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

17-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 ER was from Invitrogen. Antibodies against p53, p21, ER were purchased from Santa Cruz. Anti--actin and anti-FLAG (M5) antibodies were purchased from Sigma; an anti-chicken antibody against ER was from Abcam (14021). ER was also detected with the rabbit polyclonal ER-LBD antibody [1]. An antibody against phosphorylated form of ER 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 phosphorylated forms of Chk1 and Cdc25C were from Cell Signaling; anti-phospho-histone H3 and FITC conjugated anti--H2AX antibodies were from Upstate.

Cell Culture

HeLa cells (in which p53 pool is depleted by HPV-18E6) stably expressing ER 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 T47-D cells with tetracycline-regulated expression of ER (T47-DER) and control cells, T47-DPBI (mock) were grown in RPMI-1640, supplemented with 10% FCS, 50 g/ml gentamicin, and 10 ng/ml doxycycline [3]. p53 mutant MDA-MB-231 cells stably expressing ER 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 ER and the control cells stably infected with the lentivirus carrying the empty pLenti vector were maintained in DMEM/F12 supplemented with 10% FCS, 50 g/ml gentamicin, 8 g/ml insulin, 20 ng/ml EGF and 500 ng/ml hydrocortisone. MEFs were grown in DMEM supplemented with 10% FCS, 50 g/ml gentamicin.

Recombinant DNA

The plasmid pcDNA3-FLAG-ER was used as a template for TOPO cloning of wild-type ER and the ER 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-ER and pLenti6/V5-D-FLAG-ER-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

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 g ER plasmid, together with 2 g of 3-ERE-TATA-LUC reporter plasmid and 60 ng of b-galactosidase gene-expressing plasmid using LipofectamineTM 2 000 (Invitrogen). Cells were mock treated (EtOH) or treated with 17-estradiol or DPN for 24 h. Reporter gene activity was normalized to b-galactosidase 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 ABI-PRISM 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 M 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 S-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 ER abrogates the G2/M checkpoint following cisplatin or etoposide treatment in T47-D cells and HeLa cells

(A) Induction of ER 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 (-ER) or 0.01 ng/ml (+ER) doxycycline and mock treated (DMSO) or treated with 20 M 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 ER 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 M etoposide (Etop) in the presence of 10 nM E2 for 48 h. Cell cycle was analyzed by FACS.

(C) HeLa cells stably expressing ER 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 M 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 ER-expressing cells under the same treatment (two-tailed Student t test, p < 0.04).

(D) Western blot comparing the levels of ER in lysates from HeLa cells carrying the empty pSG5 vector (pSG5) and cells stably expressing ER (ER)

Fig. S3 Doxycycline does not affect the DNA damage response in mock T47-DPBI cells

(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 M 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 ER in the absence of doxycycline were treated with cisplatin and doxycycline at a concentration that induces ER expression in T47-DER 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 G2/M checkpoint observed in cisplatin-treated T47-DER cells was due to tetracycline-regulation of other genes than ER.

(B) Immunoblots of p-Chk1 and ER from T47-DPBI cells incubated in the presence of 10 ng/ml or 0.01 ng/ml doxycycline and treated with 20 M cisplatin or 10 M 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-DER cells was due to ER expression

Fig. S4 ER induces mitotic catastrophe in p53-/- MEFS in response to cisplatin or doxorubicin treatment

Control (Lenti) and ER-expressing (ER) p53-/- MEFS were incubated in phenol red-free-DCC-FCS-containing media for 48 h, treated with 1 M cisplatin (Cis) or 0.1 M 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, -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 M

REFERENCES

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5.Muller P, Kietz S, Gustafsson JA, Strom A (2002) The anti-estrogenic effect of all-trans-retinoic acid on the breast cancer cell line MCF-7 is dependent on HES-1 expression. J Biol Chem 277: 28376-28379

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