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Oncogenic Viruses. “There is no single mechanism by which viruses cause tumors”. 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

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Oncogenic viruses

Oncogenic Viruses

“There is no single mechanism by which viruses cause tumors”


Transformation and potential tumorigenesis
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


Cell cycle
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


Cell cycle control proteins
Cell cycle control proteins

  • Activation of cell cycle progression -cyclins, cyclin dependent protein kinases (Cdks), Cdk inhibitors

  • Inhibitors of cell cycle progression - tumor suppressors


Tumor suppressor rb
Tumor suppressor Rb

  • Rb binds to transcription factor E2F and prevents gene expression of proteins needed to go to S phase


Tumor suppressor p53
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


Oncogenic viruses may be rna or dna
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


Viruses cannot kill cell to be tumorigenic
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)


Rna transforming viruses are retroviruses so far hepc
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


Oncogenes
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.



V onc genes transducing
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)

% transformed

time


Cis acting insertions are low efficiency tumor viruses
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


Virus gene product

C-onc


Identifying c onc in mouse tumors
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


Hepatitis b
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?)


Dna transforming viruses can be found in all families
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


How are cells transformed
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





What happens to virus dna
What happens to virus DNA?

  • Oncogenes are integrated (adeno, papova) and retained

  • May require more than one viral gene (Rb and p53)


Cotransfection of adenovirus e1a and other genes on neo vector
Cotransfection of adenovirus E1A and other genes on Neo vector

focus

Plating after 4 weeks

G418 is a neomycin-type drug

Cells are transformed with E1A but only E1B/neo is maintained


Immunoblots a c and pcr d f
Immunoblots (a-c) and PCR (d-f) vector

Cells transformed but don’t need viral genes to remain


Hit and run mechanism
“Hit and run” mechanism vector

  • 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


The issue of hcv
The issue of HCV vector

  • 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?



Is death due to apoptosis
Is death due to apoptosis? vector

  • A) DNA gel sizes

  • B) ELISA - ab against nucleosome bound cytoplasmic DNA


Is expression of p53 p21 affected by core as
Is expression of p53, p21 affected by core AS? vector

  • RNA protection assay

    • Isolate mRNAs and add AS then RNAase

    • Run gel on protected fragments

      How about protein levels?


Western blot
Western blot vector


What is happening with telomerase activity
What is happening with telomerase activity? vector

Needed against senescence

  • Luciferase as a marker for gene activity


Hcv core protein expression and apoptosis genes
HCV core protein expression (+) and apoptosis genes vector

  • Hep 191 cells engineered with core gene under induction control

  • HepRXR cells w/o core


Kshv and kaposi s sarcoma
KSHV and Kaposi’s sarcoma vector

Cells transfected with GPCR

  • Virus expresses constitutive G protein coupled receptor

Blood vessels


Human umbilical vein endothelial cells huvecs
Human Umbilical Vein Endothelial cells (HUVECs) vector

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)


Vegf is a major angiogenic inducer
VEGF is a major angiogenic inducer vector

  • 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)


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