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

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****

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

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tho

usa

nd

s o

f cel

l/cm

2

72 h

Umannová et al., 2007