Narušení regulace buněčného cyklu, Narušení regulace buněčného cyklu, programované buněčné smrti či programované buněčné smrti či
mezibuněčné komunikace prostřednictvím mezibuněčné komunikace prostřednictvím organických polutantů – mechanismy organických polutantů – mechanismy
karcinogeneze? karcinogeneze?
O
O
NO2
OCH3
Sir John Percivall Pott Sir John Percivall Pott (1775):(1775):
„„first published first published description of an description of an
occupational cancer occupational cancer related to coal soot“related to coal soot“
Sir Ernest Kennaway Sir Ernest Kennaway (1931):(1931):
„„first single PAH first single PAH carcinogen“carcinogen“
Current view:Current view:PAHs are genotoxic PAHs are genotoxic carcinogens forming carcinogens forming
DNA adductsDNA adducts
Reality is not so simple:Reality is not so simple:• alternative bioactivation pathways;alternative bioactivation pathways;• tumor promoting effects of PAH metabolites;tumor promoting effects of PAH metabolites;• direct cellular effects of parental compounds;direct cellular effects of parental compounds;• to describe nongenotoxic effects of POPs, it is to describe nongenotoxic effects of POPs, it is necessary to study their mechanisms of effects necessary to study their mechanisms of effects of at cellular and molecular level.of at cellular and molecular level.
Polycyclic aromatic hydrocarbons (PAHs):Polycyclic aromatic hydrocarbons (PAHs):
Possibilities open for alternative effects of PAHs:Possibilities open for alternative effects of PAHs:
• direct alteration of signaling pathways (direct alteration of signaling pathways (mitogen-activated protein mitogen-activated protein kinaseskinases; tyrosine kinases; Ca; tyrosine kinases; Ca2+2+; ; modulation of phospholipid metabolismmodulation of phospholipid metabolism))• interation with nuclear receptors (interation with nuclear receptors (estrogen receptor-estrogen receptor-; estrogen ; estrogen receptor-receptor-; androgen receptor; peroxisome proliferator-activated ; androgen receptor; peroxisome proliferator-activated receptors);receptors);• deregulation of deregulation of cell-to-cell communication – gap junctionscell-to-cell communication – gap junctions; ; adherens adherens junctionsjunctions;;• deregulation of deregulation of cell proliferationcell proliferation and programmed cell death; and programmed cell death;• aberrant function of cell cycle checkpointsaberrant function of cell cycle checkpoints and DNA repair; and DNA repair;• epigenetic effects;epigenetic effects;• alternative biotransformation and oxidative stress;alternative biotransformation and oxidative stress;
• activation of the aryl hydrocarbon activation of the aryl hydrocarbon receptor (AhR) and related effects;receptor (AhR) and related effects;
Model chemical carcinogens Model chemical carcinogens vs. vs.
environmental pollutantsenvironmental pollutants
Organism:Organism: Name:Name: Ligand-binding:Ligand-binding: Physiological Physiological function:function:
Nematodes:Caenorhabditis elegans
AHR-1 No Neuronal development;Behavioral effects.
Insects:Drosophila melanogaster
Spineless (Ss) No Development;Regulation of homeobox genes and dendrite morphology.
Vertebrates: AhR (AhR1, AhR2)
Yes Toxicity mechanisms;Liver and kidney development;Neuronal differentiation?Circadian rhytms?
AhR AhR ==
bHLH-PAS bHLH-PAS family family proteinprotein
Activation and effects of AhR:Activation and effects of AhR:
„„Classical“ AhR-regulated genes:Classical“ AhR-regulated genes:
contain xenobiotic response elements (XRE) or dioxin
responsive elements (DRE) in their promoter region:
• phase I and II enzymes - CYP1A1, CYP1A2, CYP1B1, UDP-
glucuronosyltransferase,GST-Ya, NQO1;
• AhRR.
AhR-regulated genes involved in control of AhR-regulated genes involved in control of cell proliferation and cell death:cell proliferation and cell death:
• pro-apoptotic genes - Bax;
• immediate - early response genes – Jun,
Fos;
• cell cycle regulation – p27Kip1, p21Waf/Cip.
Contact inhibitionContact inhibition::
• the rate of proliferation of most non-transformed
adherent cells decreases with increased cell density as
they become arrested in G1 phase of cell cycle, which is
a phenomenon known as contact inhibition;
• the loss of contact inhibition can lead to deregulated
growth and is often associated with malignant
transformation;
• a release from contact inhibition is a mechanism
suggested to be an important part of effects of tumor
promoters, such as TPA or TCDD.
Majority of cells are not actively proliferating Majority of cells are not actively proliferating – they are in a quiescent G0 phase of cell – they are in a quiescent G0 phase of cell
cycle.cycle.In vitroIn vitro model of contact-inhibited cells. model of contact-inhibited cells.
Biliary epithelial cells
HepatocytesProgenitor (oval) cellsDMSODMSO 1 nM TCDD1 nM TCDD
Rat liver epithelial ‘stem-like’ WB-F344 cells
Effects of PAHs onEffects of PAHs on contact-inhibited WB-F344 cellscontact-inhibited WB-F344 cellscell n
um
bers
cell n
um
bers
% S
-ph
ase
% S
-ph
ase
Chramostová et al., 2004
Expression of dnAhR blocks the proliferative Expression of dnAhR blocks the proliferative effects of AhR ligands:effects of AhR ligands:
Andrysík et al., 2006Andrysík et al., 2007
pRb phosphorylation
Proteins involved in control of contact Proteins involved in control of contact inhibition:inhibition:
AhR ligands AhR ligands modulate modulate
expression of expression of proteins involved in proteins involved in
G1G1→→S cell cycle S cell cycle transition:transition:
Transient knock-Transient knock-down of AhR blocks down of AhR blocks cyclin A induction:cyclin A induction:
Andrysík et al., 2007
Doxycyclin - + +Time (h) 0 24 48
- + - Doxycyclin (48 h)- - + TCDD (48 h)
Cyclin A
ERK2
DN
A s
ynth
esis
(indu
ctio
n x-
fold
)
0
1.0
2.0
3.0
4.0
Weiss et al., 2008
Cyclin A/cdk2 activity control is essential for the Cyclin A/cdk2 activity control is essential for the maintenance of contact inhibition:maintenance of contact inhibition:
Andrysík et al., 2007
Andrysík et al., 2007
Vondráček et al., 2005
Cel
l num
ber
(indu
ctio
n x-
fold
)
dn-ARNTvect.
stable clonesstable clones
dn-AhR vect.
0
0.5
1.0
1.5
2.5
2.0
- + - + - + - + TCDD AhRctr.
siRNA
ARNT
DN
A s
ynth
esis
(indu
ctio
n x-
fold
)
0
0.5
1.0
1.5
2.5
2.0
Induction of cell proliferation is independent of Induction of cell proliferation is independent of the dimerization partner ARNT:the dimerization partner ARNT:
AhR
CYP1A1
CycA
wild-typeWB-F344 cells
dn-ARNTWB-F344 cells
DMSO 0
.1%
DMSO 0
.1%
TCDD 5 n
M
TCDD 5 n
M
PCB 126
100
nM
PCB 126
100
nM
Weiss et al., 2008
The story is more complex – AhR ligands disrupt also control The story is more complex – AhR ligands disrupt also control of cell-to-cell communication – cell adhesion and gap of cell-to-cell communication – cell adhesion and gap
junctional intercellular communication:junctional intercellular communication:
Nollet et al., 1999
Cx43
DMSO
TCDD
DMSO
TCDD
-actin
Cx43
dnAhR
DMSO
TCDD
The complex story gets even more complex – AhR The complex story gets even more complex – AhR ligands interact with inflammatory and growth ligands interact with inflammatory and growth regulators:regulators:
DM
SO
0.1
%TN
F-
20 n
g/m
lTCD
D 5
nM
NF-B activation in WB-F344
cells
AhR activation in WB-F344 cells
DM
SO
0.1
%TN
F-
20 n
g/m
lTCD
D 5
nM
mRNA CYP1A1
**$*
**$**
****
0,0
20,0
40,0
60,0
80,0
100,0
120,0
%
6 h
24 h
48 h
mRNA CYP1B1
*
**$$ **
**$$****
0,0
20,0
40,0
60,0
80,0
100,0
120,0
%
6 h
24 h
48 h
proliferation
******
**$ **$$
0100200300400500600700800
tho
usa
nd
s o
f cel
l/cm
2
72 h
Umannová et al., 2007
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