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Deacetylase inhibition, estrogen signalling and promoter repression.
Supplemental Material
Valproic acid and trichostatin A induce transcriptional silencing of a
subset of genes that include estrogen receptor .
George Reid1,*, Raphaël Métivier1,2, Chin-Yo Lin3, Stefanie Denger1, David
Ibberson1, Heike Brand1, Vladimir Benes1, Edison T. Liu3 and Frank Gannon1.
*
: Corresponding author. E-mail: [email protected]
1
European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg,
Germany. Phone: +49 6221 889 1102. Fax: +49 6221 889 1200.
2
Present Address: Equipe EMR, UMR CNRS, 6026 (ICM), Universite de Rennes I,
Batiment 13, 35042 Cedex, Rennes, France.
3
Genome Institute of Singapore, Genome Building, #02-01, 60 Biopolis Street,
Singapore 138672.
Deacetylase inhibition, estrogen signalling and promoter repression.
Supplemental Material
Supplemental Figure S1: VPA and TSA are cytotoxic and cytostatic and induce
apoptosis and differentiation. VPA has a cytotoxic effect on both ER- positive and
ER- negative cells. There is little difference in the cytotoxicity of VPA (IC 50 values
Deacetylase inhibition, estrogen signalling and promoter repression.
Supplemental Material
between 1 and 3 mM) and TSA (IC50 values between 1 and 3 nM) between the ER-
positive breast carcinoma cell line MCF-7, and the ER- negative cell lines MDA
MB231 and HeLa. Moreover, at 40 hours after the addition of VPA, around 25% of
cells were found to have a DNA content less than in Go/G1 (figure S1(b)), indicating
that a proportion of cells are apoptotic. VPA also induces differentiation of MCF-7
cells, as indicated by the extensive number of lipid droplets in cells treated for 48
hours with 6 mM VPA (figure S1(c)). TSA also induces apoptosis and differentiation
in MCF-7 cells (data not shown). The general growth inhibitory effect of VPA and
TSA suggests that multiple changes in gene expression occur on inhibition of
deacetylase activity.
Deacetylase inhibition, estrogen signalling and promoter repression.
Supplemental Material
Deacetylase inhibition, estrogen signalling and promoter repression.
Supplemental Material
Supplemental Figure S2: VPA and TSA induce cell cycle arrest. Flow cytometry
indicates that treatment with 6 mM VPA or with 300 nM TSA arrests MCF-7 cells in
the Go/G1 and G2/M phases of the cell cycle, in contrast to the Go/G1 arrest induced by
the pure antiestrogen ICI 182,780. The analysis with TSA was restricted to 30 hours
of treatment due to excessive cell death occurring beyond this time. Arrest in both
Go/G1 and G2/M indicates that multiple key regulatory components, in addition to
ER are affected by VPA and TSA treatment.
Deacetylase inhibition, estrogen signalling and promoter repression.
Supplemental Material
Supplemental Figure S3: Comparison of the response MCF-7 cells make to VPA
and to TSA.
Deacetylase inhibition, estrogen signalling and promoter repression.
Supplemental Material
Supplemental Figure S4: Cycloheximide blocks the antiestrogenic effect of VPA
or TSA on MCF-7 cells. The response of the estrogen responsive gene set, as
described for figure 3, was analysed with regard to the effect that concomitant
addition of cycloheximide (CHX) has on the response to VPA and to TSA. The
addition of CHX prevents VPA or TSA induced down-regulation or ERa, and in
keeping with this effect, VPA and TSA no longer have a predominantly antiestrogenic effect when de novo protein synthesis is blocked by CHX.