WEBER Cours 1
Oncogenes
GH65b19-20
I. QUIZZ
- Cancer is caused by alterations of the genome, but this is not necessarily the first event in the
development of a tumor.
- Virus infections can provoke cancer → Yes, major cause in (only) a few cancer forms.
- Bacterial infections can provoke cancer → Yes, example: Helicobacter pylori, Gastric cancer.
- Cancer is caused by random mutations, there is nothing you can do to minimize your risk →
No.
- Some cancer forms occur preferentially at a certain age → Yes, example: retinoblastoma,
osteosarcoma, breast cancer.
- Tall people have a higher risk to develop cancer than small people because they have more
cells → Yes.
- Larger animals (example elephants) have a higher risk to develop cancer than humans because
they have more cells → No.
- Cancer treatment has basically not changed since the last 50 years → No, example chronic
myeloid leukemia.
- Cancer cannot be fought by our immune system like the flu → No.
- Cancer is a recent disease that did not exist 1000 years ago → No.
II. Oncogenes
What type of the virus is responsible for cancer?
➢ Wildtype virus : replicates well, few tumors
o Substrain : replicates well, many tumors
o Substrain : no replication, many tumors
➔ Conclusion : la formation de cancer ne dépend pas de la réplication virale.
What are the differences between high- and low- tumorigenic viruses ?
On a un wild-type RSV RNA qui va subir une RT pour faire un ADNc (-). Ensuite,
l’ARN est détruit avec alkali et hybridé à de l’ARN viral d’un mutant (sans gene
src).
Conclusion : tumorigneic viruses contain an additional “src” gene that is not
present in the wild-type virus.
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The “src” gene is present in the host genome. The viral src hybridizes with DNA from many species →
conclusion : a gene causing cancer is highly conserved in birds, mice, human, etc.
How does src come into the virus?
➔ Conclusion the scr gene is “stolen” from its host.
More retroviruses in bird, cat, mouse: part of their genome replaced by cellular gene/ It is these genes
that provoke tumors.
The viral for of scr (v-src) is mutated. Regulatory part lost in viral version, protein becomes hyperactive.
In general, the retroviral oncogene is a mutated version of its original cellular version. This usually leads
to hyperactive proteins.
➢ Example the v-myc oncogene – found in retroviruses for different species: various mutated
versions of myc.
➢ Regulatory (noncoding) parts of the myc-cDNA removed, protein often fused with viral gag
protein – protein stabilized, hyperactivity.
Are these findings important for human cancer?
Are retrovirus insertions the explanation for cancer in humans?
➔ Conclusion (after 10 years of hunting): human retroviruses are very rare, and rarely cause
cancer (HTLV1,2) – best known HIV which is not directly linked to cancer. No “gene thief” (as
in mouse or chicken) found in human.
Why do we have so few retroviruses ?
Once upon a time there was an invasion in our genome. We learned to fight, and suppress the Aliens
(RNA interference, miRNA, mutation). We can still find their tracks in our genome. These “repetitive
elements” influence genome stability and gene regulation, but they do not cause cancer the way
retroviruses do in birds/mice.
➢ LINE-elements (Long Interspersed Nuclear Elements) : retrotransposons, about 100 000
truncated and 4 000 full length copies of Line-1 – together about 20% of our genome.
➢ SINE-elements (Short Interspersed Nuclear Elements) : best known → Alu repeats (300 bp
each) about 1 million copies = 10% of our genome.
Is there any way to find out whether the human homologues to bird/mouse/cat cancer genes play a
role in human cancer at all ?
You need a method for hunting – a model that shows how cells can be transformed to cancer cells.
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What causes a normal cell become a tumor cell ?
What characterize a tumor cell ?
1. Immortality
➢ A normal human fetal cell population divides between 40 and 60 time in cell culture before
senescence. A cancer cell needs to become immortal, even before it becomes aggressive
➢ En recherche, les cellules immortelles étudiées sont : NIH 3T3 fibroblasts, HEK 293 (human
Embryonal Kidney), human Lymphoblastoid Cell Line.
➢ Conclusion: immortality is necessary but not enough to develop a tumor.
2. Loss of contact inhibition
➢ In cell culture, NH 3T3 fibroblasts grow as a monolayer and stop growth when all space is
filled. When transformed cells start growing on top of each other – “foci formation”.
➢ Conclusion : immortal cell lines have passed a first critical step towards tumor formation,
and can be used to investigate genes that contribute to cancer
That’s how it may look like :
➢ With empty expression vector : some foci by “spontaneous transformation
➢ Transformation with a cloned oncogene : many foci
Tumor cells do not stop growing even if there is no space left. Cells can change their phenotypes (cells
are transformed).
Which gene in a real tumor is the cause for the cancer? How can such a gene be found?
- Experiment: shear DNA from human tumor into tiny pieces, transfect NIH3T3 cells. Integrated
DNA fragments containing oncogenes should produce foci.
- Isolate and cultivate foci, extract DNA, SB analysis with probes from cancer genes (known from
mouse, rat, cat).
- Sequence analysis: the human HRAS gene is a proto-oncogene. A point mutation converts it
into an oncogene. This point mutation exchanges one single amino acid → increase the activity
of RAS protein → RAS hyperactive.
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Point mutations of proto-oncogenes in cancer. Mutations of one of the RAN family members are
frequent in some specific cancer forms. However, activating point mutations in “proto-oncogenes” are
not too frequently characterized in human cancer (rarely the primary cause).
A second source finding human cancer genes? Looking at chromosomes
➢ In nearly all cancer forms altered chromosomes – however with some regular changes in
defined cancer forms → The Philadelphia chromosome : all the neoplastic cells in early all
cases of a chronic myeloid leukemia contain a reciprocal translocation between
chromosomes 9 and 22.
➢ Burkitt’s lymphoma (discovery 1976): there is 3 variants of burkitt’s lymphoma. All have a
reciprocal translocation with chromosome 8, band q24. The translocation places the c-myc
gene to one of the 3 Ig loci : heavy chain on chr 14 or one of the 2 Ig light chains on chr 2
and 22.
➢ In detail : it’s myc again ! The 5’-regulatory region and the 1st (noncoding) exon of c-myc
are replaced by the regulatory region of the Ig locus. The c-myc protein remains without
mutation – only its expression is changed.
C-myc (c for cancer) expression belongs to the immediate early response od stimulated cells. In Burkitt
Lymphoma, c-myc is permanently expressed : cell replication is permanently induced.
All these translocations place the coding part of a proto-oncogene under control of a hightly active
promoter = overexpression.
Cloning of the Philadelphia translocation point(s) in Chronic myeloid leukemia. Southern blot analysis
on CML cancer lines (1-17) and control cells with a c-abl probe. Sample7 (tumor) and 18 (normal
fibroblasts) are from the same patient.
Conclusion : The ABL gene is rearranged in CML. The arrangements differ among the patients.
Alternative breakpoints lead to the same fusion protein :
➢ BCR = Breakpoint Cluster Region
➢ Different BCR-ABL fusion proteins in different leukemia forms
Summary :
➢ Mutations in proto-oncogenes can contribute to multiples cancer forms.
➢ The mutations lead to overexpression of an oncoprotein or hyperactivation of the
corresponding protein.
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➢ Point mutations have been found to activate some proto-oncogenes, but larger chr
rearrangements have been discovered more frequently. These rearrangements place the
proto-oncogene from its original locus into a region with high/permanent expression or
lead to the formation of fusion proteins in which the oncoprotein gains activity or looses
its feedback control.
➢ (Retro)-viral oncogenes (v-onc) are “stolen” from the virus host and mutated.
Does a mutation in an oncogene explain how cancer occurs/develops ?
But no …
➢ Most cancer forms cannot be explained by mutations of a single proto-oncogene.
➢ With very few exceptions, inheritable cancer is not caused by inherited mutations in a
proto-oncogene.
➢ Basically, all tumors show more than one change in the genome.
➢ Many agents or conditions can cause cancer without directly being mutagenic.
