SUPPLEMENTARY MATERIAL
From
“Estrogen receptor beta decreases survival of p53-defective cancer cells after DNA damage by impairing
G2/M checkpoint signaling”
Christoforos G. Thomas, Anders Strom, Karolina Lindberg and Jan-Ake Gustafsson
Corresponding Author: chthomas@uh.edu
Breast Cancer Research and Treatment
The supplementary material includes one word (text) file containing the supplementary Materials and
Methods section, the supplementary Figure Captions section and the supplementary References section.
In addition, the supplementary material includes 4 supplementary figures.
MATERIALS AND METHODS
Chemicals, Antibodies, and Drugs
17β-estradiol (E2), doxycycline, cisplatin, doxorubicin, etoposide, propidium iodide, puromycin,
blasticidin, nocodazole, insulin, epidermal growth factor (EGF) and hydrocortisone were purchased from
Sigma. 2,3-bis(4-hydroxy-phenyl)-propionitrile (DPN) was purchased from Tocris. Purified full length
ERβ was from Invitrogen. Antibodies against p53, p21, ERα were purchased from Santa Cruz. Anti-βactin and anti-FLAG (M5) antibodies were purchased from Sigma; an anti-chicken antibody against ERβ
was from Abcam (14021). ERβ was also detected with the rabbit polyclonal ERβ-LBD antibody [1]. An
antibody against phosphorylated form of ERβ was from Santa-Cruz. An antibody against Cdc25C was
from Neomarkers; a mouse monoclonal anti-BRCA1 antibody (Ab-1, MS110) was from Calbiochem;
antibody against nuclear matrix protein p84 (PE10) was from Abcam; antibodies against Caspase-2 were
from Chemicon (clone 10C6) and Cell Signaling (9491); antibodies against cleaved Caspase-3 and
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phosphorylated forms of Chk1 and Cdc25C were from Cell Signaling; anti-phospho-histone H3 and FITC
conjugated anti-γ-H2AX antibodies were from Upstate.
Cell Culture
HeLa cells (in which p53 pool is depleted by HPV-18E6) stably expressing ERβ were grown in DMEM,
supplemented with 10% FCS and 1% antibiotic (gentamicin) in a 5% CO2 humidified atmosphere at 37oC
[2]. For control purposes, HeLa cells were stably transfected with a pSG5 empty vector. p53 mutant T47D cells with tetracycline-regulated expression of ERβ (T47-DERβ) and control cells, T47-DPBI (mock)
were grown in RPMI-1640, supplemented with 10% FCS, 50 μg/ml gentamicin, and 10 ng/ml
doxycycline [3]. p53 mutant MDA-MB-231 cells stably expressing ERβ and the control cells stably
infected with lentivirus carrying the empty pLenti vector were grown in the same media as T47-D cells.
The p53 wild-type normal mammary cell line MCF10A stably expressing ERβ and the control cells stably
infected with the lentivirus carrying the empty pLenti vector were maintained in DMEM/F12
supplemented with 10% FCS, 50 μg/ml gentamicin, 8 μg/ml insulin, 20 ng/ml EGF and 500 ng/ml
hydrocortisone. MEFs were grown in DMEM supplemented with 10% FCS, 50 μg/ml gentamicin.
Recombinant DNA
The plasmid pcDNA3-FLAG-ERβ was used as a template for TOPO cloning of wild-type ERβ and the
ERβ form with mutated DNA-binding domain (DBDmut) (amino acid residues E167 and G168, glutamic
acid and glycine, replaced by alanine) into pLenti6/V5-D-TOPO. Lentiviruses containing the plenti6/V5
empty vector or the recombinant pLenti6/V5-D-FLAG-ERβ and pLenti6/V5-D-FLAG-ERβ-DBDmut
plasmids were used for the infection of p53-/- MEFS and T47-D, MCF10A and MDA-MB-231 cells as
described previously [4].
Cytoplasmic and Nuclear Fractions
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For separation of cytoplasmic and nuclear fractions, cells were suspended in a cold buffer containing 10
mM Hepes pH 7.0, 10 mM KCI, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF. After 15 min incubation on
ice, the homogenate was mixed with 10% NP-40 and centrifuged for 30 sec. The nuclear pellet was
resuspended in a cold buffer containing 10 mM Hepes-KOH pH 7.9, 400 mM NaCI, 0.1 mM EDTA, 5%
glycerol 1 mM DTT, 0.5 mM PMSF and the nuclear extract was isolated by centrifugation.
Transient Transfections
Cells were incubated in phenol red free-DCC-containing media for 48 h and transiently co-transfected
with 2.5 μg ERβ plasmid, together with 2 μg of 3-ERE-TATA-LUC reporter plasmid and 60 ng of bgalactosidase gene-expressing plasmid using LipofectamineTM 2 000 (Invitrogen). Cells were mock
treated (EtOH) or treated with 17β-estradiol or DPN for 24 h. Reporter gene activity was normalized to bgalactosidase enzyme activity.
Quantitative Real-Time PCR
Cells were lysed and RNA was extracted by using a RNA extraction Kit (Qiagen). cDNA was synthesized
as described earlier [5]. Real-time PCR was performed with the BRCA1 forward (GGC TAT CCT CTC
AGA) and reverse (GCT TTA TCA GGT TAT) primers and SYBR-Green PCR Master Mix in an ABIPRISM 7 500.
FIGURE CAPTIONS
Fig. S1 Cisplatin activates the G2/M cell cycle checkpoint in p53 mutant cancer cells
Uninfected p53-defective MDA-MB-231 cells were cultured in phenol red-free-DCC-FCS-containing
medium for 48 h and left untreated or treated with 20 µM cisplatin or 75 ng/ml nocodazole in the
presence of 10 nM E2. The cell cycle profiles were analyzed by FACS using PI staining for DNA content
(upper panel) and phospho-histone H3 staining as a mitotic indicator (lower panel). Cells accumulate at
the G2/M phase (middle upper panel) following cisplatin treatment. An additional mild increase in the S3
phase population is shown in the same panel compared to the untreated cells in the left panel. In response
to nocodazole treatment, MDA-MB-231 cells accumulate at the G2/M phase with 31.63% showing
increased staining for phospho-histone H3 (right panels)
Fig. S2 ERβ abrogates the G2/M checkpoint following cisplatin or etoposide treatment in T47-D
cells and HeLa cells
(A) Induction of ERβ expression abrogates the G2/M checkpoint in T47-D cells. p53 mutant T47-D cells
were cultured in phenol red-free-DCC-FCS-containing media for 48 h, incubated in the presence of 10
ng/ml (-ERβ) or 0.01 ng/ml (+ERβ) doxycycline and mock treated (DMSO) or treated with 20 μM
cisplatin and 10 nM E2 for additional 48 h. Cell cycle profiles were analyzed by FACS staining with PI
for DNA content (upper panels) and phospho-histone H3 as an indicator for mitosis (H3-P, lower panels).
In the right set of panels, nocodazole (75 ng/ml) was added 3 h following cisplatin addition.
(B) HeLa cervix adenocarcinoma cells (in which the p53 is depleted by HPV-18E6) stably expressing
ERβ and cells carrying the empty pSG5 vector (pSG5) following incubation in phenol red-free-DCC-FCS
containing media for 48 h were mock treated (DMSO) or treated with 10 μM etoposide (Etop) in the
presence of 10 nM E2 for 48 h. Cell cycle was analyzed by FACS.
(C) HeLa cells stably expressing ERβ and cells carrying the empty pSG5 vector (pSG5) following
incubation in phenol red-free-DCC-FCS containing media for 48 h were mock treated (DMSO) or treated
with 10 μM Etoposide (Etop) and 10 nM E2 for 30 h. Cells were incubated with trypan blue and counted
on microscope. Assays were performed in triplicate and normalized to mock treated cells at time 0.
Values represent the mean ± S.E. and the asterisk shows a statistically significant difference between
control (pSG5) and ERβ-expressing cells under the same treatment (two-tailed Student t test, p < 0.04).
(D) Western blot comparing the levels of ERβ in lysates from HeLa cells carrying the empty pSG5 vector
(pSG5) and cells stably expressing ERβ (ERβ)
Fig. S3 Doxycycline does not affect the DNA damage response in mock T47-DPBI cells
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(A) T47-DPBI (mock cells) were initially cultured in phenol red-free-DCC-FCS-containing medium for
48 h, incubated in the presence of 10 ng/ml or 0.01 ng/ml doxycycline and treated with 20 μM cisplatin
and 10 nM E2 for additional 48 h. Cell cycle profiles were analyzed by FACS analysis. In this
experiment, T47-DPBI mock cells which do not express ERβ in the absence of doxycycline were treated
with cisplatin and doxycycline at a concentration that induces ERβ expression in T47-DERβ cells. No
difference was observed in the cell cycle profiles of the cells treated with different concentrations of
doxycycline in the presence of cisplatin. This argues against the possibility that the abrogation of G 2/M
checkpoint observed in cisplatin-treated T47-DERβ cells was due to tetracycline-regulation of other genes
than ERβ.
(B) Immunoblots of p-Chk1 and ERβ from T47-DPBI cells incubated in the presence of 10 ng/ml or 0.01
ng/ml doxycycline and treated with 20 μM cisplatin or 10 μM doxorubicin in the presence of 10 nM E2
for 48 h. The phosphorylation of Chk1 at Ser-345 in the T47-DPBI mock cells was the same in the
presence of cisplatin, irrespective of the concentration of doxycycline, indicating that the impairment of
the Chk1-mediated G2/M checkpoint observed in cisplatin-treated T47-DERβ cells was due to ERβ
expression
Fig. S4 ERβ induces mitotic catastrophe in p53-/- MEFS in response to cisplatin or doxorubicin
treatment
Control (Lenti) and ERβ-expressing (ERβ) p53-/- MEFS were incubated in phenol red-free-DCC-FCScontaining media for 48 h, treated with 1 µM cisplatin (Cis) or 0.1 µM doxorubicin (Dox) in the presence
of 10 nM E2 for 30 h, fixed and stained with antibodies against phospho-histone H3, an indicator of
mitosis, γ-H2AX, an indicator of DNA damage and cleaved caspase-3, an indicator of apoptosis. Positive
staining for all three markers is indicative of mitotic catastrophe. Scale bar, 3 µM
REFERENCES
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inhibits 17beta-estradiol-stimulated proliferation of the breast cancer cell line T47D. Proc Natl Acad
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beta in SW480 colon cancer cells. Cancer Res 69: 6100-6106
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on the breast cancer cell line MCF-7 is dependent on HES-1 expression. J Biol Chem 277: 2837628379
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