CELL CYCLE CONTROL

AND
CANCER

Prof T Ranga Rao MD

Biochemistry Department
Guntur Medical College,GUNTUR
The steps of cellular growth
and division that
we observe today
in our own cells is there in
the original eukaryotic cells
since time immemorial
 Outline

Cell cycle control

Biochemical
Cancer
Progressing from biochemical to genetic
to clinical
Three biologists share 2001’s Nobel Prize in Physiology or Medicine
for helping to unlock the secrets of the cell cycle,

Leland H. Hartwell,
USA
Paul M. Nurse,
UK
R. Timothy Hunt,
UK
 Main phases of a cell cycle

Two chromatids
per chromosome G2

One chromatid
per chromosome
S

G1
One chromatid
per chromosome
The alternative to cell cycling

G2

G0 cells are not dividing,

G1 but instead will be
G0 differentiated
 The major cell cycle checkpoints
The M checkpoint:
The G2 checkpoint:
Are chromosomes
Has all DNA
G2 aligned in the equatorial
completed division?
plane?
M

S
All three
checkpoints:
G1
Is the environment
The G1 checkpoint:
favorable?
Is the cell large enough?
 The integration at Cyclin
dependent Kinase (CDK)
Has this phosphate
been removed
Is cyclin
Has this phosphate present
been added
P
P

CDK activity is “ON” only if the answer is “yes” to all questions

 CDK-molecules and cyclins drive the
cell from one phase to the next.
The CDK-molecules can be
compared with an engine and the
cyclins with a gear box controlling
whether the engine will run in the
idling state or drive the cell forward in
the cell cycle.
 The importance of Cyclins
• Different Cyclins increase in
abundance at different stages
of the cell cycle

• Each Cyclin Dependent Kinase (CDK)

interacts with one or two specific
Cyclin(s)
 CANCER

Growth 90 % sporadic
Invasion
Spread 10% familial
 Cancer:
General
Etiology and
Pathogenesis
 Cancer
• Cells in culture and in
vivo exhibit contact-
inhibition through
feedback mechanism.

• Cancer cells lack

contact inhibition
feedback mechanisms.
Clumps or foci develop.
 Specific Cellular Functions in
Cancer: Genetic Alterations
Genetic Instability

DNA Repair

CANCER

Tumor Suppressor
Oncogenes
Genes

Interstitial Deletion Gene Amplification

Inactivating Mutation Gene Overexpression
Hypermethylation Activating Mutation
Pathways leading to cancer
 Alterations of Specific Cellular
Functions in Cancer
DNA Repair
Tumor Suppressor Oncogenes
Genes

Inactivation Activation
Proliferation

CANCER
 Definitions
• A tumor suppressor gene has a normal
function in the cell, but can be inactivated.
The normal function is putting a brake on
the cell cycle in a regulated fashion.

• A proto-oncogene has a normal function in

the cell, but can be activated into an
oncogene. The normal function is
to promote cell division in a regulated fashion.
Point mutations are often the initial
events in cancer development
• Point mutations lead to changes in
amino acid sequence

• Point mutations occur as result of

DNA replication or (repair of) DNA
damage

• Especially DNA polymerases

repairing DNA damage are
error prone
Some of the factors involved in G1
checkpoint control
p16
Apoptosis

p53
p21
? Cyclin
?

CD
D

K4
E2F pRB

G1

G2
 CDK-
Activation
&Cancer
Papilloma virus-Cancer
Papilloma virus -- interaction with
p53 and pRB
p16
Apoptosis

E6
p53
p21 ?
p53 degradation Cyclin
?

CD
D

K4
E2F E7 pRB

G1 pRB inactivation
relieves E2F of
inhibition Papilloma
M virus proteins

S
G2
 Tumor suppressors include p53,
p16, p21, and pRB
p16
Apoptosis

p53
p21 ? Cyclin
?

CD
D

K4
E2F pRB

G1

S
G2
 The role of p53

p16
Apoptosis

p53
p21 ? Cyclin
?

CD
D

K4
E2F pRB

G1

G2
 P53
and the
cell cycle

P21 is a CDK inhibitor

P53 arrests the cell cycle primarily by upregulating p21

which inactivates CDK/cyclin.
P53 can also activate apoptosis
 p53 - the most commonly
mutated gene in cancers
• Mutations in p53 almost always
somatic, very rarely inherited
• If DNA is damaged, p53 gene
product stops cell division through
the pRB pathway
• If DNA and the pRB gene is damaged,
p53 will induce apoptosis
(programmed cell death)
 p53 - continued
• p53 mutated cell can therefore
proceed through S-phase with
damaged DNA
• Radiation treatment of tumors
damages DNA.
• Tumor cells in which p53 is mutated
survives Radiation better giving the
patient a poorer prognosis.
 p53 - continued

• p53 is most often classified as a tumor

suppressor gene
• Unlike other tumor suppressors, p53
has been found with dominant negative
mutations. In these instances, one
mutation in one of the alleles is enough
to produce a phenotype
 Tumor suppressor genes

• Normally, one functional allele in a cell

is enough to have full function

• Therefore, Knudsons two-hit hypothesis

on inactivation of the second allele
 Knudsons two-hit hypothesis
• Knudsons two-hit hypothesis relates to tumor
suppressor genes.
• It states that in some cancers, like the
retinoblastoma, two hits are necessary before
the cells start uncontrolled divisions. In the
inherited cases, hit number one is found in all
cells. In sporadic cases, hit number one is
found in one or few somatic cell, and hit
number two happens at least in one cell.
 Knudsons two-hit hypothesis (2)
• Familial cancer shows phenotype only if
a second hit does happen.
• Well known familial cancers show
dominant inheritance with penetrance of
approx. 60-90%.
• How can the penetrance be that high?
• High penetrance means that loss of the
second allele happens with a high probability
in one or few of the many cells in the body.
 Tumor suppressors in summary
• TS gene products acts as brakes on the cell
cycle. Loss of both alleles leads to loss of the
brake action, therefore leading to increased
cell divisions.

• TS genes can also be involved in DNA repair

and apoptosis, with loss of both alleles leading
to increased cell divisions.
Where mitogens enter the picture

p16
Apoptosis

p53
p21 ? Cyclin
?

CD
D

K4
E2F pRB

G1

S Mitogens
G2
 Examples of mitogens

• Insulin

• Epidermal growth factor (EGF),

platelet derived GF (PDGF),
fibroblast GF (FGF),
insulin-like GF (IGF), etc.
Response of the insulin receptor kinase (IRK)
to ligand binding
• Heterotetramer (2a, 2b)

• Insulin binding leads to

change in structure (different
from other RTKs)

• Conformation change
activates b-subunit TK
activity

• b subunit phosphorylates Tyr

residues on cytoplasmic
domains as well as
downstream substrates (IRS)
Three-dimensional structures of the insulin
receptor tyrosine kinase (IRK)

IRK conformational change upon

activation loop phosphorylation.
The N-terminal lobe of IRK is
colored white and the C-terminal
lobe is colored dark grey. The
activation loop (green) contains
autophosphorylation sites Y1158,
Y1162 and Y1163, and the
catalytic loop (orange) contains
the putative catalytic base,
D1132. Also shown are the
unbound/bound ATP analog and
tyrosine-containing substrate
peptide (pink). [Hubbard, EMBO
J. 16, 5572 (1997)]
Once Tyr-Phosphorylated, the IRK activity
trigerrs a number of signaling pathways

• Phosphatidylinositol 3-
hydroxy kinase, makes
PIP2,PIP3

• Grb2, Sos, activates Ras

• Activation of PI-PLC
Fibroblast Growth Factor
Receptor Tyrosine Kinase
• The fibroblast growth factor
receptor (FGFR) family have
been linked widely to the
development of cancer and
disease.
• Dimeric assembly of 2
FGF2:FGFR1 complexes.
FGF2 is colored orange, Ig-
like domain 2 of FGFR1 is
colored green, and Ig-like
domain 3 of FGFR1 is
colored cyan. [Plotnikov et al.,
Cell 98, 641 (1999)]
 Growth factor signaling
• Binding to external parts of receptors
• activation of second messengers by
either
– integral receptor kinase
– dissociation of trimeric G-proteins
– activation of other associated enzyme
(mostly a kinase)
• Further levels in the cascade
 Growth factor signaling (2)
• Activation of a transcription factor and
subsequent changes in gene regulation is the
most important end-point of signaling.
• Steroid hormones reach this level directly, as
they bind to receptors that themselves are
transcription factors.
Any gene encoding any of these proteins have
the potential for being a protooncogene
 Proto-oncogenes vs Oncogenes

Proto-oncogenes are the normal cellular gene from

which the viral oncogenes are derived.

Mechanism of oncogenic activation:

Mutation and/or Overexpression

Slow transforming retroviruses demonstrate that

overexpression of proto-oncogenes also plays a role in
retroviral oncogenesis.
Mechanisms of oncogene activation
 Oncogenes of Acutely Transforming Retroviri
src Rous sarcoma virus Chicken
myc Avian myelocytomatosis virus Chicken
erb A, erb B Avian erythroblastosis virus Chicken
myb Avian myeloblastosis virus Chicken
ets Avian erythroblastosis virus Chicken
rel Avian reticuloendotheliosis virus Turkey
H-ras Harvey rat sarcoma virus Rat
K-ras Kirsten murine sarcoma virus Mouse
abl Abelson murine leukemia virus Mouse
raf Murine sarcoma virus Mouse
fos Mouse osteosarcoma virus Mouse
fms Feline sarcoma virus Cat
fes Feline sarcoma virus Cat
sis Simian sarcoma virus Monkey
 Types of
proteins
encodes by
oncogenes
 The classical pathway
growth factor receptor >
Grb2 >
SOS >
RAS >
MAPKKK (RAF) >
MAPKK (MEK) >
MAPK
 The RAS protein
• RAS binds either GTP or GDP
• RAS containing GTP is active,
RAS-GDP is inactive
• RAS can hydrolyze GTP to
GDP when triggered by
GTPase-Activating Proteins
(GAPs)
• RAS exchanges GDP for GTP
when interacting with a GDP-
release protein (GNRP)
 The RAS example

• Several mitogenic proteins (growth factors)

bind to receptors with tyrosine kinase activity,
thereby causing receptor autophosphorylation
• The phospho-tyrosine gets bound by the
adapter GRB2 which binds GTP-exchange
protein SOS.
• SOS thereafter is active in exchanging GDP to
GTP on RAS
 The RAS example (2)

• Active GTP-RAS binds temporarily to

RAF(MAPKKK), releasing it from
inhibitory proteins.
• Free RAF protein binds to and
phosphorylates MEK (MAPKK).
• P-MEK binds to and phosphorylates
MAP Kinase on both tyrosine and
threonine thereby activating it
 Words
• MEK is MAP-ERK Kinase,
it phospholylates both tyrosine and serine

• MAP is Mitogen Activated Protein

• ERK is Extracellular signal-Regulated

Kinases
 MAP Kinase
• P-MAP Kinase phosphorylates multiple
targets differing among cell types.

• The targets include transcription factors

such as SRF, indirectly increasing the
levels of FOS and JUN (together = AP-1).

• AP-1 increases Cyclin-D production.

 Parallel pathways
• A number of parallel pathways exist.

• Several of these have a Jun N-terminal Kinase

(JNK) as the final activated kinase

• Each of these phosphorylates isoforms of Jun,

the partner of Fos in AP-1. P-Jun is more
stable than native Jun, thereby increasing the
effective concentration of this transcription
factor.
 Oncogenes – RAS Proteins
• RAS is a protein binding either GDP or
GTP, hydrolyzing the latter to the
former when induced by interaction with
a GTPase Activating Protein(GAP).
• Mutations in RAS often impair the
ability to hydrolyze GTP, freezing
RAS in the active state.
• HRAS, KRAS and NRAS are three
examples of RAS genes.
 Oncogenes – RAS Proteins(2)
• HRAS involved in carcinoma of colon, lung,
pancreas and others.
• KRAS involved in melanoma, thyroid
carcinoma, acute myeloblastic leukemia
(AML), etc..
• NRAS involved in myeloma and leukemia, and
in neuroblastoma. Less commonly activated
than KRAS in myeloma and leukemia cancers.
 Oncogenes in summary
• Oncogenes are very rarely inherited
• Oncogenes are the activated, cancer-
causing forms of proto-oncogenes
• Any gene performing one of the steps
from growth factor binding to gene
transcription that leads to cell division
could become an oncogene
 Clinical observations
• Inherited cancers occur earlier in life than
sporadic cancers
• Inherited cancers show more foci
(independent neoplasms) than sporadic
cancers
– E.g., breast cancer with bilateral occurrence
indicates an inherited case
• More than one case of a rare cancer in a
family indicates segregation of a familial
cancer mutation
 Knowledge of cancer mutations:
any help?
• Yes! Recent news have claimed that cheap,
fast genotyping of patient tumors combined
with knowledge of which medicine work best
for which combination of mutations, will
become the next big step forward in cancer
treatment.

• The cost of such a tailored treatment will be

even higher than current costs.
 Summary
• Tumor suppressors are inactivated
– Both alleles are inactivated in the cancer cell
• Oncogenes are activated
• Inherited cancer involves inheritance of
one mutated tumor suppressor allele
• Inherited cancer shows dominant
inheritance with reduced penetrance
THANK YOU