used to determine DNA size.
Results
EGCG treatment has no effect on Brf2 methylation
Since EGCG has already been shown to inhibit cancer cell growth, I began with studying
the effects of EGCG on cancer cell growth at a deeper level. A549 cells were treated with either
0 μM or 20 μM EGCG for 24 hours, and the genomic DNA was then isolated from these cells.

4. Next, CpG methyltransferase was used in order to methylate one of the genomic DNA samples
to be used in subsequent restriction digestions as a positive control.
Figure 1: Restriction Digestion of A549 Cells Following 24 Hour Treatment with EGCG
A) Representation of the Homo sapiens Brf2 promoter sequence, with location of CpG island,
transcription start site, and restriction enzyme cut site labeled. Black lines indicate binding site of Brf2
promoters. B) A549 cells were treated for 24 hours with 0 uM or 20 uM EGCG. Following treatment,
genomic DNA was isolated and subjected to digestion with the methylation sensitive restriction enzyme
BseYI. Semi-quantitative PCR analysis followed, using primers for the Brf2 promoter, as well as GAPDH
as a control. PCR products were run on a 1% agarose gel. + indicates DNA sample that was methylated
prior to treatment using CpG Methyltransferase, to be used as a control.
Now that the DNA samples were all ready for digestion, the Brf2 promoter sequence
was entered into NEBcutter and MethPrimer in order to determine which methylation sensitive
restriction enzymes would be used, as well as the potential location of any CpG islands near the
transcription start site. Using this information, I decided to digest my EGCG treated and
untreated samples with the BseYI enzyme (Figure 1 A). Semi-quantitative PCR analysis revealed
that there is no difference between the Brf2 promoter regions in A549 cells treated with 0 μM
EGCG and those treated with 20 μM EGCG (Figure 1B). These results indicate that EGCG
treatment does not lead to a change in the methylation status of the Brf2 promoter of lung
adenocarcinoma cells.
Gunpowder green tea shows no significant change in A549 cell proliferation
While I was looking into the methylation status of the Brf2 promoter in A549 cells, I was
also interested in searching for an effect in this same cell line following treatment with
gunpowder green tea. Since our lab has previously determined the amount of EGCG in
gunpowder green tea, I was able to calculate a working concentration of tea necessary to
perform proliferation assays of A549 cells similar to those conducted with EGCG. Preliminary

5. data shows no significant change in cell growth after treating A549 cells with 9.2 μg/mL
gunpowder green tea, though more research in this direction is still required (Figure 2).
Figure 2: Effect of Treatment with Gunpowder Green Tea for 24 hours on A549 cell growth
A549 cells were either treated with 9.2 μg/mL concentration of gunpowder green tea (black), or with
RPMI growth media containing no tea (white). After 24 hours, cell proliferation was measured and
statistical analysis was performed.
Discussion
RNA pol III is responsible for regulating proper levels of cell proliferation in mammals, so
it is no surprise that it is a major focus in cancer research. The transcription factor responsible
for recruiting RNA pol III to its target promoters, TFIIIB, has recently become a target of interest
following the discovery that its Brf2 subunit behaves as an oncogene. EGCG has been
demonstrated in the past to inhibit the growth of cancer cells by blocking transcription of RNA
pol III. It is possible that this effect is seen following epigenetic changes induced by EGCG.
Using restriction digestion with a known methylation-sensitive restriction enzyme, I
have determined that EGCG shows no effect on the methylation status of the Brf2 promoter.
Semi-quantitative PCR analysis of this region following digestion with BseYI, a restriction
enzyme, shows no difference in the intensity of the bands between the 0 μM EGCG treated and
20 μM EGCG treated A549 cells. If EGCG treatment was successful in demethylating this
promoter region, the restriction enzyme would have cut the DNA at the indicated site, and PCR
amplification would yield no band on an agarose gel. Additional studies with other methylation
sensitive enzymes that cut in this promoter region are necessary to further show that the effect
I am seeing is true.
Though my research in regards to treatment with the gunpowder green tea is still new,
it is worth showing that though I expect for there is EGCG in the green tea, I have not yet been
able to show a significant loss of cell proliferation in A549 cells following 24 hour treatment [4].
I hope to continue studying the effect of other teas in this cell like, as well as the effect of those
same teas in other cancer cell lines, such as MCF-7 breast cancer cells, or H2347 lung cancer
cells.

6. References
1. Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer J Clin. 2014;64(1):9–29.
2. Lockwood WW, Chari R, Coe BP, et al. Integrative genomic analyses identify BRF2 as a novel
lineage-specific oncogene in lung squamous cell carcinoma. PLoS Medicine.
2010;7(7)e1000315
3. Cabarcas S, Schramm L. RNA polymerase III transcription in cancer: the BRF2 connection.
Molecular Cancer. 2011;10: 47.
4. Schramm L (2013) Going Green: The role of the green tea component EGCG in
chemoprevention. J Carcinog Mutagen 4: 1000142.
5. Jacob J, Cabarcas S, Veras I, Zaveri N, Schramm L. The green tea component EGCG inhibits
RNA polymerase III transcription. Biochemical and Biophysical Research Communications.
2007;360(4):778–783.
6. Mirza S, Sharma G, Parshad R, Gupta SD, Pandya P, Ralhan R. Expression of DNA
methyltransferases in breast cancer patients and to analyze the effect of natural
compounds on DNA methyltransferases and associated proteins. J Breast
Cancer. 2013;13:23–31.

7. References
1. Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer J Clin. 2014;64(1):9–29.
2. Lockwood WW, Chari R, Coe BP, et al. Integrative genomic analyses identify BRF2 as a novel
lineage-specific oncogene in lung squamous cell carcinoma. PLoS Medicine.
2010;7(7)e1000315
3. Cabarcas S, Schramm L. RNA polymerase III transcription in cancer: the BRF2 connection.
Molecular Cancer. 2011;10: 47.
4. Schramm L (2013) Going Green: The role of the green tea component EGCG in
chemoprevention. J Carcinog Mutagen 4: 1000142.
5. Jacob J, Cabarcas S, Veras I, Zaveri N, Schramm L. The green tea component EGCG inhibits
RNA polymerase III transcription. Biochemical and Biophysical Research Communications.
2007;360(4):778–783.
6. Mirza S, Sharma G, Parshad R, Gupta SD, Pandya P, Ralhan R. Expression of DNA
methyltransferases in breast cancer patients and to analyze the effect of natural
compounds on DNA methyltransferases and associated proteins. J Breast
Cancer. 2013;13:23–31.