2. Transformation and potential tumorigenesis
• Transformation - alteration in a cell’s properties that leads
to immortalization and different growth patterns that result
from alteration in cell cycle
– Loss of anchorage dependence
– Loss of contact inhibition (foci)
– Decreased requirements for growth factors
• Tumorigenesis (oncogenicity) - in vivo development of
tumors
3. Cell cycle
• M- mitosis
• G1 - cells grow
• S - DNA synthesis
• G2 - growth and preparation for
mitosis
• G1/S decision point for going to
dividing state
• Problem for DNA viruses that
need S phase machinery
4. Cell cycle control proteins
• Activation of cell
cycle progression -
cyclins, cyclin
dependent protein
kinases (Cdks), Cdk
inhibitors
• Inhibitors of cell cycle
progression - tumor
suppressors
5. Tumor suppressor Rb
• Rb binds to
transcription factor
E2F and prevents gene
expression of proteins
needed to go to S
phase
6. Tumor suppressor p53
• P53 halts progression
when DNA damaged
– to give cell time to
repair or
– triggers apoptosis of
damaged cell by
activating Bcl-2
causing mitochondria
to release cytochrome
C and activate caspase
system
• If damaged (mutated) cell
moves to S phase then it
may replicate
7. Oncogenic viruses may be RNA or DNA
• 20% of human cancers believed
to be of viral origin
• These include:
– Cervical cancer
– Burkitt’s lymphoma
– Hepatocarcinoma
– Kaposi’s sarcoma
• Virus is not only factor
8. Viruses cannot kill cell to be tumorigenic
• Therefore may depend on host
cell
• May
– Integrate as part of their
cycle (retroviruses)
– Viral ORI and genes push
cell to S phase (herpes,
papilloma)
9. RNA transforming viruses are retroviruses so
far… (hepC)
• Permissive cells are
transformed
• Integration of viral cDNA
genome
• Requires expression of
oncogenes
– cell genes (c-onc)
– modified viral versions (v-
onc) whose expression
promotes transformation
and tumors
• HepC (no DNA phase) -
chronic inflammation and repair
– Viral proteins interact with
p53 and lead to cell
proliferation and prevent
apoptosis
10. oncogenes
• Cell gene is called proto-
oncogene
– can induce transformation
only after being altered
(mutation or coming under
the control of a highly
active promoter).
– usually encodes a protein
that affects DNA replication
or growth control at some
stage of the normal
development of the
organism.
12. V-onc genes - transducing
• Virus LTR is a strong promotor
• V-onc is altered form of c-onc
• rapid onset, high efficiency
tumorigenesis (acute
transforming)
time
%
transformed
13. Cis-acting insertions are low efficiency
tumor viruses
• Nondefective viruses
• Near c-onc and LTR activation
• Insertional inactivation of
tumor suppressor genes
• Chronic-transforming
% transformed
15. Identifying c-onc in mouse tumors
• Tumor cell DNA (mouse)
• Restriction fragments used to
form circles
• PCR based on viral genome
primers
• Sequence adjacent genes and
compare to mouse genome and
human equivalents
• Identified known sites and
several new ones
16. Hepatitis B
• DNA virus with RNA
intermediate
• In tumors virus is integrated
with little gene expression
• Believed to be from chronic
liver damage/loss and
replacement causing increased
mutations
• (similar to SOS response?)
17. DNA transforming viruses can be found in
all families
• Papova - circular DNA
• Adeno, Herpes - linear
• Oncogenic efficiency is low
• Typically nonproductive
infections - nonpermissive cells
or mutant virus
• Oncogenes are normal virus
early genes (used in replication)
– Virus gets stuck in early
phase and produces high
concentrations
– No cellular homologs
18. How are cells transformed?
• Cell cycle control changes due to viral genes that
– Interact directly with the proteins in the cycle
• Bind to and inhibit or degrade
– Interfere with expression of host cell cycle control
genes
20. Amino acid sequence similarities in Rb binding
site
V P E V I D L T C H E A G F P P S D D
Q P E T T D L Y C Y E Q L N D S S E E
F N E E - N L F C S E E M - P S S D D
L X C X E
AdE1a
HPV E7
Sv40 Tag
21. HPV E7 sequences differ in low and high risk
strains
6/11 P V G L H C Y E Q L N D
16 T T D L Y C Y E Q L N D
18 P V D L L C H E Q L S D
31 A T D L H C Y E Q L P S
33 P T D L Y C Y E Q L S D
Affects binding affinity to Rb
22. What happens to virus DNA?
• Oncogenes are integrated
(adeno, papova) and retained
• May require more than one
viral gene (Rb and p53)
23. Cotransfection of adenovirus E1A and other
genes on Neo vector
Plating after 4 weeks
G418 is a neomycin-type drug
Cells are transformed with E1A but only
E1B/neo is maintained
focus
24. Immunoblots (a-c) and PCR (d-f)
Cells transformed but don’t need viral genes to
remain
25. “Hit and run” mechanism
• Virus thought to cause
mutation in cell genes
and then virus is no
longer needed
• Similar results with
CMV
• Tumors may start with
virus but leave no
evidence of infection
26. The issue of HCV
• Core protein is a transcriptional regulator of cell promoters
for p53, p 21 etc
• Can immortalize hepatocytes if engineer cell with core on
plasmid
• What is the affect on immortalized cells of eliminating
core protein?
• How can you do this?
27. • Engineer antisense plasmid
(also could use siRNA)
• What happens to cells?
• Square = AS
• Circle = untransfected
• Triangle = vector control
28. Is death due to apoptosis?
• A) DNA gel sizes
• B) ELISA - ab against
nucleosome bound cytoplasmic
DNA
29. Is expression of p53, p21 affected by core AS?
• RNA protection assay
– Isolate mRNAs and add AS
then RNAase
– Run gel on protected
fragments
How about protein levels?
31. What is happening with telomerase activity?
Needed against senescence
• Luciferase as a marker for gene
activity
32. HCV core protein expression (+) and apoptosis
genes
• Hep 191 cells engineered with
core gene under induction
control
• HepRXR cells w/o core
33. KSHV and Kaposi’s sarcoma
• Virus expresses constitutive G
protein coupled receptor
Cells transfected with GPCR
Blood vessels
34. Human Umbilical Vein Endothelial cells
(HUVECs)
Grew transformed +/- GPCR 3T3 cells
and collected medium. Added it to
HUVECS and counted (3a)
Concentration dependent (3b)
Angiogenicity - microtubule formation
HUVECs added on top of gel-like
material and conditioned
medium added (3c)
Coculture expt - gel added on top of
transfected cells and HUVECs
added on top (3d)
35. VEGF is a major angiogenic inducer
• Transfected cells w/ or w/o
GPCR and measured VEGF in
medium by ELISA (4a)
• Used antibody to VEGF to
mitogenicity (4b) grey bar =
anti-vegf; white bar = control
ab
• Repear angiogencity expts (4c-
d)