Molecular basis of cancer. P53, Rb gene transcription.

1. Dr. Ishan Y. Pandya
(PhD. Biotechnology)
E-mail: genomes.world37@gmail.com
Institute of Life Sciences, JJTU, Rajasthan, India

2. •Tumor suppressor genes
•Deletion of RB gene causing
RETINOBLASTOMA
•Functions of tumor suppressor gene products
•Role of pRb in regulating the cell cycle
•Role of p53
•Other tumor suppressor genes
•References

3. Two broad categories
1. Tumor suppressor genes
2. Oncogenes

5. Some cells lost their malignant properties,
suggesting that normal cells possess
factors that can suppress the uncontrolled
growth of a cancer cell

7. •Encodes proteins that promotes the loss of
growth control & the conversion of a cell to
a malignant state.
•As a result,they act as accelerator of cell
proliferation & tumorigenesis

9. GENE TYPE OF CANCER
APC Colon/rectum cancer
BRCA1 Breast & ovarian carcinoma
BRCA2 Breast carcinoma
INK4 Lung cancer,brain tumors ,lymphomas
p53 brain tumors,breast,colon/rectum,liver &
lung carcinomas
PTEN brain tumors,kidney & lung carcinomas
Rb retinoblastoma,bladder,breast &lung
carcinomas
Smad 1 & Smad4 colon/rectum carcinoma
WT1 Wilms tumor

10. •In humans ~2 dozen genes are implicated as tumor suppressor
genes.
•Loss of function of one or more tumor suppressor genes leads to the
transformation of a Normal cell to a cancerous cell.
•First tumor suppressor gene to be studied & eventually cloned is called
RETINOBLASTOMA.
•It is a rare childhood cancer of retina.
•Gene responsible for this disorder is named as “RB”
•This cancer can be inherited.

12. •Incidence of retinoblastoma follows 2 distinct patterns:
1.Occurs at high frequency at young age in members of certain family
2.Occurs sporadically at an older age among the members of population at large
•In retinoblastoma one member of 13th pair of chromosome was missing a small
piece from the internal portion of the chromosomes
•The deletion was both in retinal cancer cells & cells elsewhere in the body
•It is inherited as a dominant genetic trait with one normal allele & one
abnormal allele
•~10% individuals who inherit a chromosome with RB deletion never develop a
retinal cancer.

13. •The genetic basis of retinoblastoma was explained by ALFRED KNUDSON of
university of Texas in 1971.
•He proposed that the development of retinoblastoma require both the copies of
RB genes Of retinal cell be either eliminated or mutated before the cell can give
rise to a disease
•He proved that retinoblastoma show both the alleles of the gene to be mutated in
a cancer Cell
•Individuals with sporadic retinoblastoma had normal cells that lacked RB
mutation & tumor cells in which both alleles of the gene were mutated
•People with inherited form of retinoblastoma are at a high risk of developing other
types of tumors in life particularly soft tissue sarcomas.

14. •The consequences of RB mutations are not confined to a person
who inherit a mutant allele
•Mutation in RB alleles are common occurrence in sporadic breast,
prostate & lung cancer among individuals with inherited two
normal RB alleles
•A number of LABS have now directed their attention to determine
the role of the product of RB genes as they have great importance
in regulating the growth of a wide variety of tissues .

18. Protein encoded by the PTENtumor suppressor genes is an interesting example of
antagonism between ONCOGENES & TUMOR SUPPRESSOR GENE products.
PTEN Lipid phosphatase Dephosphorylates PIP3 at 3rd position of
Inositol,yielding PIP2
By dephosphorylating PIP3,PTEN
antagonizes the activities of PI 3-
Kinase & AKT
PI 3-kinase & AKT act as oncogenes by promoting
cell survival

19. •Several tumor suppressor genes encodes transcriptional regulatory proteins
Eg. The product of WT1 is a repressor that appears to suppress transcription of
a number of growth factor inducible genes.
•WT1 is frequently inactivated in WILMS tumors (a childhood kidney tumor)
•Inactivation of WT1 may thus lead to abnormal growth factor expression, which
in turn drives tumor cell proliferation
•Two other tumor suppressor genes,Smad2& Smad4,encodes transcrip-
tional factors that Are activated by TGF BETA signaling & lead to inhibition of
cell proliferation
•Conversely the loss of PTEN can contribute to tumor development as a result of
increased Levels of PIP3,activation of AKT & inhibition of programmed cell
death.

20. •The products of Rb & INK4tumor suppressor gene regulates the cell cycle at the
same point as that affected by cyclinD1.
•Rb inhibits passage through the restriction point in G1 by repressing transcription of a
number of genes involved in cell cycle progression & DNA synthesis
•Mutational inactivation of Rb in tumors thus removes a key negative regulator of cell
cycle progression
•The INK4 tumor suppressor genes also regulates the passage through the restriction point
•Inactivation of INK4 thus leads to elevated activity of CDK4,6/CYCLIN D complexes,
resulting in an uncontrolled phosphorylation of Rb

21. INHIBITION OF CELL CYCLE PROGRESSION BY Rb & p16:
Rb inhibits progression past the restriction pt in G1.Cdk4,6/cyclin D complexes promote
passage through restriction pt by phosphorylating & inactivating Rb.the activity of
Cdk4,6/cyclin D is inhibited by p 16

22. •pRb protein is encoded by RB genes
•Regulate the passage of cells from the G1 stage of cell cycle into S phase
•The transition to S phase activates many genes that encodes proteins ranging
from DNA Polymerase to cyclins & histones
•Genes of E2F family required for S phase activities are key targets of pRb
•More than 40 proteins are capable of binding to pRb
•pRb contains at least 16 different serine & threonine residues,thus have a
complex Binding

23. •Several DNA tumor viruses (SV40 & adenovirus)encode a
protein that binds to pRb ,blocking its ability to bind to E2F
•They block the negative influence that pRb has on
progression of a cell through cell Cycle.

24. •Some of the most interesting data on the RB gene have come from
studies on knockout Mice
•The mice which lacks both the alleles of RB genes(RB-/-) divide in
an uncontrolled manner & produce totally aberrant embryos.
•Role of RB genes in the formation of tumor is more evident in
mice that are heterozygous For the gene(RB+/-)
•Animals with this genotype carry out normal development but
exhibit a greatly elevated risk of developing cancer.

25. •Codes for p53 protein of MW 53000 daltons.
•Was recognized as tumor suppressor gene in 1990.
•If they are absent,they result in a rare inherited disorder called Li Fraumeni
syndrome
•Victims of this disease are afflicted with a very high incidence of certain cancers
Including Breast cancer,brain cancer & leukemia.
•Persons with this syndrome inherit one normal & one abnormal allele of the p53
tumor Suppressor genes & thus are highly susceptible to cancer
•Tumors composed of cells bearing p53 mutations are co-related with a poorer
survival rates than those containing a wild type gene
•More than a 1000 different p53 mutations have been identified among human
tumor samples.

27. •p53 is very important in preventing a cell from becoming malignant
•It activates the expression of other genes out of which one encodes for a protein
called p21
• p21 inhibits cyclin dependent kinase that normally drives the cell through G1
checkpoint
•As the level of p53 rises in a damaged G1 cell ,expression of the p21 gene is
activated & progression trough the cell cycle is arrested
•During this arrest the cell repairs the damage before undergoing DNA synthesis
•When both the copies of p53 genes are mutated,the cell can’t produce p21
inhibitor
•Failure to repair DNA damage leads to the production of abnormal cells that
have the potential to become malignant

30. •Alternatively ,p53 can direct a damaged cell along a pathway that leads to death
by apoptosis
•This is done by activating the expression of baxgenes which encodes a
protein called Bax (initiates apoptosis)
•If both the alleles of p53 are inactive,the cell with damaged DNA fails to be
destroyed
•If a normal p53 gene is introduced into a cancer cell,the genetically engineered
cell often undergoes apoptosis
•Level of p53 in a healthy G1 cell is very low.however in a damaged cell the concen-
tration of p53 Rises rapidly.
•The increase in p53 levels is not due to increased expression of gene but due to a
decrease in the protein’s degradation
•p53 degradation is facilitated by a protein called MDM2,which binds to p53 &
escorts it out of the nucleus to the cytosol where the p53 is ubiquinated & destro-
yed

33. •A protein kinase called ATM is normally activated following
DNA damage
•It phosphorylates p53
•Phosphorylated p53 cant act with MDM2 due to which the existing
p53 is stabilized in the nucleus & allows them to activate the
expression of genes such as p21 & bax.
•Some tumor cells contain a wild type p53 genes but extra copies of
MDM2.
•This prevents the p53 level from building to required levels to stop
the cell cycle or induce apoptosis following DNA damage

34. •Mice that lack a gene encoding MDM2 die at an early stage of development as
their cells Undergoes p53 depended apoptosis
•This observation illustrated an important principle in cancer genetics
•“Even if a crucial gene such as RB or p53 is not mutated or deleted,the function
of that gene can be affected as a result of alterations in other genes whose
products are a part of the same pathway as the crucial gene”
•Because of its ability to trigger apoptosis ,p53 may play a major role in treat-
ment of cancer by radiation & chemotherapy
•It was assumed for many years that cancer cells are more susceptible to drugs &
as they divide more rapidly

35. •An alternate theory suggest that normal cells are more susceptible
to drugs or radiations Because once they sustain genetic damage,
they either arrest the cell cycle or undergoes Apoptosis
•If cancer cells lose p53 function ,they often can not be directed
into apoptosis & they become highly resistant to further treatment
•This is the main reason why the tumor cells without p53 gene
respond much more poorly to radiation & chemotherapy than
tumors that possess a wild type copy of the gene

36. •During normal development secreted signals such as Wnt,TGF Beta & Hedgehof
(Hh) are frequently used to direct cells to particular developmental fates
•Hh is a good eg. Of a signaling pathway implicated in cancer induction
•In skin & cerebellum ,one of the human Hh proteins, sonic hedgehof,stimulates
cell division by binding to & inactivating a membrane protein called Ptc1
•Loss of function mutations in Ptc1 permits cell proliferation in the absence of an
Hh signal; thus Ptc1 is a tumor suppressor gene
•People who inherit a single working copy of this gene are at a high risk to develop
skin & brain cancer
•Mutation in other genes of Hh pathway are also associated with cancer

37. •Besides mutations in RB & p53 ,mutation in a number of other tumor suppressor
genes Are detected in few types of cancer
Eg; Familial adenomatous polyposis coli(FAP) is an inherited disorder in which
individuals develop thousands of premalignant polyps (adenomas)from epithelial
cells that line the colon wall
•If not removed ,the cells with in the polyps are very likely to progress to a fully
malignant stage
•This was due to a deletion of a small portion of chromosome 5 which is identified
as the site for tumor suppressor gene called APC
•If both the alleles of APC are deleted,it causes the cell to lose growth control &
proliferate to form a polyp rather than differentiating into a normal epithelial cell
of the intestinal wall

39. •Mutated APC genes are not only found in inherited forms of colon
cancers but also in 80% Of sporadic colon tumors
•Suggesting that the genes plays a major role in the development of
this disease
•APC genes encodes a protein that binds to a number of different
proteins
•Mechanism of action is complex
•APC restrains cell growth by interfering with the transcription of
genes (eg.myc) that stimulates cell proliferation

40. •After an intensive effort of lab studies in 1994,two genes BRCA1 & BRCA2,were
identified As being responsible for the majority of inherited cases of breast
cancers
•BRCA1 also predisposes a women to develop an ovarian cancer(having a very high
mortality rate)
•Both genes have two distinct functions:Transcription factor & a part of cell’s
DNA repair Machinery
BRCA1 + BRCA2 + PROTEIN Rad 51
This complex repairs DNA strand breaks
occurring Due to exposure to radiations
Failure to repair the damage leads to
The activation of p53

41. •CELL & MOLECULAR BIOLOGY GERALD KARP
•CELL BIOLOGY COOPER
•MOLECULAR CELL BIOLOGY LODISH
•MOLECULAR BIOLOGY ALBERTS
•GOOGLE

42. THANK YOU