Moetaz El-DomyatiProfessor & Chairman of Dermatology,

STDs and Andrology Department,Faculty of Medicine, Al-Minya University,

Egypt.

Apoptosis, DNA Damage and Repair

DNA Damage and Efficacy of Repair Mechanisms

Lines of defense against the induction and persistence of DNA damage:

Preventing DNA damage: by detoxifying peptides, proteins, and oxy-radical scavengers, e.g. vitamin E and C.

DNA repair pathways: minimal DNA damage already present in the genome may be sensed by DNA damage response pathways, and the damage may be removed to reduce the possibility for inducing mutations.

Apoptosis: the damaged cell may be eliminated by spontaneous death.

DNA Repair Pathways

§ Nucleotide excision repair (NER) pathway

§ Mismatch repair (MMR) pathway

§ Base excision repair (BER) pathway

§ Double strand breaks (DSBs) repair pathway

Apoptosis is a Greek word that means falling of Apoptosis is a Greek word that means falling of leaves from a tree.leaves from a tree.

Apoptosis is a genetically encoded program that Apoptosis is a genetically encoded program that results in cell death.results in cell death.

Necrosis (Necrotic death)Necrosis (Necrotic death) Cell deathCell death

Apoptosis (Programmed cell death)Apoptosis (Programmed cell death)

Necrosis Massive cell injury and is often Necrosis Massive cell injury and is often accompanied by inflammation.accompanied by inflammation.

Apoptosis Physiological, programmed Apoptosis Physiological, programmed suicide that occurs in response to either external suicide that occurs in response to either external or internal stimuli, that result in elimination of or internal stimuli, that result in elimination of unwanted cells, for maintaining proliferative unwanted cells, for maintaining proliferative homeostasis.homeostasis.

- Cell shrinkageCell shrinkage- Organelles remain intactOrganelles remain intact- Intact membrane Intact membrane

permeabilitypermeability- No inflammationNo inflammation- PhysiologicalPhysiological

-Cell swellingCell swelling

- Swell or lyseSwell or lyse

-Altered permeabilityAltered permeability

-Provoke inflammationProvoke inflammation

-PathologicalPathological

ApoptosisApoptosis NecrosisNecrosis

Apoptotic cell identificationApoptotic cell identification Routine histopathology.Routine histopathology.

Hoechst 33342 or Hoechst 33258 Hoechst 33342 or Hoechst 33258 staining and videomicroscopy staining and videomicroscopy (nuclear chromatin clumping).(nuclear chromatin clumping).

Fluorescent microscopy (acridine Fluorescent microscopy (acridine orange), agarose gel electrophoresis, orange), agarose gel electrophoresis, flowcytometry assays.flowcytometry assays.

TUNEL stainingTUNEL staining

EMEM

P53 is a well known tumour P53 is a well known tumour

suppressor gene located on short suppressor gene located on short

arm of chromosome 17, it is arm of chromosome 17, it is

considered as the “guardian of considered as the “guardian of

the genome”, and as a regulator the genome”, and as a regulator

of apoptosis.of apoptosis.

P53 helps to maintain genomic P53 helps to maintain genomic

integrity by inducing G1 arrest, integrity by inducing G1 arrest,

of cell cycle, allowing DNA of cell cycle, allowing DNA

repair, or if the DNA damage is repair, or if the DNA damage is

extensive, it induces apoptosis. extensive, it induces apoptosis.

P53

P53 gene is a 53 Kd P53 gene is a 53 Kd

nuclear phosphoprotein, it nuclear phosphoprotein, it

has 2 forms of expression:has 2 forms of expression:

wildwild (dispersed)(dispersed)

Mutant Mutant (compact)(compact)

P53 (Cont.(

The B-cell leukemia/lymphoma gene (Bcl-2) is an The B-cell leukemia/lymphoma gene (Bcl-2) is an

important effector gene during the apoptotic process.important effector gene during the apoptotic process.

It is a proto-oncogene located at site of translocation It is a proto-oncogene located at site of translocation

between chromosome 14 and 18.between chromosome 14 and 18.

Bcl-2 protein prolongs cell life by inhibiting Bcl-2 protein prolongs cell life by inhibiting

apoptosis.apoptosis.

Bcl-2

Terminally differentiated cells lose Bcl-2 expression. In Terminally differentiated cells lose Bcl-2 expression. In

the skin, basal keratinocytes express Bcl-2 and suprabasal the skin, basal keratinocytes express Bcl-2 and suprabasal

keratinocytes don’t .keratinocytes don’t .

Bcl-2 (Cont.(

Bcl-2 is found mainly Bcl-2 is found mainly

on the mitochondrial on the mitochondrial

membrane and membrane and

endoplasmic reticulum endoplasmic reticulum

(cytoplasmic).(cytoplasmic).

Recently, a number of Bcl-2 family members have been Recently, a number of Bcl-2 family members have been

identified:identified:

Bcl-2, Bcl-xl, Bcl-w and Mcl-1 Bcl-2, Bcl-xl, Bcl-w and Mcl-1 inhibit inhibit

apoptosis.apoptosis.

Bax, Bak, Bik and Bad and Bcl-xs Bax, Bak, Bik and Bad and Bcl-xs activate activate

apoptosis.apoptosis.

Bcl-2 (Cont.(

Pro-apoptotic Anti-apoptotic- P53- Fas- Bcl-2 Family:- Bax- Bak- Bcl-xs- Bad- Bid- Bik- Bok- Caspase (CP) Family:- Initiator CPs (CP 8,9,10,12)- Effector CPs (CP 3,6,7)

- Bcl-2 Family:- Bcl-2- Bcl-xL- Mcl-1- Bcl-w- A1- Bfl-1- NR13

Pro- and Anti-apoptotic Genes and Proteins

The ratio of antiapoptotic The ratio of antiapoptotic

Vs proapoptotic levels has Vs proapoptotic levels has

been suggested to determine been suggested to determine

the inherent susceptibility of the inherent susceptibility of

a given cell to respond to a given cell to respond to

apoptotic signals.apoptotic signals.

Fas/Fas-LFas/Fas-L The extrinsic pathway mediated by Fas The extrinsic pathway mediated by Fas

(Factor of Apoptosis Stimulus), which is a 45-(Factor of Apoptosis Stimulus), which is a 45-kDa glycosylated type 1 transmembrane kDa glycosylated type 1 transmembrane receptor and a member of TNF superfamily, receptor and a member of TNF superfamily, ligates to its ligand (Fas-L) resulting in ligates to its ligand (Fas-L) resulting in formation of a death-inducing signaling formation of a death-inducing signaling complex (DISC), which contains the Fas-complex (DISC), which contains the Fas-associated death domain protein (FADD), associated death domain protein (FADD), activating initiator caspase (cysteine activating initiator caspase (cysteine aspartase) 8 and 10, that in turn activates the aspartase) 8 and 10, that in turn activates the executioner caspases 3,6,7.executioner caspases 3,6,7.

BaxBax Bax (Bcl-2 Associated X-protein) is a pro-Bax (Bcl-2 Associated X-protein) is a pro-

apoptotic gene that was discovered as a apoptotic gene that was discovered as a first gene in apoptotic cascade initiated first gene in apoptotic cascade initiated by p53 gene.by p53 gene.

It’s a cytoplasmic pro-apoptotic Bcl-2 It’s a cytoplasmic pro-apoptotic Bcl-2 family member.family member.

Proapoptotic Stimuli

Death receptorDeath receptorFas/Fas LigandFas/Fas LigandTNF/TNFRTNF/TNFR

StressStressHeat ShockHeat ShockUltra VioletUltra Violet

DNA DamageDNA DamageChemotherapyChemotherapyRadiationRadiation

Growth factorGrowth factoror Cytokineor CytokineDepletionDepletion

Cell Death SignalCell Death Signal

p53p53

Activation of CaspasesActivation of Caspases

Cleavage of Death substratesCleavage of Death substrates

ApoptosisApoptosisDegradation of Chromosomal DNADegradation of Chromosomal DNA

Bcl-2Bcl-2

The Caspases Family

P53P53 has been implicated in the control of cell cycle, has been implicated in the control of cell cycle,

DNA repair and synthesis, cell differentiation, DNA repair and synthesis, cell differentiation,

genomic stability and programmed cell death.genomic stability and programmed cell death.

Mutant form can act as a dominant oncogene, whereas Mutant form can act as a dominant oncogene, whereas

wild-type P53 has characteristics of a recessive wild-type P53 has characteristics of a recessive

tumour-suppressor gene.tumour-suppressor gene.

Mutation analysis have indicated that even normally Mutation analysis have indicated that even normally

looking sun-exposed skin may have mutant form of looking sun-exposed skin may have mutant form of

P53.P53.

These changes in P53 expression observed after These changes in P53 expression observed after treatment may play a role in mediating the treatment may play a role in mediating the effects of such treatment modalities on the effects of such treatment modalities on the epidermis, as well as prevention of actinic epidermis, as well as prevention of actinic neoplasia by adjusting any disturbance in the neoplasia by adjusting any disturbance in the proliferation/apoptosis balance observed in proliferation/apoptosis balance observed in photoaged facial skin.photoaged facial skin.

(El-Domyati et al., 2003 & 2007)

The P53 tumour-suppressor gene is the classic The P53 tumour-suppressor gene is the classic example of the tumour-suppressor genes that can act example of the tumour-suppressor genes that can act as an oncogene, since it is found mutated in more than as an oncogene, since it is found mutated in more than 50% of human cancers.50% of human cancers.

Mutations of P53 gene have been reported in non-Mutations of P53 gene have been reported in non-melanoma skin cancers like BCC, SCC, as well as melanoma skin cancers like BCC, SCC, as well as Actinic Keratosis suggesting that loss of P53 function Actinic Keratosis suggesting that loss of P53 function is an early event in skin carcinogenesis.is an early event in skin carcinogenesis.

)Ponten and Lundeberg, 2003(

This alteration in proliferation/apoptosis balance could play an active This alteration in proliferation/apoptosis balance could play an active role in tumorigenesisrole in tumorigenesis..

Mutations in oncogene lead to “gain of function”, Mutations in oncogene lead to “gain of function”, which signals the cell to keep on dividing and which signals the cell to keep on dividing and dividing without restraint.dividing without restraint.

In contrast, mutation in tumour-suppressor genes In contrast, mutation in tumour-suppressor genes leads to “loss of function” in mechanisms by which leads to “loss of function” in mechanisms by which cell ordinarily prevents itself from replicating if it has cell ordinarily prevents itself from replicating if it has sustained potentially dangerous damage to its DNA. sustained potentially dangerous damage to its DNA.

Thus, Thus, wild typewild type of P53 of P53 is considered a tumour is considered a tumour suppressor gene.suppressor gene.

Whereas, Whereas, mutatedmutated form of P53 can act form of P53 can act like a dominant like a dominant oncogene.oncogene.

New New diagnosticdiagnostic and and prognosticprognostic strategies could be strategies could be undertaken based on the detection of tissue P53 and undertaken based on the detection of tissue P53 and apoptosis in cancer patients.apoptosis in cancer patients.

Future therapy based on the Future therapy based on the renewalrenewal of P53 of P53 function, or function, or developmentdevelopment of drugs that of drugs that mimicmimic tumour-suppressor function, or tumour-suppressor function, or inhibitinginhibiting mutations mutations acquired activity, or inducing apoptosis would be of acquired activity, or inducing apoptosis would be of utmost importance to the approaches focusing on utmost importance to the approaches focusing on restoration of tumour suppressor function and restoration of tumour suppressor function and tumour management.tumour management.