Patrick Balaguer

1
Cell based-assays to detect endocrine disruptors

• Patrick Balaguer
• INSERM U540
• Endocrinologie Moléculaire et Cellulaire des
  Cancers
• Montpellier France

2
Reporter gene technology
3
Reporter gene procedure

• synthesis of the reporter gene plasmid
• transient or stably transfection of the plasmid
• study of the expression of the reporter protein

4
Criteria of a reporter gene

• Not present in the cells
• Easy to measure, rapidity
• Sensitivity
• Non radioactive
• Detection in intact living cells

5
Reporter genes

• Chloramphenicol acetyl transferase (CAT)
• Alkaline phosphatase (SeAP)
• ?Galactosidase (bGAL)
• Luciferases (LUCs)
• Green Fluorescent Protein (GFP)

6
Criteria of a reporter gene
7
Transient transfection

• Very versatile, easy to perform
• Several plasmids could be transfected
• High percentage of transfected cells
• Variable transfection efficiency
• Require an internal reference
• Short term effects (24 to 72 hours)

8
Stable transfection

• Inherited genotype
• Chromatin environment of the transgene
• Stable expression
• Reproductibility and reliability
• Low percentage of cells stably transfected
• A limited number of plasmids could be integrated

9
Stable transfection procedure

• Transfection by a plasmid carrying luciferase and
  resistance gene
• First selection by drug resistance
• Second selection by luciferase expression
• Subcloning by extensive dilution

10
Stable transfection procedure
11
(No Transcript)
12
Luminescent image (5 mn) (without ligand)
13
Luminescent image (5 mn) (with ligand)
14
Luminescent image (5 mn) (without ligand)
15
Luminescent image (5 mn) (with ligand)
16
Luminescent image (5 mn) (without ligand)
17
Luminescent image (5 mn) (with ligand)
18
RLU
19
Luminescence detection in 96-well plates

• T 0h Cell seeding
• (150 ml, 20,000 cells)
• T 8h ligands addition (50 ml)
• T 24h Luciferin addition (0,3 mM in culture
  medium).
• Living cell detection (2s par puits)

20
Estrogen reporter cell lines

• MCF-7 (ERa endogenous) Vit-tk-luc. MVLN (Pons
  M, 1990).
• MCF-7 (ERa endogenous) ERE-bGlob-luc. MELN
  (Balaguer P, 1999).
• T47-D (ERa endogenous) ERE3-TATA-luc. ER calux
  (Legler J, 1999).
• T47-D (ERa endogenous) ERE3-TATA-luc. (Wilson
  VS, 2004).
• MCF-7 (ERa? endogenous) Vit-tk-luc. MMV-Luc
  (Willemsen P, 2004).
• HEK293 (ERa or ERb) ERE2-MMTV-AP. a and b
  (Barkhem T, 1998).
• HeLa (ERa or ERb) ERE-bGlob-luc. HELN a and b
  (Balaguer P, 1999).
• HEK293 (ERa or ERb) ERE3-TATA-Luc. (Schreurs R,
  2002).
• CHO (ERa or ERb) RO-Luc. (De Gooyer M, 2003).

21
MELN bioluminescent cell line
Ligands
MCF-7
endogenous estrogen receptor a
reporter gene stably integrated
22
Xenoestrogens screening in MELN cells
23
Then stably transfected by hERa???????? ? cell
lines HELN-hERa, HELN-hER?
24
Selective ERa / ERb agonists
EE2 ERa Gen ERa EE2 ERb Gen ERb
ERa
ERb
25
Selective ERa / ERb antagonists
Raloxifen ERa RU486 ERa Raloxifen ERb RU486
ERb
ERb
ERa
26
Then stably transfected by DAB-hERa???
DAB-???? ? cell lines HELN-DAB-hERa,
HELN-DAB-hER?
27
(No Transcript)
28
Then stably transfected by rtE? ? cell lines
HELN rtER
29
Zearalanol a
30
(No Transcript)
31
(No Transcript)
32
(No Transcript)
33
Steroid receptors
RXR partners
PR, GR, MR (ERs, AR)
PPARa, PPAR?, PPARg, T3Ra, PXR, CAR, (VDR)
RXR

34
Environmental samples screening in MELN
Cells. Urban site water and sediment.
1 ml dextrait méthanol 2 L deau ou 10 g de
sédiments
water 5 pmoles eqE2/L. sediment 1600 pmoles eqE2
/kg.
35
Inhibition test of MELN activation by His-ERa
(LBD)
Pure compounds or environmental samples giving
80 transactivation E2 0,03 nM NPm 1
µM
His-ERa 15 nM
80
80
MELN
36
Nonylphenol mixture (1 mM) is not captured by
His-ERa?? 15 nM)
37
Inhibition test of MELN activation by
His-ERa (environmental samples)
Urban site water High affinity compounds
Urban site sediment Low affinity compounds
38
Purification of xenoestrogens by His-ER?
Environmental sample
XE
XE
Immobilisation de His-ERa??n nickel Sepharose
XE
39
Purification of xenoestrogens by His-ER?
Purification of xenoestrogens
40
Purification of xenoestrogens by His-ER?
Elution of xenoestrogens
41
Purification of xenoestrogens by His-ER?
transactivation (100 E2 1 nM)
42
Environmental samples screening in MELN (ERa) and
HAhLP (AhR) Cells. Urban site sediment.
sediment 1.6 nmoles eq E2 /kg and 200 nMoles eq
dioxin /kg .
43
(No Transcript)
44
MELN xenografts
One month after implantation in presence of E2,
MELNs cells will form a tumor and can be used as
estrogenic biosensor.
45
(No Transcript)
46
In vivo detection in MELN xenograft
E2 EC50 5 µg/kg/j
47
MELN microimplantation in mice

• ? Choice of the implantation site
• adipose tissue (pesticide accumulation)
• estrogen dependent organs (breast, uterus)
• ? rapidity of the obtention of the biosensor

48
Special thanks to the team
49
Special thanks
To the collaborators Selim Aït-Aïssa and
Jean-Marc Porcher, Verneuil-en-Halatte,
France. Claude Casellas, Montpellier,
France. Jean-Pierre Cravedi and Daniel Zalko,
Toulouse, France. Dino Moras and Marc Ruff,
Strasbourg, France. Pierre Germain and Hinrich
Gronemeyer , Strasbourg, France. Nicolas Olea,
Granada, Spain.