Cancer,
Oncogenes, Tumor Suppressor Genes
Cancer is formed by tumor cells
Cancer - A group of diseases
Cancer is a disorder that occurs at a
cellular level
Cancer occurs when genetic
alterations result in the unregulated
proliferation of cells
Most cancers derive from a single
abnormal cell.
Scanning electron micrographs reveal the organizational and morphological
differences between normal and transformed 3T3 cells
Loss of contact inhibition by cancer cells in cell culture.
Steps in the process of metastasis
Cell transformation
Physical factors
chemical factors
biological factorsaccumulation of
gene mutation
transformationnormal cell cancer cell
immortalization
invasion
metastasis
the development of cancer is resulted from
interaction between hereditary factors and
environmental factors.
Two general classes of genes play
key roles in cancer induction
1. proto-oncogenes: positive control gene,
gain-of-function mutations, act in a
dominant manner
2. Tumor suppressor genes: negative control
gene, loss-of-function mutations, act in a
recessive manner.
正负调控信号平衡的重要性
细胞正常正调信号
负调信号
抑制增殖,促进分化、
成熟、衰老和凋亡
促进细胞生长增
殖,阻止分化
Section Ⅰ oncogene
What is an oncogene?
an oncogene is any gene that encodes a
protein able to transform cells in culture or
to induce cancer in animals, including v-onc
and c-onc. Most oncogenes are mutant
forms of normal genes (proto-onc) involved
in the control of cell growth or division.
Peyton Rous
(1879-1970)
• 1911,Rockefeller institute
• Suggested that spontaneous
chicken tumor caused by a
virus—Rous sarcoma virus
• 1966, Nobel Prize in
Physiology or Medicine
Discovery of oncogene began from the Rouse sarcoma virus research
Rous sarcoma
Experiment by Rouse
Isolate chicken sarcoma
Grind tissue cells
Centrifuge
Separate supernatant
Filter sterilization
Inoculated it on healthy chickens
Experimental results and conclusions by Rouse
Result: The recipients developed tumors
of the same type as seen in the original
animal
Conclusions: Chicken tumor can be
transmitted between individuals; media
may be filterable virus
Confirm that the retroviral with RNA genome has
led to chicken tumors, which was named the
Rouse sarcoma virus (RSV)
1969,oncogene theory: Carcinogenesis is
caused by the oncogene
1970, the first oncogene src was isolated and
identified from RSV
1971, to determine that
the Src in the 3'end of RSV
genome
Progress after Rouse
Genome structures of two forms of RSV
Str
uct
ure
and g
ene p
roduct
s of
an inte
gra
ted r
etr
oviral
genom
e.
RSV: strong tumorigenesis role, lead to
tumors after 1-2 weeks of animals
infection, strong transformation effects
on cultured cells
slow-transforming retrovirus: weakly
tumorigenesis role, lead to tumors after
several months of animals infection, no
transformation effects on cultured cells
•Two types of RSV:
The resulting problem
What is the origin of Virus oncogene ?
Answer for this question
1976,Bishop
1.Prepare the radioisotope labeled v-src gene probe
nuclease S1
V-src cDNA
Full length cDNA of
wild RSV
Genome RNA of
slow-acting
retrovirus
+
2. Hybridization of v-src gene probe and chicken
cell genomic DNA
Result:In the genome of normal chicken cells, found
the homologous sequences of v-src—c-src, and then to
find that the split gene c-src exsit in from primitive
eukaryote genome to normal human genome.
Conclusion: c-src is inherent normal gene within the
cell genome.v-src is originated from the c-src—
Proposed the concept of proto-oncogene, which
specifically refers to the corresponding sequences in
normal cells, oncogenes are mutant forms of proto-
oncogene that is activated in cancer cells
Experimental results and conclusions by Bishop
The relationship between the v- src and the c-src
proto-oncogene from which it has been derived
Next question
How do virus get proto-oncogene
src from normal cells and then
convert it into a oncogene?
Retroviral life cycle
蹲睛视被砻挛醮扯艋溲茔舂嗤兔驰绌滏述秽腭睫鲺该剧苑唾叫伤排驯嘟竿乃踱袒扒葑矽悖恬獍
病毒癌基因v-src来源于宿主细胞的C-SRC基因
Retroviral Oncogenes (partial list)
Oncogene(v-onc) Prototype Retrovirus
src Rous sarcoma virus
myc Avian myelocytomatosis virus
erb A, erb B Avian erythroblastosis virus
myb Avian myeloblastosis virus
H-ras Harvey rat sarcoma virus
K-ras Kirsten murine sarcoma virus
abl Abelson murine leukemia virus
fes Feline sarcoma virus
sis Simian sarcoma virus
Isolation and identification of c-onc
Tumor retrovirus is only responsible
for less than 20% of human cancers.
Transformation is due to the
abnormal genes (i.e. onc) generated
for a variety of reasons (chemical,
biological and physical)
?
?
Oncogenes
arise in
humans
through two
distinct routes:
viral route and
inherited route
distribute widely from Yeast to Human beings;
Because most proto-oncogenes are basic to animal life,
they have been highly conserved over eons of
evolutionary time and have low expression level;
Whose products participate in cellular growth
controlling pathways; have not carcinogenic function in
normal cells and play important roles in cell growth,
development and differentiation;
When inappropriately activated, these genes
convert into oncogenes and lead to fault in growth
control and contribute to the development of cancer.
Characteristics of proto-oncogenes
Proto-oncogenes
Classification of proto-oncogenes (family)
1. src family src,abl,fgr,fes,yes,fps, tyrosine protein kinase
lck,kek,fym,lyn,tkl
2. ras family H-ras,K-ras,N-ras GTP binding proteins(p21)
3. myc family c-myc,N-myc,L-myc ,fos DNA binding proteins
4. sis family sis PDGF derived protein(p28)
5. myb family myb, myb-ets Transcription factor
Family Oncogen Function
Several families of oncogene
• How do the proto-oncogenes turn into ‘enemies within’?
• What are the functions of oncogene products?
Two questions about oncogene
At least four mechanisms can produce
onc from the corresponding proto-onc:
Point mutation
Chromosomal translocation
Promoter/enhancer insertion
Gene amplification
Point mutation
in a proto-onc that results in a constitutively
active protein product
single base change leads to one code
change, which further lead to one AA
change, which further lead to increase of
oncoprotein activity
Normal H-ras
GGC GTC
Cancer H-ras
Gly Val
e.g. Point mutation in c-H-Ras in bladder cancer: 12th coden (13,61)
Mutant Ras can continuously transduce
growth signal and cause cell transformation。
Transduce
growth signal
Mutant Ras
protein
maintain
active status
This mutation affect the conformation of ras protein and
diminish its activity as a GTPase. The lower activity of
GTPase could result in chronic stimulation of the activity
of ras protein, which can result in a number of effects on
cellular metabolism for ras mediate a wide variety of
signal transduction pathways.
Promoter/enhancer insertion
The strong promoter / enhancer in the
retrovirus LTR can activate some
proto-oncogene and its overexpressing
when retrovirus integrate into cell
chromosome.
Chromosome translocation is that
one part of one chromosome
exchange with another chromosome .
Chromosome translocation can
cause changes in gene structure
and gene expression.
Proto-oncogene can be activated
and overexpression if it is close to
a strong promoter / enhancer.
Chromosomal translocation
Burkitt淋巴瘤是1964年Epstein首先从非洲儿童
Burkitt淋巴组织中分离出EB病毒而命名。病变特
点为肿瘤常发生于颌骨、颅面骨、腹腔器官和中
枢神经系统等。目
前认为本病的发
病与EB病毒感染
密切有关,90%的
病人瘤细胞有异
常核型
Chromosomal translocation in Burkitt’s lymphoma
As a result of a translocation between chromosomes 8
and 14, the c-myc gene is placed adjacent to the gene
for part of the antibody heavy chain (CH), leading to
overproduction of the Myc transcription factor in
lymphocytes and hence their growth into a lymphoma.
慢性粒细胞白血病是一种影响血液及骨髓的恶性肿
瘤,它的特点是产生大量不成熟的白细胞,这些白
细胞在骨髓内聚集,抑制骨髓的正常造血;并且能
够通过血液在全身扩散,导致病人出现贫血、容易
出血、感染及器官浸润等。
慢性粒细胞白血病的病因仍未
明确,但认为费城染色体与该
病密切相关,大约有90至95%
的病人出现费城染色体。
Chromosomal translocation in chronic myeloid leukemia
Philadelphia chromosome
The conversion of the Abl proto-oncogene into an oncogene in patients with CML.
How Bcr-Abl protein causes chronic myelogenous leukemia.
Gene amplification
of a DNA segment including a proto-onc,
so that numerous copies exist, leading to
overproduction of the encoded protein
This anomaly may take either of two forms: the
duplicated DNA may be tandemly organized at a
single site on a chromosome, or it may exist as
small, independent mini-chromosome-like
structures.
DNA amplifications in stained chromosomes take two forms
Four ways in which a proto-onc can be
made overactive to convert it into an onc
The major character of cancer cells is
uncontrolled growth, function of oncoproteins
is to promote cell growth and transformation
Growth factors act on the cell cycle and
mitosis via transmembrane signal
transduction
How do the proto-oncogenesturn into ‘enemies within’?
4. Transcription
factors
Growth gene
3. Intracellular
signaling protein
1. Growth factors
2.Growth factor receptor
FUNCTION OF ONCOGENE PROTEINS
Growth factor
◆ GF: polypeptides, which can bind to its
receptor and promote cell growth, such
as EGF, FGF, PDGF
◆ oncogenes: e.g. sis (P28) ~ B chain of
PDGF, overexpression of sis can induce
cell transformation .
Growth factor receptor
◆ RTK, GPCR
◆ e.g. c-erb B ~ EGFR, can not bind to
GF due to gene mutation, can
continuously promote cell growth and
cell transformation.
Oncogene-encoded defective EGF receptor
吉非替尼—易瑞莎
EGFR=ErbB-1=HER1
Intracellular signal molecules
non-receptor TPK: such as src, abl, fes,
they bind with cytokine-R/GF-R,
phosphorylate downstream protein and
transfer growth signal.
Regulation of Src activity and its activation by
an oncogenic mutation. (a) Domain structure of
c-Src and v-Src. (b) Effect of phosphorylation
on c-Src conformation.
Csk: C-terminal Src kinase
Mec
han
ism
of
au
toin
hib
itio
no
f S
rc
Imatin
ibfo
r th
era
py o
f C
ML
G-protein: Ras family (K-ras, N-ras, H-ras ),
play a central role in signal transduction
Ser/Thr protein kinase: such as raf, mos,
they are downstream the ras and
phosphrylate its downstream protein.
Val→Glu, V600E
Zelboraf (vemurafenib) for advanced melanoma
Transcriptional factor
◆Including: c-myc, c-myb, c-fos, c-jun
◆ they are final member of growth
signal transduction pathway.
◆ immediate-early genes
◆ their protein form transcriptional
factor (homodimer/heterodimer)
The path from growth factors
to cell division leads through
the enzyme cascade that
activates MAPK;
phosphorylation of the
nuclear transcription
factors Jun and Fos; and the
activity of the transcription
factor E2F, which promotes
synthesis of several enzymes
essential for DNA synthesis
Regulation of cell division
by growth factors
Protein products of oncogenes
Oncoproteins
Resemble the normal products of proto-
oncogenes
Devoid of important regulatory elements
Independent on growth factors or other signals
Some anticancer drugsand drug targets in the Ras–MAPK signaling pathway.
Section II
Tumor Suppressor Genes
A kind of negative regulatory gene ,the
normal function of these genes is to suppress
the proliferation-stimulating activities of
cellular oncogenes.
That have a negative, suppressing effect on
tumor creation and thus help to prevent
formation of tumors.
1. Definition of tumor suppressor gene
2. Evidence for tumor suppressor gene
Cell fusion experiment
Single chromosome transfection experiment
two hit hypothesis
Detection of LOH (loss of heterozygosity)
Normal cell
fusion
hybrid cell (normal,but instability)
passage
tumor cell
chromosome 11 loss
Import chromosome 11
Cell normal
tumor cell
Knudson: two hit hypothesis
The genetic mechanisms underlying retinoblastoma
Hereditary versus Sporadic Retinoblastoma
Retinoblastoma(视网膜母细胞瘤)is a juvenile eye
cancer that is caused by a mutation in the Rb gene
located on chromosome 13 of humans.
3. Cells proliferate in the of cell cycle
cyclin dependent PK (CDK): 1~6, activated by cyclin, inhibited by CKI,
cyclins: activate CDK, A, B, C , D, E, etc.
different cyclin-CDK complex regulate different phase of cell cycle
CDK inhibitor protein (CKI): inhibit CDK, p15, p16. p21, p25, p27, etc.
Cell proliferation in cell cycle way
3 types of proteins regulate cell cycle directly
The control of the cell cycle
Whether cell
cycle can start and
run depends on two
critical check
points:
The former is more
important. If cell can
pass G1/S, the cell
can divide
G1/S and G2/M
Cyclin-Cdk complexes of the cell-cyclec ontrol system
Variations in the activities of specific CDKs
during the cell cycle in animals
The inhibition of a cyclin-Cdk complex by a CKl
4. Tumor suppressor gene p53 and Rb function
to inhibit cell cycle
① Retinoblastoma susceptibility gene (Rb):
the first identified tumor suppressor gene
forms of RB :
Rb gene locate in 13q14, has 27 extrons, 4.7kb mRA
encode nuclear protein P105
Active unphosphorylated/ hypophosphorylated form
Inactive hyperphosphorylated form
function: control cell cycle via interaction with
transcription factor E2F
RB is a negative regulator of progression in the cell
cycle at the G1 to S transition
PRb functions as a switch for conversion of
mitogenic and antimitogenic signals at the
transcription level in the cell cycle, which is mediated
by phosphorylation status.
The pathway by which Rb controls cell cycle
Figure. Regulation of Rb
and E2F activities in late G1.
Interaction of E2Fs with
nonphosphorylated Rb
protein initially inhibits E2F
activity. When signaling from
mitogens is sustained, the
resulting initiate the
phosphorylation of Rb,
converting some E2F to the
active form, which activate
target genes whose products
are essential for S phase.
In G0/G1, un/hypo-phosphorylated RB binds to a E2F and inactivates E2F.
At the end of G1, RB is phosphorylated, releasing (activating) E2F.
Active E2F stimulates the expression of genes required for the entry into S-phase (DNA replication).
At the end of M, RB is dephosphorylated. Active RB binds E2F and inhibits the expression of genes required for DNA synthesis.
② p53 gene
• p53 gene located in 17p13, is
another gene with tumor
suppressor activities. This
protein contains 393 amino
acid residues and a single
amino acid substitution can
lead to loss of function of the
gene. P53四聚体形式
Three regions of p53 protein:
N-terminus (1-80) transcriptional activation domain. Sites for phosphorylation.
Core section (102-290) which binds to DNA in a sequence specific manner.
C-terminus(319-393) by sequences involved in the oligomerization of the protein. Sites for phosphorylation.
1. p53 activates p21 when damaged DNA emerge, cell cycle stops at G1 phase
2. Inhibiting the activity of helicase
3. DNA replication or repaired with replication factor A.
4. If above processing donesn’t work, p53 will induce cell apoptosis (programmed cell death)
This genetic function of this gene is to prevent cell division of cells with damaged DNA.
How DNA damage arrests the
cell cycle in G1 by P53 protein
Figure. Damage to DNA
activates p53. The outcome
depends on the stage of the
cell cycle. Early in the
cycle, p53 activates a
checkpoint that prevents
further progress until the
damage has been repaired.
If it is too late to exercise
the checkpoint, p53
triggers apoptosis.
When p53 is inactive (loss-of-function), this
regulatory pathway does not function
• The RB gene product is always phosphorylated
(inactive), cells continue to replicate DNA and
divide
• About 50% of human cancers can be
associated with a p53 mutation including
cancers of the bladder, breast, cervix, colon,
lung, liver, prostate, and skin. p53 related
cancers are also more aggressive and have a
higher degree of fatalities.
If p53 gene mutation
Regulation of passage from G1 to S
by phosphorylation of pRb
Properties Oncogene Tumor suppressor gene
Mutational events involved One Two
in cancer
Function of mutation Gain of function Loss of function
(“dominant”) (“recessive”)
Germ-line inheritance No Yes
Somatic mutations Yes Yes
Effect on growth control Activate cell proliferation Negatively regulate
growth promoting genes
Effects of gene transfection Transform partly abnormal Suppress malignant
fibroblasts (e.g., NIH3T3) phenotype in malignant cells
Genetic alterations Point mutations, Deletions,
gene rearrangements point mutations
amplification
Properties of Oncogenes and Tumor Suppressor Genes
Test 3
It is known that norepinephrine, as a ligand for the β3-adrenergic
receptor (one member of the GPCR family) in adipocyte plasma
membranes,up-regulate the expression of the uncoupling protein 1(UCP1)by activating PKA, please explain the molecular mechanism.
Based on this, one experiment is designed to study the effect of
norepinephrine treatment on mouse adipocytes which cultured in vitro
and to observe: whether UCP1 gene expression is up-regulated at
transcription and translation levels, respectively, before and after
treatment. What molecular biology techniques can be used to detect
UCP1 gene expression? Please list the techniques and describe the
experimental principle and technical process, respectively.
NOTE: 1. time mark: 20171221
2. the question must be copied on the answer paper before
you answer.