in the Teaching Material of
Medical Biotechnology Master’s Programmes
at the University of Pécs and at the University of Debrecen
Identification number: TÁMOP-4.1.2-08/1/A-2009-0011
CELL CYCLE AND CANCER
Zoltan Balajthy
Molecular Therapies- Lecture 12
in the Teaching Material of
Medical Biotechnology Master’s Programmes
at the University of Pécs and at the University of Debrecen
Identification number: TÁMOP-4.1.2-08/1/A-2009-0011
Learning objectives of chapter 12 and 13 . The purpose of this chapter is to describe the processes and regulations of both cell cycle and cell death, explain the unregulated cell division, and to point out the
therapeutic intervention in cancer at molecular levels.
Topics in chapter 12.
12.1. Interpretation of cell cycle
Constitutive and Inducible Cell Cycle Kinase Inhibitors
12.2. Mitogen-activated Protein (MAP) Kinase Cascade
Transcriptional Events in G1 Phase of Cell-cycle
Mechanisms of Gene-suppression by the Retinoblastoma Protein 12.3. Biochemical Events of Cell-cycle – in M Phase
12.4. Cancer Causing Genes in Mitotic Signal Pathway Proto-oncogenes and Oncogenes
12.5. Different Families of Receptor Tyrosine Kinases (RTK)
Proto-oncogenes are normal genes that can become oncogenes
12.6. Therapeutic Targets
Monoclonal antibodies and specific inhibitors
Checkpoints controll The conventional cell cycle is modified to indicate :
- G1 activities, those preparatory for S phase, may begin during the previous cycle, concurrently with G2 and mitotic events.
- early preparation for mitosis (G2) may overlap with S
- exit from G1 into the G0 quiescent state and reentry into the cycle
The critical points:
C (indicating competence), V (end of entry) R (restriction point, end of progression)
12.1. The Functional Cell Cycle
Cyclin-CDK Regulators of Cell-Cycle
G1 S G2 M G1
INTERPHASE MITOSIS KINESISCYTO-
Methaphase Anaphase cyclin D
CDK
cyclin E CDK
START
cyclin A CDK
cyclin B
CDK APC
G2/M
START G2/M
Metaphase - Anaphase
G0 P27
CDK:cyclin-dependent protein kinases, APC: anaphase promoting complex, P27:cyclin-dependent protein kinases inhibitor,
cell divison cycle: CDC,
cyclin-dependent protein kinases: CDK
cyclin-dependent protein kinases inhibitor: CDI
, CDK1
CKIs
DNA damade, starvation or factor inducible
DNA-damage inducible
Constitutive CKI Constitutive
CKI APC
Constitutive and Inducible Cell Cycle Kinase Inhibitors
, and are check points at the „s”, „gm”, and „m” stable states
12.1. Mitogen-activated Protein (MAP) Kinase Cascade
SOS: guanine nucleotide exchange factor GAP: GTPase activating protein
ERK: extracellular signal-regulated kinases
RAF, MEK, ERK: protein kinase phosphorilation cascade FOS: transcription factor
MEK
ERK
FosP P
Raf
P P P P
P P Shc
SOS GRB2
Receptor tyrosine kinase
L
ERK
P P RAS
GDP
RAS GTP
GDP Pi
target
Active Inactive
GAP
GTP Incoming
signal
Incoming signal
Outgoing signal
Fatty acid and farnesyl modification of ras
SH3 SH3
MEK
ERK ERK
FosPP PP
Raf Raf Raf Raf
P PPP PP PP PP
P P PP PP Shc
SOS GRB2
SOS GRB2
Receptor tyrosine kinase
L L
ERK ERK
P P PP PP RAS
GDP
RAS GTP
GDP Pi
target
Active Inactive
GAP
GTP Incoming
signal
Incoming signal
Outgoing signal
Fatty acid and farnesyl modification of ras
SH3 SH3
CDK inhibitors
CDK inhibitors CDK inhibitors
Dihydrofolate reductase Thymidine kinase Thymidilate synthase DNA polymerase
E2F: transcription factor E2F1 EGF: epidermal growth factor
CDK: cyclin-dependent protein kinase Rb: retinoblastoma protein
D1, A, E: Cyclin D1, A és E
DNS replication machinery
Start of S phase
pozitív erősítés
transzkripció leállítás
Transcriptional Events in G1 Phase of Cell-cycle
DNS replication machinery
E2F Rb HDAC
Pol II
Off
Pol II
On
Ac Ac
Ac
Ac Ac
Ac Ac Ac Ac
Ac Ac Ac
E2F
AcRb
HDAC
G1 CDKactive p
p
p p
HAC
TF
Ac Ac
Ac
Cyclin E, A Cyclin E
Active CDK2
Cyclin E, A
E2F Rb HDAC
Pol II
Off
Pol II
On
Ac Ac
Ac
Ac Ac
Ac Ac Ac Ac
Ac Ac Ac
E2F
AcRb
HDAC
G1 CDKactive p
p
p p
HAC
TF
Ac Ac
Ac
Cyclin E, A Cyclin E
Active CDK2
Cyclin E, A
Mechanisms of Gene-suppression by the Retinoblastoma Protein
11.3. Biochemical Events of Cell-cycle – in M Phase
T
T Y
cyclin B
T
T Y
CKI p
cyclin B CDK p
p
p p
p T
T Y
cyclin B CDK
p T
T Y
CDK
C i
y kl n
T
T Y
CDK p p Inhibitory
P-ation
Stabilizing
P-ation CDC 25
CKI
Proteolytic degradation
APC
Inactive protein kinase Active protein
kinase
Proteolytic degradation
Substrats Substrats
p
Histone H1 Lamin Vimentin caldesmon
Chromatin condensation Cytoskeleton rearranged Mitotic spindle formation Nuclear membrane dissolved
Cell shape changes
Substrats phosphatases
p +
G2 M G1
CDK
T
T Y
cyclin B
T
T Y
CKI p
cyclin B CDK p
p
p p
p T
T Y
cyclin B CDK
p T
T Y
CDK
C i
y kl n
T
T Y
CDK p p Inhibitory
P-ation
Stabilizing
P-ation CDC 25
CKI
Proteolytic degradation
APC
Inactive protein kinase Active protein
kinase
Proteolytic degradation
Substrats Substrats
p
Histone H1 Lamin Vimentin caldesmon
Chromatin condensation Cytoskeleton rearranged Mitotic spindle formation Nuclear membrane dissolved
Cell shape changes
Substrats phosphatases
p +
G2 M G1
CDK
12.4. Cancer Causing Genes in Mitotic Signal Pathway
Shc
SOS GRB2
Receptor tyrosine kinase
L
RAS GTP
SH3 SH3
L
RAF
MEK
ERK
Fos
ERKNUCLEUS P
Transcription factor (activator) Protein kinases phosphorilation cascade
Gene expression
Proteins that stimulate the cell cycle
G protein
Receptor
NUCLEUS Transcription factor (sucs as p53) Protein kinases
phosphorilation cascade
Gene expression
Proteins that inhibit the cell cycle
G protein
GTP GTP
Growths-stimulating pathway Growths-inhibiting pathway L
Defective or missing transcription factor, such as p53, can’t activate transcription
Protein overexpressed Protein absent X
Cell cycle overstimulated Cell cycle not inhibited Effect of abnormalities X
Shc
SOS GRB2
Receptor tyrosine kinase
L
RAS GTP
SH3 SH3
L
RAF
MEK
ERK
Fos
ERKNUCLEUS P
Transcription factor (activator) Protein kinases phosphorilation cascade
Gene expression
Proteins that stimulate the cell cycle
G protein
Receptor
NUCLEUS Transcription factor (sucs as p53) Protein kinases
phosphorilation cascade
Gene expression
Proteins that inhibit the cell cycle
G protein
GTP GTP
Growths-stimulating pathway Growths-inhibiting pathway L
Defective or missing transcription factor, such as p53, can’t activate transcription
Protein overexpressed Protein absent X
Cell cycle overstimulated Cell cycle not inhibited Effect of abnormalities X
Proto-oncogenes and Oncogenes
Proto-oncogenes code for growth factors.
Mutation cause overpro- duction of growth factors.
Proto-oncogenes code for growth factor
receptors.
Mutant receptor no longer requires growth factor binding.
Proto-oncogenes code for proteins that
transmit external
stimuli for cell division.
Stimulation no longer needed.
Proto-oncogenes code for transcription factors.
Factors always bind to their target gene promotor.
Possible Biochemical Mechanisms of Protooncogene - oncogene Conversion
- promoter insertion - enhancer insertion
- chromosomal translocation - gene amplification
- single point mutation or deletion
Out the 5 mechanisms described above, the first 4 involve an increase in amount of the product of an oncogene due to increased transcription but no alteration of the structure of the product of the oncogene. Thus it appears that increased amounts of the product of an oncogene may be sufficient to push a cell becoming malignant. The fifth mechanism, single point mutation, involves a change in the structure of the product of the oncogene but not necessarily any change in its amount. This implies that the presence of a structurally abnormal key regulatory protein in a cell may be sufficient to tip the scale toward malignancy.
EGFR (HER 2,3,4
TGF-a EGF
IGF-1R
P P
P P IGF-1 IGF-2
PDGFR
PDGF
VEGF-A VEGF-B
VEGFR
HER2
None
EGFR/HER2
P P
P P
Receptor Heterodimerization and Activation
12.5. Different Families of Receptor Tyrosine Kinases (RTK) Recognize a Diverse Set of Different Ligands
Transmembrane Lipophilic Segment
Intracellular Protein Tyrosine Kinase Domain
Extracellular Binding Domain
EGFR-s (HER3,4)
TGF-a EGF
EGFR
Kinase Catalytic Site Cytosolic
Domein Extracellular
Domein
Receptor Homodimerization Ligand-binding Site
Ligand
P P
P P
ATP ADP
Ligand Binding Activates RTKs by Dimerization
Proto-oncogenes are Normal Genes That Can Become Oncogenes
EGFR HER2HER2 EGFR
Plasma membrane Val
Oncogenic
Mutation (Val → Glu)
(Deletion)
Glutamine
Ligand-independent Receptoer Oncoprotein Activating mutations
in RTKs take several forms but all lead to ligand-independent dimerization and thus activation.
Proto-oncogene receptor protein
ErbB Neu
mAbs
L
X X
Signal transductiom
X
L
X X
Signal transductiom
X
L Tyrosine kinase
inhibitors (TKI)
TKI TKI
L L
✝ ✝
Cell death Toxin-induced
cell death
L L
RNA interference
Protein synthesis
X
Neu, EGFR-targeting Methods
Expression of HER2 Receptor on the Surface of Normal and Tumor Cells Herceptin Binds to the HER2 Receptor on the Surface of Tumor Cells
Normal expression of HER2 receptor
Increased expression of HER2 receptor
Herceptin attaches itself to HER2 receptor Herceptin flags the cancer cells for destruction by the immune system
1, blocking signalization on cell surface receptor
2, inhibition of TK activity of TKR
3, inhibition of protein kinases phosphorilation cascade
4, activation of growths-inhibiting pathway (Rb) or restoration of p53 function
5, inhibition of angiogenesis 6, inhibition of methastasis
In addition to the classical form of cancer treatment: like surgery, chemotherapy and radiotherapy, there are new therapeutic targets at molecular level against cancer cells.
1
2 TKI
3
Shc
SOS GRB2
SOS GRB2
Receptor tyrosine kinase
L L
RAS GTP
SH3 SH3
L L
RAF RAF
MEK MEK
ERK ERK
Transcription factor (activator) Protein kinases phosphorilation cascade
Gene expression
Proteins that stimulate the cell cycle
G protein
GTP GTP
Growths-stimulating pathway
Protein overexpressed
NUCLEUS Transcription factor (sucs as p53)
Gene expression
Rb
E2F 4
TKI
12.6. Therapeutic Targets
Blocking of Oncoproteins of EGFR and Mitogen-activated Protein Kinase Signalization via Monoclonal abs. and Specific Inhibitors
MEK
ERK
Fos
Raf
SOS GRB2
RAS GDP
Ligand binding blocked by antibodies
TKI: Tyrosine kinase inhibitor
SOS: guanine nucleotide exchange factor GAP: GTPase activating protein
ERK: extracellular signal-regulated kinases RAF, MEK, ERK: protein kinase
phosphorilation cascade FOS: transcription factor
Fatty acid and farnesyl modification of ras
SH3 SH3
P P
X X inactive
inactive
inactive inactive
L L
Signal transduction cascade blocked
Gene activation and cell cycle progression inhibited TKI TKI
Ras farnesyltransferase Inhibitors(BMS-214662)
MEK inhibitor
CI-1040
Raf inhibitor
BAY 43-9006
Tyrosine kinase inhibitor
MEK
ERK ERK
Fos
Raf Raf
SOS GRB2
RAS GDP
Ligand binding blocked by antibodies
TKI: Tyrosine kinase inhibitor
SOS: guanine nucleotide exchange factor GAP: GTPase activating protein
ERK: extracellular signal-regulated kinases RAF, MEK, ERK: protein kinase
phosphorilation cascade FOS: transcription factor
Fatty acid and farnesyl modification of ras
SH3 SH3
P P
X X inactive
inactive
inactive inactive
L L
Signal transduction cascade blocked
Gene activation and cell cycle progression inhibited TKI
TKI TKI TKI
Ras farnesyltransferase Inhibitors(BMS-214662)
MEK inhibitor
CI-1040
Raf inhibitor
BAY 43-9006
Tyrosine kinase inhibitor
