Narušení regulace buněčného cyklu, programované
buněčné smrti či mezibuněčné komunikace
prostřednictvím organických polutantu -
mechanismy karcinogeneze?
IP
'•»'#:'■*5,.::? ►».>•",• >'.%*
Polycyclic aromatic hydrocarbons (PAHs):
Sir John Percivall Pott
(1775):
,first published description
of an occupational cancer
related to coal soot"
I
Sir Ernest Kennaway (1931): „first single PAH carcinogen"
Reality is not so simple:
•  alternative bioactivation pathways;
•  tumor promoting effects of PAH metabolites;
•  direct cellular effects of parental compounds;
•  to describe nongenotoxic effects of POPs, it is necessary to study their mechanisms of effects of at cellular and molecular level.
PAH
CVPIAI/Efwiide hydrola«
CONJUGATE FORMATION
gluctironlde
Sulfite
O-metbylated catechol
* OXIDATIVE DNA DAMAGE
■10'
DNA ADDICTS
PAH ^qiiinone
* OXIDATIVE DNA DAMAGE
ADDI'CT FORMATION
* amino acids GSH RNA DNA
Model chemical carcinogens
vs. environmental pollutants
Possibilities open for alternative effects of PAHs:
•  direct alteration of signaling pathways (mitogen-activated protein kinases; tyrosine kinases; Ca2+; modulation of phospholipid metabolism)
•  interation with nuclear receptors (estrogen receptor-a; estrogen receptor-ß; androgen receptor; peroxisome proliferator-activated receptors);
•  deregulation of cell-to-cell communication    gap junctions; adherens junctions;
•  deregulation of cell proliferation and programmed cell death;
•  aberrant function of cell cycle checkpoints and DNA repair;
•  epigenetic effects;
•  alternative biotransformation and oxidative stress;
• activation of the aryl hydrocarbon receptor (AhR) and related effects;
c. bHLH/PAS Class I
bHLH
dimeraaiion DNA binding
T
PAS
secondary
dfeiwrtzatlon _I_
Tran s activation
AhR
bHLH-PAS family protein
AhR Ah RR
HIF-a I PAS
5IM 1&2
Class II
ARNT1 &2
JľJ
^
Xenobiotic response
B
m
^    Hypoxia signalling
B
B
Neurogenesis
Repression
B
General partner factor
BMAL1 & 2
B
Circadian rhythm
Organism:	Name:	Ligand-binding:	Physiological function:
Nematodes: Caenorhabditis elegans	AHR-1	No	Neuronal development; Behavioral effects.
Insects: Ďrosophila melanogasfer	Spineless (Ss)	No	Development; Regulation of homeobox genes and dendrite
Vertebrates:	AhR (AhRl, AhR2)	I    Yes   J	^Toxicity mechanisms^ development; Neuronal differentiation? Circadian rhytms?
Activation and effects of AhR:
E pi [he I in] hyperplasia         QTumor promotion)
Induction of drug metabolizing enzymes
.Altered ER signaling
Porphyria
Deregulated lipid metabolism
Decreased serum thyroxine
Wasting
leta holism of arachidoniĽ acid to biologically active products
Persistent thy no id hormone receptor activation
EGT receptor down-regulation
Lipid pcroxidalior Immunosuppression inhibition of gluconeogenesis Teratogenes is/em hry Otu x ki ty
Utilization of
brown adipose tissue.
Vi lamin A depletion
Cardiac dysfunction
Xenobictic response genes
„Classical" AhR-regulated genes: contain xenobiotic response elements (XRE) or dioxin responsive elements (ĎRE) in their promoter region:
• phase I and II enzymes - CYP1A1, CYP1A2, CYPIBI, UĎP-glucuronosy/transferase, GST- Ya, NQOl; AhRR.
AhR-regulated genes involved in control of cell proliferation and cell death:
•  pro-apoptotic genes - Bax;
•  immediate - early response genes - Jun, Fos;
•  cell cycle regulation - p27KiP1, p21w°f/aP.
Majority of cells are not actively proliferating -
they are in a quiescent GO phase of cell cycle.
In vitro model of contact-inhibited cells.
Progenitor           Contact inliHePa+ocy+cs
TP A or TCĎĎ.
cell numbers
i
*>
O
é
8         S
^
\
)Rm$tmsm&
c	M O	š         S         S O                     O                    O
%	m	
\	1               f           c	
i		
%	_________1—-----\-----,	
<&		
%	__________________H    »—}—'	
i	--------------------r     —j	
v	h   -f-.	
^		
>	\     —\-	
-£	\	
T*	H         *	
		
O             M            -ŕ-            Ofr             í»
O        14
D               N)               Jh               0>               <X>
O            IO
\
S     é      8      8
o            o           O            O            O
>
>^^^^-
^^^^^g-
fcftfeKftKK^
>^^^^
HfeKKK^at^
4
o      m      u      m      a
M       -^       *
o     w     ■**     en     o»     -±     -*      _*     ^     _*
O      M       -^      OJ      00
IO
s
8
fc^^H
o
Ö
%> S-phase
Expression of dnAhR blocks the proliferative effects of
AhR liqands:
			
p53| —	— — —	—	• i
			
phospho-p53	m		
			
			
		<#<	**•
	c
	0
	u
DC	*4-0
C	0s
<	
C	L. a)
"O	n
	E
	D
	C
	ä)
	u
250 i
200
150
100
50-
IJŕCXE (325-329) bHLH    PAS  I____________Q-rich
AHR   |"Š^:i;
j    m        ju
A495-805 ^;i;.;;:j:;fij:.i
8(15
DMSOTCDD    Fla     BbF    BaA    BaP
wild type
1.2% ± 0.3
dnAhR
0.9% ± 0.4
O en
Proteins involved in control of contact inhibition:
Cdkl-cyclin B Cdk1-cyclin A
AhR ligands modulate
expression of proteins
involved in 61—>S cell
cycle transition:
Transient knock-down
of AhR blocks cyclin A
induction:
	wild type				dnAhR
			U^		
Myelin A cdk2	--h				------
	m m — m «q	^^			— — — — — _
					
p27	_•.-.—				««•«»«••»«
					
pRB		x	i		•_______— -
	i		i		
ß-actin	-----------------V-		/		
/>* <*é<f<ľ			r	/V <* «* ^ ^	
control
AhR        DsRed         AhR         DsRed
SÍRNA       siRNA       siRNA        siRNA
AhR
CYP1A1 cyclin A
ERK2
-
M
m    é
3
/V/V/V/^/V
Cyclin A/cdVZ activity control is essential for the maintenance of contact inhibition:
B)
wild type
dnAhR
histone H1

800
J:     600 o
"o     200
*       *
*       I   I
■■■■■II   ■■!
Andrysik et al., 2007
&
Time (h) Doxycyclir	\ 0    24    48 +     +	DNA synthesis (induction x-fold) -i      K)      CO      Ji. ^   o    o    o    o	■	1	1
Cyclin A-^	_    - —				
					
ERK2-^					
Weiss et al.,	2008		+		-    Doxycyclin (48 h) +    TCDD (48 h)
Vondráček et al., 2005
Histone H1
Cdk2 activity
pRb
2 2»1                  ** DM93    PCB 126
*
cdk4 activity
t  a»
a
It   m
u
DMSŮ      PCB 12Ö
Induction of cell proliferation is independent of the
dimerization partner ARNT:
2.5
<2 ^ 2.0 c/)   o CD \"
f S 1.5
^   °
<|1.0
Q .E
0.5
Weiss et al., 2008
DU
+
+ ctr.
sírna
2.5
-9. 2.0
(D
X
1   §
o -g
1.5
1.0
0.5
+ TCDD
AhR          ARNT
vect.   dn-ARNT dn-AhR    vect.
stable dones     stable dones
AhR CYP1A1 CycA
/nj'       V      jfc     _**      «5        J&
#    (5^   &' & é?   r*
^*   \°    «°   <y* \°    «
^ 3
wild-type WB-F344 cells
dn-ARNT WB-F344 cells
The story Is more complex - AhR ligands disrupt also control of cell-to-cell communication - cell adhesion and gap junctional intercellular
communication:
cytoplasm
£j|Tyrosine Kinases 'W[c-metR. EGF.R, cerbfl
s£k[tyrosine Ptvosphatas
/   S  [ptp-^/k, lar-ptp
---------^v^pssy
ß-catemn or plakoglobln
aE-catenin
a-actiron
'et et al., 1999
	
phosho-FAK(120kDa)	I—— —I
E-cadherin (120kDa)	—  —   ~" II
FAK(116kDa)	\* ^" —" I
ß-catenin (98kDa)	—---------------
y-catenin (82kDa)	L« -I
ß-actin (42kDa)	k*~l|
		
	-	-«—y-catenin (82kDa) -— E-cadherin (120kDa)
	•                 --------------	
DMSO TCDD (5nM) 3M4NF(10|iM) | BNF(10jiM)	M    i     i   + i   +   +   i i   +    i    i I i    i    +   i +   i     ii +   i    +   i	
I

Call 1 cytosol
Diameter of channel = 1,5 nm
Cell 2 cytosol
Connexon
Longitudinal secttori of connsxon

No passage of large? molecules
Passaged ions and smetl molecules
2^1 nm
Connexor	
t	';
	
Gap   w Junction	
	
Gap        Ä Junction	

	ĎMSO			ĎMSO	
				1      .	
	TCĎĎ			TCĎĎ	
					
				• •	
					
		2='S2 Í=ppo
Cx43	m m m m m m	
_____________<	/<? * «* <? <#	
The complex story gets even more complex - AhR ligands interact with inflammatory and growth regulators:
I I
800 700 600 500 400 300 200 100 0
NF-kB activation in WB-F344 cells
AhR activation in WB-F344 cells
<y   £   &

Xm

<0

/
proliferation
**               **
**
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ii íl íl u ii ňll II
D 72 h
*jr  £4?   -*•«
^ <
jr*-    *ř<ť   **V*   *ř«ť    r<ť
*- ,# £' 4* & V

^
#
<p
\v
\u
\u
«?
120,0 100,0 80,0 60,0 40,0 20,0 0,0
mRNA CYP1A1
***                                      ** l--------1
6h 24 h
48 h
^
^
IT1RNACYP1B1
124 h 148 h