Transcript Cell Transformation

IDENTIFYING
GENOTOXIC AND NON GENOTOXIC CARCINOGENS
WITH CELL TRANSFORMATION ASSAYS
P. VASSEUR, M.A. MAIRE, C. RAST, S. ALEXANDRE, H. BESSI.
University of Metz, CNRS , France
Duluth, 19-21 May 2010
 Context, Definitions, History of CTA
 SHE cells
Protocol
 Mechanisms
 The Balb 3T3, C3H10T1/2 cell lines
 Performances of cell transformation assays (CTA)
 Analyses SHE results
 Conclusion
Duluth, 19-21 May 2010
Context
Long term rodent carcinogenicity assay : expensive, time consuming
→ not required for the evaluation of chemicals (EU),
except the genotoxic ones produced at high tonnage
Short term in vitro and in vivo genotoxicity assays have been used
as surrogates to predict carcinogenicity.
Yet,
a number of chemical carcinogens to humans and mammals are
negative in genotoxicity assays, but positive in cell transformation assays (CTA)
In 2007, OECD has recommended the development of guidelines for cell
transformation assays for in vitro detection of chemical carcinogens
Duluth, 19-21 May 2010
OECD Environment, Health and Safety publications Series on
Testing and Assessment N°31, 2006.
Detailed Review Paper (DRP 31) on Cell Transformation Assays
for Detection of Chemical Carcinogens.
Environment Directorate, November 28, 2006, 170 p.
http://www.olis.oecd.org/olis/2007doc.nsf/LinkTo/NT00002F0A/$FILE/JT03230941.PDF
http:www.oecd.org/document/12/0,2340,en_2649_34377_1898188_1_1_1_1,00.html
Duluth, 19-21 May 2010
Cell Transformation
Cell transformation is the induction of phenotypic alterations
in cultured cells that are characteristics of tumorigenic cells.
These phenotypic alterations can be induced by exposing mammalian
cells to carcinogens.
Transformed cells that have acquired the characteristics of malignant cells
have the ability to induce tumors in susceptible animals (Berwald and
Sachs, 1963, 1965).
Duluth, 19-21 May 2010
MULTISTAGE TRANSFORMATION OF
SYRIAN HAMSTER EMBRYO (SHE) CELLS
BY CHEMICAL CARCINOGENS
Earle (1943) : Morphological changes in cell culture were
associated with the oncogenicity of these cells in vivo
Berwald and Sachs, 1963, 1965
Primary or
secondary
SHE cells
+
BaP or 3MC
then
subcultured
Subcutaneous
Injection into
newborn hamster
Foci of
rapidly
dividing cells
Tumours
Further development and validation of SHE assay
DiPaolo et al., 1969
Pienta et al., 1977
Barrett et al., 1979
Newbold et al., 1982
Chouroulinkov & Lasne, 1976 ,…
demonstrated the ability of chemical carcinogens
from different chemical classes to induce
morphological transformation (MT) in vitro
Morphological transformation (MT)
= Changes exhibited by transformed cells related to neoplasia and
associated with behaviour and growth control modifications :
. alteration of cell morphology
. disorganized pattern of colony growth
. acquisition of anchorage-independent growth (Combes et al., 1999)
Later on, transformed cells become able to :
. grow in semi-solid agar
. produce autocrine growth factors
. evolve to tumorigenicity when injected into appropriate hosts
. divide indefinitely (immortalized), which is associated with other
alterations like aneuploïd karyotype and altered genetic stability.
Phenotypic changes / SHE CTA
• Changes in
normal
transformed
- cytoskeleton
- morphology of cells
& colonies
Characteristic
phenotype
of transformed cells :
- a random growth pattern of spindle shaped cells,
- a piling up of cells in a criss-cross pattern (a loss of growth inhibition and
of cell-cell orientation at confluency)
Normal SHE colony
Morphologically transformed SHE colony
Morphologically transformed SHE colony
Chemical carcinogens classified in two groups
. Genotoxic carcinogens
able to initiate cells to carcinogenesis through direct interaction with DNA,
resulting in DNA damages and/or structural/numerical chromosomal
aberrations which can be detected by genotoxicity tests.
. Non-genotoxic carcinogens
carcinogenic agents devoid of direct interaction with DNA.
The indirect modifications to DNA structure, amount or function may induce
altered gene expression and/or signal transduction.
Generally, non-genotoxic carcinogens refer to carcinogens negative in genotoxicity
assays performed to measure endpoints such as gene mutations and chromosomal
damages (chromosomal aberrations, micronuclei).
The multistage process of carcinogenesis in vivo
Normal
Initiation
Foci
Promotion
Benign lesion
Progression
Malignant lesion
1. Genotoxicity
Mutagens
Viruses
Radiations
Initiation
2. Epigenetic events ……
Factors of
Cell growth, division
Transcription
Promotion
Progression
Invasion
1. Genotoxicity
Mutagens
Viruses
Radiations
2. Epigenetic events ……
Factors of
Cell growth, division
Transcription
Efficient
controls
at
every steps,
Inhibition of DNA repair
Activation of protooncogenes
Inactivation of tumor-suppressor genes
of antimetastasis genes
1. Genotoxicity
Mutagens
Viruses
Radiations
2. Epigenetic events ……
Factors of
Cell growth, division
Transcription
Efficient
controls
at
every steps,
BUT, IF INACTIVATED
Inhibition of DNA repair
Activation of proto-oncogenes
Inactivation of tumor-suppressor genes
of antimetastasis genes
1. Epigenetic events 2. Genotoxicity ……
Histone desacetylation
Hypo/hypermethylation
Inhibition of DNA repair
Activation of proto-oncogenes
Inactivation of - tumor-suppressor genes
of
- antimetastasis genes
Acetylation
Histone acetyl transferase
(HAT)
open chromatin
transcription,
gene activation
Desacetylation
Histone desacetylase
(HDAC)
+
Methylation
Histone methyl transferase
+
HMT
Methyl Binding Proteins
(MBP)
compacted chromatin
blocage transcription
gene inactivation
tumor suppressors genes p53, p16
Hypoacetylation H4
observed in early steps
of carcinogenesis
Acetylation
Histone acetylases
Histones H3, H4
Silencing of
tumor suppressor genes
active, open chromatin
normal state
Histone
desacetylase
Desacetylation
Hypermethylation H3 H4,
Overexpression HMT
H3, H4
in a variety of
Histone methyl
neoplasia
transferases
DNA hypermethylation
of promoter sequences
→ transcriptional silencing DNA
Methylation
DNA methyl
transferases
SAM
methionine
Hypomethylation
also tumorigenic
Iacobuzio-Donahue, Ann Rev Pathol. Mech. Dis.2009.
Nickel
Hypoacetylation H4
observed in early steps
of carcinogenesis
Acetylation
(Sutherland et al.
Ann NY Acad Sc, 2003)
Histone acetylases
Histones H3, H4
Silencing of
tumor suppressor genes
active, open chromatin
normal state
Histone
desacetylase
Desacetylation
Hypermethylation H3 H4,
Overexpression HMT
H3, H4
in a variety of
Histone methyl
neoplasia
transferases
DNA hypermethylation
of promoter sequences
→ transcriptional silencing DNA
Preferential binding
to methylated
CpG sites
PAH (tobacco smoke)
AFB1
Methylation
DNA methyl
transferases
SAM
methionine
Hypomethylation
also tumorigenic
Arsenic, Alcohol
Cadmium
Effect dose dependent
(Herceg, Mutagenesis, 2007)
The MT phenotype of colonies expresses changes in
the expression of genes involved in cell cycle control,
proliferation and differentiation.
resulting from genotoxicity and non-genotoxic mechanisms
leading to :
- alteration of DNA repair
- disturbance in signal transduction
- histone desacetylation, DNA hypermethylation & hypomethylation
- modulation of gene expression → disturbance of cell cycle control,
proliferation and differentiation (Alexandre et al., 2003)
Histone desacetylation, DNA hypermethylation & hypomethylation
- oxidative stress (Jiung et al., 1999, Zhang et al. 2000)
inflammation
- imbalance of cell proliferation/apoptosis
- changes in intercellular communication (Cruciani et al. 1997)
- telomerase activation
….
- immunosuppression
Disturbance in signal transduction
from cell environment
nucleus
Lipophilic hormones
Nuclear
receptors
Syntheses,
replication,
Nuclear
mitosis
transcription
Non lipophilic Membrane
growth
+ receptor
factors
cascade
factor
phosphorylation / dephosphorylation
transient activation of a number of intermediates
Signal transduction
kinase cascade
Disturbance in signal transduction
from cell environment
nucleus
Growth + Receptor
factor
Syntheses,
replication, mitosis
A signal transduction pathway may be disrupted, activated
or blocked, by analogs that substitute or interfere with
some intermediates or the receptor itself.
An activation may be permanent, instead of transient, leading to a
sustained response
( ex : cell cycle dysregulation, increased rate of mitosis)
The tumor promoter TPA
12-O-Tetradecanoylphorbol-13-acetate
substitutes to diacylglycerol (DG)
and activates the PKC pathway
DG
Signal
Phorbol ester
TPA
C
OH
Phorbol ester
Kinase C
Inactive
protein
Phosphorylated protein
Ca++
active
cell response
Oxidative stress is involved in acrylonitrile (ACN)-induced
morphological transformation
8-oxodGuo in SHE cells
in SHE cells
4,5
4
3,5
3
2,5
2
1,5
1
% MT colonies
2,5
0,5
Vit.
Con E
Vit.
E
0
ACN
2
1,5
1
0,5
0
ACN
-tocopherol
0
25 50
75
0
5
25 50 75 µM
5
5
5 µM
Zhang et al., 2000. Carcinogenesis 21, 727-733.
DEHP, Di-(2-ethylhexyl)phthalate, a non genotoxic carcinogen
induces SHE cell transformation at doses inhibiting apoptosis
(50 µM) in serum-deprived cells
CONTROL
APOPTOTIC
Consequence :
survival of abnormal cells
Mechanisms :
Surexpression of the
antiapoptotic gene bcl-2
DEHP 10 µM
Repression of the
protooncogene c-myc
Maire et al., 2005.
Toxicol Lett, 158, 237-245
DEHP 50 µM
DEHP inhibits apoptosis via surexpression of bcl-2 (antiapoptotic)
→ change in bax/bcl-2 ratio
bcl-2 (500 pb)
Bcl-2 (26 kDa)
and represses the protooncogene c-myc expression
(Maire et al., 2005, Toxicol Lett,158, 237-245)
Protocol of the SHE assay
Obtention of SHE cells (feeder and target cells)
Hamster
embryos
13 days
Gestation
5000 rads
primary
cultures
non
differentiated
cells
feeder cells
target cells
7 days
SHE cells tested
at clonal density
150 cells / dish
storage
- 196°C
of exposure
Fixation,
coloration
scoring
25-45 colonies / dish
Cloning : 1 cell → 1 colony of hundred(s) cells )
Cloning efficiency (> 20%) : 150 cells → 25-45 colonies
Normal colony
Scoring of coded plates,
under stereomicroscope
Transformed
colony
Experimental design (continued…)
*Preliminary experiment for dose-range finding
*Definitive test
- 5 dose levels, vehicle control and positive control (BaP)
- Cytotoxicity evaluated by clonal efficiency
- Nb target cells adjusted in order to obtain 20-45 colonies/dish
- 40 dishes per concentration (or 10/conc. x 4 experiments)
- Transformation frequency and cloning efficiency established
from 1000 scored colonies per concentration
- Statistical analyses
- Criteria of acceptance fulfilled
Experimental design (continued…)
- statistical analysis for comparison between vehicle control
and concentration level (Fisher’s exact test or 2) and
positive dose-response trend (Cochran-Armitage test)
- positive response is declared when :
. 2 positive (successive) concentrations, at least
. or one positive concentration plus positive trend
-criteria for acceptance fulfilled
. 20% cloning efficiency in controls
. Nb transformed colonies in the range 25-45/dish
Experimental design (continued…)
Test medium : DMEM (without phenol red)
with fetal calf serum (12-15%), 10% CO2
pH / exposure
physiological pH : 7.0 - 7.35
exposure 7 days
or
LeBoeuf’s modification (1986) pH : 6.7
exposure to the tested chemical 24 h or 7 days
In parallel to SHE cell MT assay ,
Development of cell transformation assays (CTA)
on mouse established cell lines
* Balb 3T3, clone A31 Kakunaga, 1972
Yamasaki, 1985
* C3H10T ½ Chen and Heidelberger, 1969,
Reznikoff et al., 1973
Cell Transformation Assays (CTA)
SHE
•
•
•
•
diploïd, normal cells
metabolically competent
secondary cultures
low level of spontaneous
transformation
• short term (7 days) exposure
• mimics the first stages of the
neoplastic transformation
• Balb 3T3, C3H10T1/2
•
•
•
•
aneuploïd cell lines
limited metabolic ability
infinite life span
high level of spontaneous
transformation
• long term (> 4 weeks)
• mimic the late stages of the
neoplastic process
The multistage process of carcinogenesis in vivo (a)
(a)
Normal
Foci
Initiation
Benign lesion
Promotion
SHE
From Combes et al., 1999. ATLA 27, 745-767.
Malignant lesion
Progression
Balb/c 3T3,
C3H 10T1/2
Morphologically transformed and non-transformed foci of
BALB/c 3T3 cells (foci induced by 1 µg/ml 3-methylcholanthrene)
Non-transformed
Transformed
Photo Dr H Yamasaki.
Morphologically transformed and non-transformed foci of
C3H 10T1/2 cells (treatment with 1µg/ml 3-methylcholanthrene for 24h)
Type I
normal
Type II
Type III
Photo Dr J. Landolph.
PERFORMANCES
OF THE CELL TRANSFORMATION ASSAYS
- OECD Comparison with commonly used short-term genotoxicity tests
for assessing carcinogenic potential
Salmonella (Ames) test (mutagenesis assay)
Mouse lymphoma L5178Y cell mutagenesis assay
HPRT mutagenesis assay
In vitro chromosomal aberrations
In vivo chromosomal aberrations
In vivo micronucleus test
Data set
Nb chemicals
Rodent
Carcinogens
Non
Carcinogens Organic Inorganic
SHE :
264
191
73
203
61
BALB:
186
127
59
165
21
C3H :
141
117
24
121
20
Data banks
- IARC, NTP, GENETOX, CCRIS, CPDB/Gold and Zeiger (1997)
- Heidelberger et al. (1983), Matthews et al.(1993), Leboeuf et al. (1996)
and many other published articles …
SHE results on 64 metals and inorganic compounds
Asbestosis, ceramic fibres,
cadmium, nickel, chromium compounds, …
SHE
Results
+
-, ?, eq
Rodent
carcinogen
Rodent
non
carcinogen
50
6
56
3
5
8
53
11
64
Elias et al. Carcinogenesis, 10-11, 2043-2052, 1989 ;
Elias et al.,Toxicology In Vitro, 14, 409-422, 2000;
Elias et al., J. Toxicol Environ Health, 65, 2007-2027, 2002;
Elias et al, Ann. Occup. Hyg., 46, 53-57, 2002. …
Comparison with rodent carcinogenicity
Definitions
In vitro
In vivo
Carcinogen Non-carcinogen
+
a
b
-
c
d
Concordance = % agreement with in vivo exp. (a+b)/(a+b+c+d)*100
Sensitivity
= % carcinogens that are positive (a/a+c)*100
Specificity
= % noncarcinogens that are negative (d/b+d)*100
Positive Pred.= % positive calls that are carcinogens (a/a+b)*100
Negative Pred.=% negative calls that are noncarcinogens (d/c+d)*100
False negative = c/ a+c
False positive = b/ b+d
Performance of CTA
relative to rodent bioassay
SHE
BALB
C3H
264
149
96
Concordance
86%
68%
73%
Sensitivity
91%
75%
72%
Specificity
74%
53%
80%
False negative
9%
25%
28%
False positive
26%
47%
20%
(Inconclusive
(10%)
(28%)
(30%)
n=
Not included)
Performance of CTA
relative to rodent bioassay
n=
SHE
pH 6.7
88
SHE
pH 7.0
204
BALB
C3H
149
96
Concordance
Sensitivity
74
66
85%
92%
68%
75%
73%
72%
Specificity
85
66%
53%
80%
False negative
33
8%
25%
28%
False positive
15
34%
47%
20%
(Inconclusive
(2)
(12%)
(28%)
(30%)
Not included)
Carcinogens CTA positive
Genotoxic
Direct alkylating agents
lactones, epoxides
aldehydes
alkylsulfonates
Indirect acting alkylating agents
N-nitroso compounds
Halogenated aliphatic hydrocarbons
Indirect acting, DNA covalent binding,
Intercalating agents,
Polycyclic aromatic hydrocarbons
Aromatic amines, nitroarenes
mycotoxins
Non genotoxic
Steroïds
Phthlates & HPP (fibrates) (SHE)
Polyhalogenated biphenyls
Halogenated aryl (insecticides)
PCDD
Biotoxins, cyanotoxins
Tumor promoters (TPA,
okadaïc acid…)
False negatives in CTA
SHE
BALB 3T3
C3H
Aniline
Anthraquinone
Arochlor 1254
DDT
Ethinyl estradiol
Ethyl alcohol
d-Limonene
Metaproterenol
Methylcarbamate
Nitrilotriacetate NTA
5-nitro-o-toluidine
Pyridine
Tetrahydrofuran
TEHP tris(2ethylhexyl) phosphate
2-Aminoanthracene
Chlorinated aliphatic hydro
Inorganics
mono, di, tetra, hexachloroethane
Clofibrate
1,2-epoxybutane
Ethinyl estradiol
d-Limonene
Monuron
2-4-Dinitrotoluene
Phthlates
Butylbenzyl phthlate,
DEHP
Procarbazine
TEHP
lead acetate
potassium dichromate
nickel chloride
sodium arsenate
Organics BrdU
Phenobarbital
Propyleneimine
Styrene
Thioacetamide
Divergent responses
Diethylstilbestrol
DEHP
Hexamethy phosphoramide
5-nitro-o-toluidine
Many known or suspected aneugens induce CT in SHE cells
SHE
Acrylamide
Asbestos
Benzene
Benomyl
Cadmium chloride
Chloral hydrate
Colcemid
DES
Econazole nitrate
Griseofulvine
Hydroquinone
Pyrimethamine
Vincristine
+
+
+
+
+
+
+
+
+
+
+
+
+
In vitro
ABS
In vivo
MN
Rodent
Carcinog
+
+
+/-
+
+/+
-/?
+/?
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
?
+
Performances of short-term genotoxicity tests on the
chemicals of the data set
Ames
n = 252
MLA
170SHE
In vitro
ABS
184
In vivo
MN
158
Concordance
Sensitivity
51%
74%
64%
56%
37%
86%
65%
57%
Specificity
81%
34%
63%
52%
False negative
63%
14%
35%
43%
False positive
19%
66%
37%
48%
(Inconclusive
21%
29%
15%
31%
Not included)
Non genotoxic (S. typhi) carcinogens SHE positive
Ames negative
Diethylstilbestrol
Acetamide
Diethylthiourea
Acrylamide
Dimethylhydrazine
Actinomycine D
Estradiol
Amitrole
Ethionine
Auramine
Ethylbenzene
Benzene
EGBE Butyl glycol
BrdU
Hexachlorobutadiene
Butylhydroxytoluene
Hexamethylphosphoramide
Butylbenzylphthlate
Hydroquinone
Catechol
Methylpyrilene, HCl
Chlordane
Methyl eugenol
Chlorothalonil
Methylclofenapate
Cinnamyl anthranilate
Mezerein
Clofibrate
Monuron
Cyclosporine
N-nitroso ethylaniline
Decabromodiphenyloxide Okadaïc acid
Dieldrin
Oxymetholone
Diethanolamine
Procarbazine, HCl
DEHP
Progesterone
Reserpine
Safrole
Sulfamethoxazole
TPA phorbol ester
Thiourea
Trichlorophenol
TEHP tris(2ethylhexyl) phosphate
Wyeth 14043 (HPP)
One embryo
20-50 tests
One female (m ≈5-8 embryos) : 160-400 tests
Renewal of target cells every year
Yet, cells and kits are now available and provided by
some companies
Training necessary
6-8 weeks required for a confirmed result
Quite performant as alternative to rodent carcinogenicity assays.
CTAs, in vitro assays necessary for non genotoxic carcinogens !
The use of toxicogenomics & proteomics will help in mechanistics for a
better knowledge of :
. the link between gene expression, cytoskeleton alterations,
neoplastic cell transformation,
. the pattern(s) of genomic changes common to some categories
of non genotoxic carcinogens
. a base set of gene expression changes (if existing) typical of
non genotoxic carcinogens
Thank you
Duluth, 19-21 May 2010