2. APOPTOSIS AND ITS RELATION TO CANCER Engin ULUKAYA (MD, PhD) Uludağ University, Department of Biochemistry, 16059 Bursa / TURKEY

3. Talk about.... 1. APOPTOSIS 2. DEREGULATION OF APOPTOSIS IN MALIGNANCIES 3. POTENTIAL ROLE OF APOPTOSIS IN CANCER TREATMENT

4. APOPTOSIS

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

6. 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

8. 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

9. 2) Translocation of phosphatidylserine 3) ATP-dependency 4) Internucleosomal DNA fragmentation (ladder pattern) 5) No apoptosis at +4 o C 6) No inflammation

10. CELL SURFACE DEATH RECEPTORS Calbiochem, Inc

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

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

13. THE APOPTOTIC PATHWAY TriggersModulatorsEffectorsSubstratesDEATH. 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

14. AN APOPTOTIC CELL IN CULTURE Collins JA, et al. 1997

15. From the archive of Dr Ulukaya

16. DEREGULATION OF APOPTOSIS IN MALIGNANCIES

17. Transfection studies in rat fibroblasts Ras Apoptosis Tumor growth c-myc Apoptosis Tumor growth 1

18. Igney and Krammer 1999 2

19. CASPASES CAN BE INHIBITED BY VIRUSES... CrmA... Baculovirüs p35... Ebstein Barr Virüs BHRFI proteini... Ebstein Barr Virüs LMP-1 proteini 3

20. APOPTOSIS-RELATED CELLULAR PROTEINS INVOLVE IN THE PROGRESSION OF MALIGNANCIES... p53... pRb... Fas... Mdm2... c-myc... c-Jun... Bcl-2 family 4

21. Bcl-2 FAMILY - Bcl-2 - Bcl-X L - Mcl-1 ******************* - p35 (Baculovirüs) -BHRF1 (Ebstein-Barr Virüsü) - LMW5 HL (“African Swine Fever Virus”) - p19 (E1B) (Adenovirüs) - Bax - Bcl-X S - Bak - Bad *************** -???? Anti-apoptoticPro-apoptotic

22. Bcl-X L Bad Bcl-X L Bax Bcl-2 Bax Bcl-2 Bad CELL SURVIVAL CELL DEATH

23. . 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

24. "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 OncoTech, Inc

25. Is Cancer Puzzling ? Question 1...

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

27. POTENTIAL ROLE OF APOPTOSIS IN CANCER TREATMENT

28. Things to do.... Determination of the Apoptosis- Related Proteins (1)

29. . 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)

30. Measurement of the Cytotoxic (Apoptosis- Inducing) Effects of Chemotherapeutic Agents on Individual Cancer Tissue Specimens Removed from Cancer Patients Things to do.... (2)

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

33. METHODS FOR THE CHEMOSENSITIVITY TESTING 1... Clonogenic assay 2... Thymidine Incorporation Assay 3... Tissue Explant Assay 4... MTT assay 5... Fluorescence Assay 6... DISC Assay 7... The ISCO* ATP-Tumor Chemosensitivity Assay (ATP-TCA) *ISCO, International Society of Chemosensitivity Testing in Oncology

34. From the archive of Dr Ulukaya

35. Kindly supplied from Dr I Cree

36. ... 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)....

37. 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%

38. 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

39. SUMMARY It 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 of patient’s response to chemo- or radio- therapy as well as of survival rates Chemosensitivity testing, thereby individualised chemotherapy on the basis of patient-specificity, seems to be promising in the succesful treatment of malignancies. This testing, thereby, may revolutionize the way we use anti- cancer drugs in near future