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 death
APOPTOSIS
Cells 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-dependency
4) Internucleosomal DNA fragmentation
(ladder pattern)
5) No apoptosis at +4
o
C
6) No inflammation

CELL SURFACE DEATH RECEPTORS
Calbiochem, Inc
CASPASES
Caspase-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 PATHWAY
Triggers Modulators Effectors Substrates DEATH
. 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
AN APOPTOTIC CELL IN CULTURE
Collins JA, et al. 1997
From the archive of Dr Ulukaya
DEREGULATION OF
APOPTOSIS IN
MALIGNANCIES
Transfection studies in rat fibroblasts
Ras
Apoptosis
Tumor growth
c-myc
Apoptosis
Tumor growth
1
Igney and
Krammer
1999
2
CASPASES CAN BE INHIBITED BY
VIRUSES
... CrmA
... Baculovirs p35
... Ebstein Barr Virs BHRFI proteini
... Ebstein Barr Virs LMP-1 proteini
3
APOPTOSIS-RELATED CELLULAR
PROTEINS INVOLVE IN THE PROGRESSION
OF MALIGNANCIES
... p53
... pRb
... Fas
... Mdm2
... c-myc
... c-Jun
... Bcl-2 family
4
Bcl-2 FAMILY


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





- Bax
- Bcl-X
S

- Bak
- Bad
***************
-????
Anti-apoptotic Pro-apoptotic
Bcl-X
L
Bad
Bcl-X
L
Bax
Bcl-2
Bax
Bax Bax
Bcl-2
Bad

CELL
SURVIVAL
CELL 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

OncoTech, Inc
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 the 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)

Measurement of the Cytotoxic (Apoptosis-
Inducing) Effects of Chemotherapeutic
Agents on Individual Cancer Tissue
Specimens Removed from Cancer Patients
Things to do ....
(2)
In Other Words...
Designation of Patient-Specific Chemotherapy
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)

*I SCO, I nternational Society of Chemosensitivity Testing in Oncology

From the archive of Dr Ulukaya
ATP-Tumour Chemosensitivity Assay
Tumour
1mm
3
Fragments
Overnight enzyme
dissociation
Wash cells,
count and
estimate viability
Plate at 20,000
cell/well
Incubate for 5-7 days,
extract ATP and read
in a luminometer
Kindly supplied from Dr I Cree



... 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

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 patients 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