This document discusses proto-oncogenes, oncogenes, and tumor suppressor genes. Proto-oncogenes are normal genes that regulate cell growth, while oncogenes are mutated proto-oncogenes that contribute to uncontrolled cell growth. The discovery of oncogenes began with Rous sarcoma virus, which was found to contain the viral oncogene v-src. The tumor suppressor gene p53 plays a key role in DNA repair and apoptosis, preventing cancer formation. When mutated, p53 cannot regulate the cell cycle properly, allowing damaged DNA to be passed on and tumors to form.

2. Introduction
Proto-oncogenes: A gene which codes for protein that help in the regulation
of normal cell growth.
 Stimulate cell division
 Prevent cell differentiation
 Regulate programmed cell death
Oncogenes: sequence of DNA that have been altered or mutated from its
original form, proto-oncogenes.
 Unregulated cell division
 Slower rate of cell differentiation
 Increased inhibition of cell death

3. Discovery of Oncogenes
 Discoverd by Peyton Rous 1909, worked at the
Rockefeller institute for medical research began
a series of experiments that started with a
chicken that had a lump on its leg, which was a
soft-tissue sarcoma.
 When Rous ground up the tumour and injected
into other chickens, also causes sarcoma in it.
 Active agent was identified as virus-retrovirus-
was called as Rous sarcoma virus

5.  Analysis of RSV in 1970 showed retrovirus with
small single stranded RNA gene, src which is
responsible for cancer-inducing properties of
RSV and considered as Oncogenes.
 In 1975, scientists discovered that healthy cells
also carry normal version of src (c-src) genes,
which plays an important role in normal cell
development.
 When it gets mutated will converted into
virulant (v-src)

6. SRC: Proto-oncogene tyrosine-protein kinase
 Cell morphology, motility
 Proliferation and survival
Structure:
 14-c myristoyl group, SH3 andSH2 domain, protein
tyrosine kinase domain and c-terminal regulatory tail
 Chief phosphorylation site of src include tyrosine
416 result in activaion from autophosphorylation and
tyrosine 527 result in inhibition from
phosphorylation by c-terminal src kinase.

7. Tyrosine kinase signalling and
oncogenesis
 Normal cells receive and interpret external signals which
influence their growth, shape, metabolism and movement.
Complex interactions between molecules inside the cell
transmit these signals and initiate appropriate cellular
responses.
 Protein phosphorylation on Tyrosine (Tyr) residues has
evolved as an important mechanism to coordinate this
intracellular signalling process in multicellular organisms.

9. Inactivation:
 Y416 is autophosphorylation site in the activation loop and
negative regulation in tyrosine site by phosphorylation at
Y527 in the c-terminal end.
 c-src is auto inhibited through intramolecular association
between the SH2 and Y527, and SH3 and poly proline motif
in the linker.
Activation:
 Activation is by either dephosphorylation of Y527 to
release the SH2 domain or by binding of peptide ligand to
recognise SH2 leading to autophosphorylation of Y416.

10. When Src goes wrong???
Under normal circumstances, Src is predominantly inactive in cells,
being switched on only at specific times.
 fine balance between phosphorylation and dephosphorylation is
disrupted, changes can occur in Src activity with drastic results.
 Several cancers, including colon and breast cancer, have been
associated with an increase in Src activity.
 v-Src, from the transforming virus, Rous Sarcoma Virus. v-Src was
found to lack the region of the cellular protein (c-Src) that contains
Tyr527, making it continually active.

11.  In late stage colon cancers, mutations have been reported in the src
gene that cause the loss of the region containing Tyr527, leading to
Src over-activity.
 Proteins that regulate Src have also been found at abnormal levels
in cancer cells, including both those that activate and those that
inactivate Src.
 Proteins such as PTPalpha, SHP-1 and PTP1B that activate Src by
dephosphorylating Tyr527 have been detected at elevated levels in
various cancer cells, including epidermal and breast carcinoma cells.

12. Therapeutic applications
Control over the metabolic alterations :
 A decreased oxidative phosphorylation, increased glucose
consumubsequent lactate production are the remarkable features of
oncogenic activation .
 Over the last decade, evidence has accumulated that the oncogenes myc,
AKT, nuclear factor kB (NF-kB) and the tyrosine kinase receptors
(insulin-like growth factor 1, epidermal growth factor, EGF; IGF-1; Her-2;
etc.), which turn on phosphatidylinositol 3-kinases (PI3Ks), mammalian
target of rapamycin (mTOR) pathway and the Ras, RAF–Mitogen-
Activated Protein Kinase (MAP kinase) along with hypoxia induced factor
(HIF), can activate the transcription of numerous genes that encode the
proteins that mediate the various metabolic pathways .

13. Myc and HIF-1 transcription factors
enhances the rate of transcription of some
of the GLUT transporters and hexokinase
2, increasing both glucose uptake and its
retention in the cell .
Increases the oxidative phosphorylation
and regulate the metabolic pathway.

14. Tumor suppressor gene:
A protective gene that normally limits the growth of tumors. When a tumor
suppressor gene is mutated (altered), it may fail to keep a cancer from growing.
 The first insight into the activity of tumor suppressor genes came from somatic cell
hybridization experiments, initiated by Henry Harris and his colleagues in 1969.
 The fusion of normal cells with tumor cells yielded hybrid cells containing chromosomes
from both parents.
 The first tumor suppressor gene was identified by studies of retinoblastoma, a rare
childhood eye tumor.

15.  Protein that halts cell cycle progression in response to
DNA damage may no longer sense damage or trigger a
response. Genes that normally block cell cycle progression
are known as tumor suppressors.
 Tumor suppressors prevent the formation of cancerous
tumors when they are working correctly, and tumors may
form when they mutate so they no longer work.
 One of the most important tumour suppressor gene is p53,
which plays a key role in the cellular response to DNA
damage.

17. Mechanism of p53:
Involved in two process
Repairing DNA damage
Apoptosis

18.  DNA repair, which also depends on p53, whose
second job is to activate DNA repair enzymes.
If the damage is fixed, p53 will release the cell,
allowing it to continue through the cell cycle.
 If the damage is not fixable, p53 will play its
third and final role: triggering apoptosis
(programmed cell death) so that damaged DNA
is not passed on.

19. p53 in cell cycle:
p53 involved in the checkpoints.
Cell cycle check points:
There are a number of checkpoints, but the three
most important ones are:
 The G1 checkpoint, at the G1/S transition.
 The G2 checkpoint , at the G2/M transition.
 The spindle checkpoint, at the transition from
metaphase to anaphase.

20.  Key to the DNA damage response is a protein called p53, a famous
tumor suppressor often described as “the guardian of the
genome.”
 p53 works on multiple levels to ensure that cells do not pass on
their damaged DNA through cell division.
 it stops the cell cycle at the checkpoint by triggering production
of Cdk inhibitor (CKI) proteins. The CKI proteins bind to Cdk-
cyclin complexes and block their activity for DNA repair.

22.  cancer cells, p53 is often missing, nonfunctional, or less active than normal.
For example, many cancerous tumors have a mutant form of p53 that can
no longer bind DNA.
 Since p53 acts by binding to target genes and activating their
transcription, the non-binding mutant protein is unable to do its job.
 When p53 is defective, a cell with damaged DNA may proceed with cell
division. The daughter cells of such a division are likely to inherit mutations
due to the unrepaired DNA of the mother cell.
 Over generations, cells with faulty p53 tend to accumulate mutations, some
of which may turn proto-oncogenes to oncogenes or inactivate other tumor
suppressors.

23. Apoptosis
The cells between your embryonic fingers died in
a process called apoptosis, a common form of
programmed cell death.
 In programmed cell death, cells undergo
“cellular suicide” when they receive certain cues.
Apoptosis involves the death of a cell, but it
benefits the organism as a whole.

24. Cells that undergo apoptosis go through a
different and much more orderly process.
They shrink and develop bubble-like
protrusions (technical name: “blebs”) on
their surface.
 The DNA in the nucleus gets chopped up
into small pieces, and some organelles of
the cell, such as the endoplasmic reticulum,
break down into fragments.

25.  They release signals that attract debris-eating
(phagocytic) immune cells, such as macrophages.
Also, the fragments of the dying cell display a
lipid molecule called phosphatidylserine on their
surface.
 Phosphatidyserine is usually hidden on the inside
of the membrane, and when it is on the outside,
it lets the phagocytes bind and "eat" the cell
fragments.

26. Pathways :
Intrinsic and extrinsic pathways
Extrinsic pathway:
 In this pathway, phosphatidyserine is the death signal, which
activates the pro-caspase 8(inactive) to caspase 8.
 Activated caspase 8 , which activated the procaspase 3 to
caspase3.
Intrinsic pathway:
 p53, which activates the bax, and bax activates cyt C, binds
to apaf1 to form apoptosome.
 Activation of apoptosome activates procaspase 9 to active
form of caspase 9

28. Applications of p53-Based Cancer
Therapy
Reactivating Mutant p53:
 PhiKan083 is a carbazole derivative found from in silico screening of
the crystal structure of p53.
 By binding mutated p53, PhiKan083 raises the melting temperature of
mutated p53, which results in the reactivation of its function.
 PRIMA-1 -Restored sequence-specific DNA binding and active the
conformation.
 CP-31398- Restore the protein folding of mutated p53 to a more
natural conformation that permits a wild-type function

29. p53 Stabilization:
 MDM2 is an E3 ubiquitin ligase which controls p53
degradation. Many tumors overexpress MDM2, even
tumors without p53 mutations.
 Nutlins are cis-imidazoline compounds that act as
antagonists of the MDM2-p53 interaction.
 MI-219 is another small molecule that inhibits the
MDM2-p53 interaction. MI-219 also activates the
p53 pathway in cells with wild-type p53. Apoptosis
and cell cycle arrest were observed in xenograft
tumors which resulted in tumor regression .

30. Classes of Drugs for p53 Stabilization:
 Tenovin was found by a cell-based drug screen to
activate p53. Tenovin acts as an inhibitor of the
protein-deacetylating activities of SirT1 and
SirT2.
 The intraperitoneal administration of tenovin-6
has been demonstrated to induce a regression of
xenograft tumors in a mouse mode

31. p53 Gene Therapy:
 p53-based gene therapy was reported in 1996. A
retroviral vector containing the wild-type p53 gene
under the control of an actin promoter was injected
directly into tumors of nonsmall cell lung cancer
patients.
 After development of a replication-defective
recombinant p53 virus (Ad5CMV-p53) , many clinical
trials have been performed, including one in
esophageal cancer patients .

32. Conclusion
 Cytoplasmic p53 can activate a transcription-
independent apoptotic program. The next generation
of p53-based cancer therapeutic approaches should
therefore be developed to take advantage of this
cytosolic function.
 This may be safer than regulating the transcription
modulation of wild-type p53, which can induce both
prosurvival and proapoptotic effects in tumor cells.

33.  Recent reports have showed that p53 regulates the process of
self-renewal of neural stem cells and hematopoietic stem cells.
 The cancer stem model insists that tumors are maintained by a
small population of cancer stem cells that can divide both
symmetrically and asymmetrically.
 Mammary stem cells with the targeted mutation of p53 have been
reported to show the same properties as cancer stem cells. The
reactivation of p53 restored the asymmetric cell division of
cancer stem cells and induced tumor growth inhibition .

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