Molecular characterization of apoptosis induced by PDT: implication of tumor

suppressor genes as prospective markers

A. Fekih, A. Major, F. Ludicke, A. Campana, andI. Irminger-Finger

Department of Obstetrics and Gynaecology, andBiology of Aging Laboratory, Department of Geriatrics, University of Geneva, Switzerland

What Is PDT?

Photodynamic therapy (PDT) is a revolutionary medical technology that uses lasers to activate light-sensitive pharmaceuticals to treat cancer and other diseases in a non-surgical, minimally invasive way.

How does it work ?

• Porphyrin absorption maximum at a wavelength corresponding to incident laser light.

• The excited singlet porphyrin can decay back to the ground state with release of energy in the form of fluorescence.

• Singlet can be converted into the triplet excited state (3P*) which is able to transfer energy to another triplet.

p0

1px

E

Fluo

resc

ence

INTERSYSTEN CROSSING

3px

Phos

phor

esce

nce

1O2

3O2

OXYGEN

Jablonski diagram

What are Photosensitisers

Photosensitisers are substances that absorb light energy and transform this energy in chemical reactions. Many different photosensitisers are currently under development for potential drug use, 8 of which in clinical trials.

Laser wavelengths for activation of photosensitive drugs used in PDT?

Each PDT drug, because of its unique chemical composition, requires a different activating wavelength of light. All current PDT drugs generally fall in the 630nm to 750nm range, the visible red to infrared range. 5-ALA, for example, is activated at 410nm to 630nm.

What is apoptosis

• programmed cell death characterized by– membrane changes– protein degradation– nuclear shrinkage– DNA fragmentation

What is necrosis

A pathological process caused by the progressive degradative action of enzymes generally associated with severe cellular trauma. It is characterized by mitochondrial swelling, nuclear flocculation, uncontrolled cell lysis, and ultimately CELL DEATH.

Morphological difference between apoptosis and necrosis

• Physiological process• The integrity of plasma membrane is

preserved for a long time• Condensation of the chromatin• Apoptotic bodies• Absence of inflammatory reaction• Phagocytosis by adjacent cells• One cell is affected• Intra-cellular organelles are intact

initially, then condensed• Loss of contact with the surrounding

cells and the extra-cellular matrix.

• Pathological process• Swelling then lysis of the cell• Rapid loss of the integrity of the plasma

membrane• Light compaction of the chromatin• No apoptotic bodies• Important inflammatory reaction

affecting neighboring cells• Destruction of mitochondria and

lysosomes• Exocytosis.

APOPTOSIS NECROSIS

Biochemical difference between apoptosis and necrosis

• Induced by physiological stimuli• Active and regulated process• Needs energy• Transcriptional activity associated• Translational activity associated• DNA fragmentation.

• Induced by non-physiological stimuli• Passive and non regulated process• Does not need energy • No transcriptional activity• No translational activity• Random DNA fragmentation.

APOPTOSIS NECROSIS

Genes reported to be implicated in PDT

• Bax, bik, bak• Bcl-X, Bad, P53• C-jun, hrk

• The apoptotic machinery can be broadly divided into two classes: sensors (FAS, p53, myc…) and effectors (bcl-2, bax, caspases…) 2

• Caspases are the ultimate effectors of apoptosis.

Pro-apoptotic genes1

Anti-apoptotic genes1

(1Novartis apoptosis pathway)

(2D. Hanahan, 2000)

•Bcl-2, Bcl-X, Rb•Mcl-1, A1, Brag-1, Bfl-1

Bax

p53pro-apoptotic genes anti-apoptotic genes

bax, bik,bcl-X, badp53, c-jun

hrk

bcl-2, bcl-X,Rb, Mcl-1

A1, brag-1,bfl

altered gene expression

bcl-2

bcl-2

bcl-X p53

Apoptotic pathways are complex

Activates

inhibited DNArepair

NUCLEUS

Possible pathway for ROS induced apoptosis5 AlA Light+

Emission of photons

NECROSIS APOPTOSIS

Loss of tumor suppressor genes

ROSROSReactive Oxigen Species Tumor suppressor

genes functioning

Tumor suppression pathways

Tumor suppressor genes are found to be implicated in

• DNA repair • Cell cycle arrest• Senescence• Apoptosis

Loss of tumor suppressor gene functions in tumor cells

• p53 loss in more than 50% of tumors• RB loss in many tumors• BRCA1 loss in breast and ovarian cancers

Breast cancer susceptibility gene products BRCA1 and BARD1

BARD1 interaction

Nuclear localizationsignals

Rad51 interaction

Granin motif

BRCTdomain

BRCA1interaction

Ankyrin repeats

BRCT domain

PCNA

p53 RNA-Pol II?

RHA

GOAL

• Defining genes that are implicated in PDT induced apoptosis

• Defining parameters controlling the extends of apoptosis and necrosis

• Fine-tuning of PDT

46 k D

66 k D

5 ALA - + - + + -UV - - + + + -Doxorubicin - - - - - +

p53 expression after PDT

53 kD

53 kD46 k D

66 k D

p53 -/- cell

TAC 2 cells

53 kD66 k D

46 k D

HeLa cell

Relative amount of p53 expression

Exp 1 Exp 3 Exp 5

p53TAC 2

HeLa

0

50

100

Exp 1 Exp 3 Exp 5

p53TAC 2

HeLa

Exp 1: 0 Exp 2: 5 ALA Exp 3: 1’ UV

Exp 3: 5 ALA+1’UV Exp 4: 5 ALA+2’UV Exp 6: Doxorubicin

BARD1 expression after PDT

TAC 2 cells

p53 -/- cell

HeLa cell

5 ALA - + - + + -UV - - + + + -Doxorubicin - - - - - +

97 kD

97 kD

97 kD

Relative amount of BARD1 expression

Exp 1 Exp 2 Exp 3 Exp 4 Exp 5 Exp 6

p53TAC 2

HeLa

0

50

100

150

Exp 1 Exp 2 Exp 3 Exp 4 Exp 5 Exp 6

p53TAC 2

HeLa

Exp 1: 0 Exp 2: 5 ALA Exp 3: 1’ UV

Exp 3: 5 ALA+1’UV Exp 4: 5 ALA+2’UV Exp 6: Doxorubicin

Quantification of apoptosis by AnnexinV staining in HeLa cells

HeLa + Doxorubicin HeLa + 1' UV

HeLa + 1' UV + 5 ALA HeLa + 2' UV + 5 ALA

annexin v

DN

A st

ain

Quantification of apoptosis by AnnexinV staining in embryonic stem cells

RD + 1'UVRD + Doxo

RD + 1' UV + 5 ALA RD + 2' UV + 5 ALA

annexin v

DN

A st

ain

Quantification of apoptosis by AnnexinV staining in TAC 2 cells

TAC 2 + Doxorubicin TAC 2 + 1' UV

TAC 2 + 1' UV + 5 ALA TAC 2 + 2' UV + 5 ALA

annexin v

DN

A st

ain

repair

eliminationapoptosis

ROS

Possible apoptotic pathway in PDT

ROSROS

necrosisdeath by damage

p53p53

BARD1BARD1BARD1

accumulation of damage

Conclusion• PDT induced apoptosis and necrosis are cell type

specific • Extend of apoptosis and necrosis depends on

– dose of ALA– time of light application

• Apoptotic potential depends on functional tumor suppressor genes

• Clinical application: PDT fine tuning should be the goal