APOPTOSIS AND ITS RELATION TO CANCER

Engin ULUKAYA (MD, PhD)Uluda University, Department of Biochemistry, 16059 Bursa / TURKEY

Talk about....1. APOPTOSIS

2. DEREGULATION OF APOPTOSIS IN MALIGNANCIES

3. POTENTIAL ROLE OF APOPTOSIS IN CANCER TREATMENT

APOPTOSIS

Cells are born, live for a given period of time and then die Bowen, 1998 --- Physiological cell death-- Cell suicide-- Cell deletion-- Programmed cell deathAPOPTOSISCells are born, live for a given period of time and then die Bowen, 1998

WHERE can APOPTOSIS be ENCOUNTERED ?

... Growth of Embrio... Tissue Homeostasis... Immunology... Chronic viral diseases... Neurodegenerative diseases... Reperfusion injury... Insuline-dependent Diabetes... Atheroschlerosis... Miyokard Infarction... AIDS... Development and Treatment of Malignancies

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GENERAL FEATURES OF APOPTOSIS ... Occupation of death receptors ... Dimerization of Bcl-2 family members ... Release of cytochrome c ... Activation of caspases ... Activation of DNase 1) A number of activities take place

2) Translocation of phosphatidylserine 3) ATP-dependency4) Internucleosomal DNA fragmentation (ladder pattern)5) No apoptosis at +4 oC 6) No inflammation

CASPASESCaspase-1 (ICE)Caspase-2 (ICH-1, Nedd-2)Caspase-3 (CPP32, Apopain, Yama)Caspase-4 (ICH-2, TX, ICEre)Caspase-5 (ICErel, TY)Caspase-6 (Mch2)Caspase-7 (ICE-LAP3, Mch3, CMH-1)Caspase-8 (FLICE, Mch5, MACH)Caspace-9 (Mch6, ICE-LAP6)Caspase-10 (Mch4)

SUBSTRATES for CASPASES ... PARP ... DNA-PK... pRb... Lamins... NuMA... Fodrin... -Aktin... Mdm2... Cyclin A2... Presenilin... Others

THE APOPTOTIC PATHWAYTriggersModulatorsEffectorsSubstratesDEATH. FADD. TRADD. FLIP. Bcl-2 family. Cytochrome c. p53. Mdm2. Caspases. Many cellular proteins. DNA. Growth factor Deprivation. Hypoxia. Loss of adhesion. Death receptors . Radiation . Chemotherapy

From the archive of Dr UlukayaApoptotic Cells (Electron Microscopy Image)

From the archive of Dr UlukayaApoptotic and Necrotic Cells (Fluorescence Microscopy Images)ApoptosisNecrosis

DEREGULATION OF APOPTOSIS IN MALIGNANCIES

Transfection studies in rat fibroblasts Ras ApoptosisTumor growthc-mycApoptosisTumor growth

CASPASES CAN BE INHIBITED BY VIRUSES... CrmA ... Baculovirs p35... Ebstein Barr Virs BHRFI proteini... Ebstein Barr Virs LMP-1 proteini3

APOPTOSIS-RELATED CELLULAR PROTEINS INVOLVE IN THE PROGRESSION OF MALIGNANCIES ... p53... pRb ... Fas... Mdm2... c-myc... c-Jun... Bcl-2 family4

Bcl-2 FAMILY

- Bcl-2 Bcl-XL Mcl-1******************* p35 (Baculovirs)BHRF1 (Ebstein-Barr Virs) LMW5 HL (African Swine Fever Virus) p19 (E1B) (Adenovirs)

- Bax- Bcl-XS Bak Bad***************????Anti-apoptoticPro-apoptotic

Bcl-XLBadBcl-XLBaxBcl-2BaxBaxBaxBcl-2BadCELL SURVIVALCELL DEATH

. Increased Bcl-2 --------------------------------- Poor prognosis . Increased FasL --------------------- Decreased CTL number

. FasL induction (with Doxorubicin)----------------Determines chemosensitivity

. Overexpression of Bax---------------- Improve the efficacy of chemotherapy

. p53 antibodies ------------------- Resistance to chemotherapy with cisplatin + 5-Fluorouracil Various Expression Levels of Apoptosis-Related Proteins Determine Patient-Specific Malignancy ? 5

"Right now we lump patients together and treat them with the same drugs and then deal with their variable response to treatment. We're essentially treating different diseases with the same medicine.

Richard Klausner, 1997

Is Cancer Puzzling ?Question 1 ...

Does Apoptosis Held a Key Position in the Treatment of Cancer ?Question 2 ...

POTENTIAL ROLE OF APOPTOSIS IN CANCER TREATMENT

Things to do .... Determination of Both Apoptosis Itself and Apoptosis-Related Proteins(1)

. p53 gene status--------------- Modulates the chemosensitivity . p53 level ---------- Predictor for the response to chemo- or radiotherapy (Advanced Head and Neck Carcinomas, Epithelial Ovarian Ca)

. Mutant p53 --------- Overall shortened survival (Breast Ca)

. Ratio of Bcl-2/Bax -------------------------- Prognostic factor (Hematologic Malignancies, Colon Ca) . Bcl-2 alone -------- Prognostic factor (Advanced Over Ca)

Assaying of Cytotoxic (Maybe Apoptosis-Inducing) Potential of Chemotherapeutic Agents on Individual Cancer Tissue Specimens Removed from Cancer PatientsThings to do ....(2)

In Other Words...Designation of Patient-Specific Chemotherapy

SOME METHODS FOR THE CHEMOSENSITIVITY TESTING1... Thymidine Incorporation Assay2... Tissue Explant Assay 3... MTT assay4... Fluorescence Assay 5... DISC Assay6... The ISCO* ATP-Tumor Chemosensitivity Assay (ATP-TCA)

*ISCO, International Society of Chemosensitivity Testing in Oncology

Kindly supplied from Dr Cree

ATP-Tumour Chemosensitivity Assay

Tumour

1mm3 Fragments

Overnight enzymedissociation

Wash cells, count andestimate viability

Plate at 20,000cell/well

Incubate for 5-7 days,extract ATP and readin a luminometer

... A working tumor chemosensitivity assay (TCA) could be of immense benefit to the pharmaceutical industry, oncologists and their patients (Cree and Kurbacher, 1997)

... ATP-TCA can be used to select patients who might benefit from specific chemotherapeutic agents alone or in combination (Cree et al, 1999)

In the literature (1)....

In the literature (2)....Chemotherapy guided by the ATP-TCA ... Retrospective clinical correlation in breast carcinoma (Cree et al, 1996): 97% assay evaluability, 76% accuracy, 27% imrovement in clinical response rate

... A greater benefit with regard to both ORR and PFS in platinum refractory patients (Kurbacher et al, 1998): Overall survival, 97 weeks / 69 weeks; Response rate, 64% / 37%

TWO GREAT BENEFITS Exclusion of chemotherapeutic agents which are not likely to be effective, thereby avoidance of their potential toxicity Selection of chemotherapeutic agents with the greatest likelihood of clinical effectiveness for improved response rates and prolonged survival

SUMMARYIt is considered that defective apoptosis is a feature of malignant development

Induction of apoptosis in malignancies is to be aimed

Detection of apoptosis-related proteins may be of importance in the prediction for response to chemo- or radiotherapy as well as for survival

Chemosensitivity testing, thereby individualised chemotherapy, seems to be promising in the succesful treatment of malignancies on the basis of patient-specificity