oncogenes and tumour suppressor genes

Question 1

major functional changes in. cancer

Answer 1

Increased growth (loss of growth regulation, stimulation of environment promoting growth e.g. angiogenesis)
Failure to undergo programmed cell death (apoptosis) or senescence
Loss of differentiation (including alterations in cell migration and adhesion)
Failure to repair DNA damage (including chromosomal instability)

Question 2

growth factor signalling, oncogenes and tumour suppressor genes

Answer 2

Many oncogenes are normally components of growth factor signalling pathways that when mutated produce products in higher quantities or whose altered products have increased activity and therefore act in a dominant manner
Many tumour suppressor gene products act as a stop signal to uncontrolled growth, may inhibit the cell cycle or trigger apoptosis

Question 3

describe oncogenes

Answer 3

make cells divide, driving cell division forward

in cancer, pick up mutation mean they are permanently active

= gain of function

Question 4

describe tumour suppressor genes

Answer 4

counteract the oncogene

loss of function

Question 5

rou’s protocol for inducing sacroma in chickens

Answer 5

tumour developed weeks later

taking new sarcoma, filtrates produced could induce tumour in other chickens

cycles repeated indefinitely. carcinogenic agent small enough to pass through filter

filter used excluded bacteria it was not small enough to exclude viruses

rous concluded that virus must be responsible for induction of tumour formation

Question 6

capture of c-src by retrovirus

Answer 6

during evolution, virus can acquire fragments of genes from host at integration and process results In creation of oncogenes

oncogene product was characterised as a 60kDa intracellular tyrosine kinase

can phosphorylate cellular proteins and effect growth

exception to central dogma DNA-RNA protein

Question 7

the oncogene hypothesis

Answer 7

Bishop and Varmus used different strains of Rous sarcoma virus in their research, they:

Identified the v-src oncogene as responsible for causing cancer.

Used hybridization experiments, and they found that the c-src gene was present in the genome of many species.

They then showed that the host cell c-src gene was normally involved in the positive regulation of cell growth and cell division.

Following infection, however, the v-src oncogene was expressed at high levels in the host cell, leading to uncontrolled host cell growth, unrestricted host cell division, and cancer.

Proto oncogenes are normal genes that can control growth

Various agents, including radiation, chemical carcinogens, and, perhaps, exogenously added viruses, may transform cells by “switching on” the endogenous oncogenic information.

Question 8

viral oncogenesis

Answer 8

Approximately 15%-20% of all human cancers are caused by oncoviruses

Viral oncogenes can be transmitted by either DNA or RNA viruses.

DNA viruses can cause lytic infection leading to the death of the cellular host or can replicate their DNA along with that of the
host and promote neoplastic transformation

DNA Viruses
Encode various proteins along with
environmental factors can initiate
and maintain tumours

RNA Viruses
Integrate DNA copies of their genomes
into the genome of the host cell and as
these contain transforming oncogenes 
they induce cancerous transformation 
of the host

Question 9

activation of oncogenes

Answer 9

over 100 identified oncogenes

examples of oncogenes for every type of protein involved in a growth factor signal transduction pathway

these genes captured by animal retroviruses are altered in human cancer, activation can involve mutations, insertions amplifications and translocations

Question 10

describe activation of oncogenes

Answer 10

mutation
amplification/duplication
translocation

Question 11

porto-oncogenes encode components of the growth factor signal transduction pathway

Answer 11

4 types of proteins are involved in the transduction of growth signals
Normally
Growth factors
Growth factor receptors
Intracellular signal transducers
Nuclear transcription factors

Growth factors, signal transduction and cancer
The majority of oncogene proteins function as elements of the signalling
pathways that regulate cell proliferation and survival in response to growth
factor stimulation

Oncogene proteins act as growth factors (e.g.EGF),
growth factor receptors (e.g. ErbB) and intracellular signalling molecules (Ras and Raf).
Ras and Raf activate the ERK MAP kinase pathway, leading to the induction of additional
genes (e.g. fos) that encode potentially oncogenic transcriptional regulatory proteins

To date-over 100 identified oncogenes

Question 12

intracellular signal transducers

RAS oncogene family

Answer 12

ras genes identified from 2 cancer causing viruses

RAS proteins are small GTPases that are normally bound to GDP in neutral state

oncogenic activation of ras is seen in about 30% human cancers

most commonly mutated oncogene

Question 13

intracellular signal transducers 2

RAS oncogene family

Answer 13

1. Binding of extracellular growth factor signal
2. Promotes recruitment of RAS proteins to the receptor complex
3. Recruitment promotes Ras to exchange GDP (inactive
   Ras) with GTP (active Ras)

4. Activated Ras then initiates the remainder of the 
signalling cascade (mitogen activated protein kinases)

5. These kinases ultimately phosphorylate targets, such as
   transcription factor to promote expression of genes
   important for growth and survival

Ras hydrolyzes GTP to GDP fairly quickly, turning itself “off”

Question 14

intracellular signal transducers 3

Answer 14

Consequence of each of these mutations is a
loss of GTPase activity of the RAS protein
normally required to return active RAS to
the inactive RAS GDP

Question 15

transcription factors

MYC oncogene family

Answer 15

The MYC oncogene family consists of 3 members,
C-MYC, MYCN, and MYCL, which encode c-Myc, N-Myc,
and L-Myc, respectively

Originally identified in avian myelocytomatosis virus (AMV)

The MYC oncoproteins belong to a family of transcription factors that regulate the transcription of at least 15% of the entire genome

Major downstream effectors of MYC include those involved in ribosome biogenesis, protein translation, cell-cycle progression and metabolism, orchestrating a broad range of biological functions, such as cell proliferation, differentiation, survival, and immune surveillance

Question 16

MYC oncogene family 2

Answer 16

The MYC oncogene is overexpressed in the majority of human cancers and contributes to the cause of at least 40% of tumours

It encodes a helix-loop-helix leucine zipper transcription factor that dimerizes with its partner protein, Max, to transactivate gene expression

Generally MYC is activated when it comes under the control of foreign transcriptional promoters. This leads to a deregulation of the oncogene that drives relentless proliferation.

Such activation is a result of chromosomal translocation

Question 17

activation of MYC in Burkitts lymphoma

Answer 17

Epstein Barr virus is associated with Burkitt’s lymphoma (BL)

BL is a high grade lymphoma that can effect children from the age of 2 to 16 years

In central Africa, children with chronic malaria infections have a reduced resistance to the virus. This is known as classical African or endemic BL

All BL cases carry one of three characteristic chromosomal translocations that place the MYC gene under the regulation of the Ig heavy chain. Therefore c-myc expression is deregulated

In BL three distinct, alternative chromosomal translocations involving chromosomes 2, 14 and 22

In all three translocations a region form one of these three chromosomes is fused to a section of chromosome 8

Question 18

chromosomal translocation is responsible for the activation of other oncogenes

Answer 18

Chronic myelogenous leukaemia (CML) accounts for 15-20%
of all leukaemias

95% of CML patients carry the Philadelphia chromosome,
that is the product of the chromosomal translocation
t(9;22)(q34;q11) generating the BCR-ABL fusion protein

As a result of this translocation the tyrosine kinase activity
of the oncogene ABL is constitutive leading to abnormal
proliferation

Therapeutic strategies for CML include Imatinib (Gleevac) a tyrosine kinase inhibitor-96% remission in early-stage patients

Question 19

discovery and identification of tumour suppressor genes

Answer 19

In 1969 Henry Harris and his colleagues performed somatic cell hybridization experiments

Fusion of normal cells with tumour cells yielded hybrid cells containing chromosomes form both parents. These cells were not capable of forming tumours

Genes derived from the normal parent acted to inhibit or suppress tumour development

The first tumour suppressor gene was identified by studies of retinoblastoma, a rare childhood eye tumour

Question 20

logic of tumour suppressor genes

Answer 20

Discovery of oncogenes, an explanation for proliferation

Like other well designed control systems biological systems follow a similar logic-component promoting a process
must be counterbalanced by others that oppose the process-tumour suppressor genes

Question 21

tumour suppressor genes

Answer 21

Body has mechanisms to ‘police’ processes that regulate cell numbers
Tumour suppressor gene products act as stop signs to uncontrolled growth, promote differentiation or trigger apoptosis
Therefore they are usually regulators of cell cycle checkpoints (e.g. RB1), differentiation (e.g. APC) or DNA repair (e.g. BRCA1)
Loss of tumour suppressor gene function requires inactivation of both alleles of the gene
Inactivation can be a result of mutation or deletion
Tumour suppressor genes are defined as recessive genes
Sometimes referred to as ‘anti-oncogenes’

Question 22

the retinoblastoma gene, Rb

Answer 22

Retinoblastoma is a rare childhood cancer (1 in 20,000) that develops when
immature retinoblasts continue to grow very fast and do not turn into
mature retinal cells.

An eye that contains a tumour will reflect light back in a white colour.
Often called a “cat’s eye appearance,” the technical term for this is leukocoria.

Two forms of the disease, familial (40%) and sporadic (60%)

The hereditary mutation is on chromosome 13 (13q14),
the retinoblastoma 1 (Rb1) gene

Question 23

discovery of retinoblastoma

Answer 23

The existence of the RB1 gene was predicted in 1971 by Alfred Knudson

Whilst studying the development of retinoblastoma he proposed that the development of retinoblastoma requires two mutations, which are now known to correspond to the loss of both of the functional copies of the Rb gene - “two-hit” hypothesis

“Loss of heterozygosity“ often used to describe
the process that leads to the inactivation of the
second copy of a tumour suppressor gene
a heterozygous cell receives a second hit in
its remaining functional copy of the tumour
suppressor gene, thereby becoming homozygous
for the mutated gene.

Mutations that inactivate tumour suppressor
genes, called loss-of-function mutations, are
often point mutations or small deletions that
disrupt the function of the protein that is
encoded by the gene

Question 24

the retinoblastoma protein RB structure

Answer 24

The Rb gene family includes three members: Rb/(p105/110), p107 and Rb2/p130
-collectively known as pocket proteins

pRb is a multi functional protein (110kDa) with over 100 binding partners

A transcriptional co factor that can bind to transcription factors

RB functions in diverse cellular pathways, such as apoptosis and the
cell cycle, it has also become clear that RB regulates these pathways
through the stimulation or inhibition of the activity of interacting proteins.

Therefore, an important starting point for understanding RB function is its
structure, which acts as a scaffold for these multiple protein interactions

It’s main binding partner is the E2F transcription factor,
interacting with the large pocket

Other viral oncoproteins can bind to Rb

Question 25

retinoblastoma protein RB and cell cycle

Answer 25

• main function of Rb is to regulate the cell cycle by inhibiting the G1 to S phase transition
• 2 important proteins involved in cell cycle are: cyclins and their associated cyclin dependent kinases
• passage of a cell through the cell cycle is regulated cyclins and cyclin dependent kinases

Question 26

retinoblastoma protein RB and cell cycle 2

Answer 26

Cyclin D is the first cyclin to be synthesized and drive progression through G1 together with cdks4/6

The G1 checkpoint leads to the arrest of the cell cycle in response to DNA damage

A key substrate for cyclin D is RB protein

Cyclin D and E families and their cdks phosphorylate RB

Question 27

retinoblastoma protein RB, function, phosphorylation and activity

Answer 27

Rb protein regulates the activity of the E2F transcription factor crucial for the expression of genes required for S phase

Rb activity is regulated by phosphorylation

When the Rb tumour suppressor is active it can inhibit cell proliferation

When Rb is dephosphorylated/hypophosphorylated it is active and remains bound to E2F

When Rb is active it blocks the progression of to S phase

When Rb is hyperphosphorylates , in response to extracellular physiological signals it is inactive
Upon phosphorylation of RB, E2F is released and migrates to the nucleus to induce transcription

When RB is inactive cell cycle progression from G1 to S occurs

Question 28

inactivation of Rb - loss of function

Answer 28

Rb can be inactivated by phosphorylation, mutation, or viral oncoprotein binding

In retinoblastoma, pRb is functionally inactivated by mutations
or partial deletions

Viral inactivation found in small DNA tumour viruses
mainly by disrupting E2F binding or destabilisation of Rb
Adenovirus - E1A
Papilloma - E7
Polyoma – Large T antigen

In cancer cells RB phosphorylation is deregulated throughout
cell cycle. As a direct consequence E2F transcription factors can
induce the deregulation of the cell cycle

Without RB on watch , cells move through G1 into S
and are not subjected to usual checks

Question 29

P53 tumour suppressor

Answer 29

The p53 gene was the first tumour suppressor gene to be identified

The p53 protein is at the heart of the cell’s tumour suppressive mechanism and has been nicknamed the ‘guardian of the genome’

It is involved in sensing DNA damage and regulating cell death/apoptosis
as well as other pathways

p53 is mutated in 30-50% of commonly occurring human cancers

Frequent mutation of p53 in tumour cell genomes suggests that tumour
cells try to eliminate p53 function before they can thrive

p53 specializes in preventing the appearance of abnormal cells

Question 30

p53 tumour suppressor 2

Answer 30

Protein has an amino transactivation domain, a central DNA binding domain, a tetramerization domain and a carboxyl regulatory domain

Can bind to around 300 different gene promoter regions-main role as a transcription factor

Question 31

regulation of P53 by MDM2

Answer 31

Normally levels of p53 protein are low in cells

These levels are kept low by MDM2 protein, a ubiquitin ligase (also an oncogene)

In unstressed normal cells both p53 and MDM2 move between the nucleus and cytosol

MDM2 binds p535 to form a complex in the nucleus where MDM modifies the carboxyl terminus of p53 and
targets it for degradation by the proteasome

WT p53 has a short 20 min half life

Question 32

activation of p53 tumour suppressor

Answer 32

Stress signals are able to activate p53

Signals are sensed by mainly kinases that then phosphorylate p53

Phosphorylation of p53 disrupts the interaction between it and
MDM2

e.g. ionizing radiation signals through two kinases ATM/ATR
activate oncogenes such as ras induce activity of p14arf
responsible for sequestering MDM2.

P53 can thus regulate genes involved in DNA damage repair,
apoptosis and cell cycle arrest

Question 33

p53 mutation/therapeutic strategies

Answer 33

Mutational inactivation is considered to be one of the most common molecular mechanisms behind the dysfunction of p53.

Extensive mutation search revealed that more than half of human cancers carry loss of function mutations of p53

Among them, 95% of mutations were detectable within the DNA-binding domain

Role of p53 a s star player in suppressing tumorigenesis makes it a promising therapeutic target

Different strategies aimed at:

• Correcting p53 mutation and restoring wild-type p53 function by targeting its regulators

Question 34

therapeutic strategies

Answer 34

Gene therapy obvious approach

Many vectors and retroviruses have been examined

Retroviruses integrate in a stable form into the genome of infected cells. It has been demonstrated that
retrovirus-mediated gene transfer of the wild-type TP53 gene into both human lung tumour cell lines and xenograft models could lead to the inhibition of tumour cell growth

Alternative strategies- use of inhibitors PRIMA-1, Restores mutant p53 by
modifying the thiol groups in the core
domain of the protein

Nutlin- is a potent MDM2 antagonist

RITA binds to p53 and can restore
mutp53 activity

Inhibitors of CRM1 result in nuclear
accumulation of p53

Question 35

genetic analysis and personalised medicine

Answer 35

A detailed readout of the molecular faults in a patient’s tumour, and new generation of drugs that precisely target them

Classifies tumours according to their genetic make-up instead of where they grow in the body

People with the ‘same’ cancer can have different forms of the disease so responses to treatment vary

Cancers growing in different parts of the body may also share the same genetic faults so respond to similar
treatments