Download V. How virusES cause cancer

Cancer
I.
SIGNAL TRANSDUCTION
A. For a cell to respond to extracellular signals either material or a signal must be
passed into the cell
1. Signal transduction has the following characteristics
a) An extracellular molecule, called a ligand, bind to s protein on the outer
surface of the cell
(1) The protein the ligand binds to is referred to as the cell's receptor and is
generally quite specific as to what type of ligand it will bind to
b) The signal is transmitted to the cell's cytoplasm by the properties of the
receptors (e.g., activate its cytoplasmic domain)
2. Overview
a) The transmission of a signal involves the interaction of the extracellular
ligand (e.g., peptide hormone) with a transmembrane protein
(1) A transmembrane protein has domains on both sides of the membrane
(2) This may activate enzymatic activity of the intracellular domain
(3) This may lead to changes that allow binding / activation of cytoplasmic
enzymes (e.g., G-proteins)
3. Amplification
a) The transduction of the signal into the cell usually results in activation of an
enzyme catalyst
(1) This enzyme can than carry out many reactions producing products referred to
as secondary messengers
(a) There will be many more secondary messengers than there were primary
messengers (i.e., the ligand that bound to the extracellular matrix of the
receptor)
4. Events
a) Peptide hormone binds transmembrane hormone receptor
(1) This will allow docking of G-protein or activation of tyrosine kinase activity
associated with the intracellular domain
(a) Upon docking, the G-protein hydrolyzes GTP, releasing the phosphate,
but binding the GDP
(b) Once activated by binding GDP it exhibits tyrosine kinase activity
b) Phospholipase C and adenylate cyclase are activated by phosphorylation by
kinases
(1) Phospholipase C cleaves the membrane lipid phosphatidyl inositol into inositol
triphosphate and the membrane bound diacylglycerol
(2) Adenylate cyclase converts ATP into cAMP
c) Cytoplasmic calcium levels increase
(1) cAMP lets calcium into the cell
(2) Inositol triphosphate causes calcium to be released from the endoplasmic
reticulum
d) Increased calcium level have several effects
(1) Activates calmodulin
(2) With the help of diacylglycerol activated protein kinase C
e) Cyclic AMP activates cAMP-dependent protein kinase A
(1) This enzyme phosphorylates cAMP-response element binding proteins
(2) The cAMP-response element binding protein enters the nucleus and binds to
cAMP-response elements
II.GENERAL
A. Definition
1. Cancers arise when a single somatic (body) cell loses control of growth and
goes under rapid proliferation
B. Somaclonal theory of cancer formation
1. Cells that make up a solid tumor are progeny, or clones, derived from a single
aberrant ancestor cell
C. Morbidity and mortality
1. 500,000 die of cancer each year
2. 30% of Americans alive today will get cancer
3. 1 million new cases per year
a) 60% of these new cases will die in 5 years
D. Cancer may strike any part of the body
1. Adenoma
a) Glandular tissue
2. Blastoma
a) Germ and germ precursor cells
3. Carcinoma
a) Epithelial tissue
4. Hepatoma
a) Liver
5. Leukemia
a) Leukocytes
6. Lymphoma
a) Lymphoid tissue
7. Melanoma
a) Skin
8. Myeloma
a) Muscle
9. Sarcoma
a) Connective tissue
III.CORRELATION
BETWEEN CANCER AND VIRUSES
A. 1911 -- Peyton Rous
1. Filterable extracts of a sarcoma could be injected into healthy chickens to
induce cancer
a) Later was found to contain a RNA virus
(1) Virus now referred to as Rous Sarcoma Virus
B. 1930 -- Richard Shope
1. Species specific effect of Papillomavirus noted
a) Caused warts in wild rabbits
b) Caused tumors in domestic rabbits
C. Human oncogenic viruses
1. Human T cell leukemia virus type I (HTLV-I) causes adult T-cell leukemia
2. Burkitt's lymphoma is caused by the Epstein-Barr virus
3. Nasopharyngeal cancer is caused by the Epstein-Barr virus
4. Hepatitis B virus causes hepatocellular carcinoma (liver cancer)
5. Papillomavirus causes cervical cancer
IV.MAMMALIAN
CELL CULTURE MODELS
A. Definitions
1. Primary cells
a) Cells taken directly from a living organism
b) Mortal
(1) Have a limited life span
(2) Divide 50 times before dying
2. Immortal cells
a) Live and reproduce forever
b) Arose from mutated cultured cells
B. Characteristics of non-cancerous cells grown in culture
1. Flattened morphology
2. Anchorage dependent growth
a) Must touch bottom of Petri plate
b) Don’t grow on soft agar
3. Confluent monolayer
a) One to several cell layers thick
4. Contact inhibition
a) Replication ceases upon contact with neighboring cells
C. Viral transformation
1. Tumor virus infections could result in 2 markedly different infections, which
corresponded to those described for in bacteriophage lambda
a) Lytic
(1) Lyse host cells
b) Lysogenic
(1) Integrate into host's chromosome
c) Transformation
(1) Host cells take on characteristics of tumor cells
D. Characteristics of transformed cells
1. Round-globular morphology
2. Not contact inhibited
a) Pile up on one another to form a clump called a focus (pl., foci)
3. Anchorage independent and will grow on soft agar
E. Transformation is only a convenient model
1. Adenovirus causes flu in humans and cancer in mouse 3T3 cells
V.
HOW VIRUSES CAUSE CANCER
A. General
1. Conversion of a cultured cell into a tumor cell by a virus is called transformation
2. In transformed cells the tumor virus integrates randomly into host cells genome
a) Retroviruses copy their RNA genome into DNA using viral reverse
transcriptase
b) Integrated virus is referred to as a provirus
c) Once integrated, host RNA polymerase may transcribe viral genes and the
resulting mRNA may be translated by host ribosomes
B. Search for viral oncogene
1. Overall strategy
a) Find the difference between a tumor virus and a mutant of that virus that can
not cause cancer
2. Specific strategy
a) Make radioactive DNA copy of wild type tumor virus RNA genome
(1) Isolate viral RNA genome
(2) Add reverse transcriptase and radioactive nucleotides
b) Fragment DNA copy and the RNA genome of the mutant (non-tumor
causing) viral RNA genome
c) Hybridize them
(1) The genes these viruses have in common should hybridize together
(a) The DNA copy of the wild-type (tumor causing) virus should be
complimentary to the RNA of the mutant (non-tumor causing) virus, except
for the gene responsible for transformation
d) Isolate single stranded DNA
(1) Single stranded DNA will be DNA that did not hybridize
(2) This will contain the gene that the wild type (tumor causing) virus contains
which is missing in the mutant (non-tumor causing) virus
C. Many viral oncogenes are captured cellular genes
1. Isolated viral oncogenes hybridize with host cell DNA
2. Oncogenes are not “bad” genes found only in tumors, but essential genes
whose normal function has been altered in some manner
a) The normal version of the potentially bad gene is called a proto-oncogene
b) The viral copy of the gene is usually indicated by a “v” (e.g., v-sis), while the
host cell version is indicated by a “c” (e.g., c-sis)
VI.HOW
ONCOGENES CAUSE CANCER
A. Cancer is a disease of cellular communication
1. Cell division is highly regulated
a) A peptide hormonal message binds to a cell receptor
b) Signal transduction brings the signal inside the cell
c) A secondary messenger is formed (inositol triphosphate)
d) Secondary messengers activate kinases
e) Kinases activate DNA binding proteins
f) DNA binding proteins bind to regulatory sequences of genes which code for
enzymes needed for cellular replication
2. Oncogenes are involved in every step
3. Approximately 40 proto-oncogenes have been isolated
B. Simian sarcoma virus
1. Carries a human gene for the platelet derived growth factor
a) Normal function of platelet derived growth factor
(1) Released by platelets to stimulate tissue repair at would sites
(2) Connective tissue cells have receptor for this hormone and divide in its
presence
(3) Platelet derived growth factor proto-oncogene is called c-sis
b) Simian sarcoma virus carries a version of the gene for platelet derived
growth factor called v-sis
2. Simian sarcoma virus will infect connective tissue cells and the v-sis gene will
be constituitively expressed
a) Now a cell secretes a growth hormone for which it also responds to by
dividing
3. Suggests that the simian retrovirus captured the cellular gene
C. Avian erythroblastosis virus
1. Carries part of the human gene for the epidermal growth factor receptor
a) Normal function of epidermal growth factor receptor
(1) This receptor receives the epidermal growth factor and tells the cell to divide
(2) Encoded for by the c-erbB gene
b) The Avian erythroblastosis virus contains an incomplete copy of this gene
called v-erbB
2. The incomplete gene codes for an incomplete receptor (lacking the extracellular
domain) which continually transduces a signal in the absence of a growth
hormone
VII.PROTO-ONCOGENES
CODE FOR NORMAL PROTEIN
A. c-ras oncogene
1. The ras proto-oncogene encodes for a G-protein
a) The G-protein is a protein kinase that activates phospholipase C
b) It is activated when receptor change conformation due to hormone binding,
thus allowing GTP to bind the valine of the G protein
c) The G-protein is turned off when the GTP is cleaved
2. The ras oncogene has a one base pair mutation
a) The twelfth codon normally codes for a valine amino acid, but the mutation
results in a glycine amino acid at that position in the G-protein
b) Once activated, the mutant G-protein will be stuck on
3. Twenty percent of tumors have mutated ras gene
B. Some proto-oncogenes are normal, but over expressed
1. Viral insertion
a) c-myc gene
(1) c-myc encoded protein interacts with DNA to release constraints of cell
replication
(2) c-myc gene is a frequent place for provirus integration
(a) Virus integrates between the operator and promoter, thus blocking
negative control of transcription
2. Translocation and transversions
a) Proto-oncogene may be moved in front of an active promoter
(1) Burkitt's lymphoma involves a translocation of an oncogene on chromosome
8(?) to a antibody heavy gene enhancer on chromosome 14(?)
(2) Parathyroid cancer is caused by an inversion on chromosome 11 that moves
an oncogene next to the parathyroid hormone promoter
3. Mutation
a) Promoter or operator region may be mutated
4. Gene amplification
a) Some tumors have 3000 myc genes
VIII.SEARCH
FOR HUMAN ONCOGENES
A. Experimental protocol
1. Cut human chromosome with HindIII
2. Ligate it to a selectable marker
a) This marker should be selectable in E. coli
3. Transfect tissue culture
4. Isolate DNA from foci
5. Cut with SauIIa
a) Any restriction enzyme except HindIII will do
6. Ligate into lambda arms
7. Transform E. coli
8. Grow E. coli under selectable conditions
9. Isolate DNA from lambda
10. Recheck ability to form foci
IX.MORE
THAN ONE ONCOGENE IS NEEDED
A. Requirements for transformation
1. Cells must be immortal
2. Cells must divide uncontrolled
B. ras and myc oncogenes
C.
D.
E.
F.
G.
H.
X.
1. ras oncogene transforms 3T3 cells (immortal) but not primary cells
2. myc oncogene could not transform 3T3 cells
Adenovirus
1. Contains 2 oncogenes, both of which are needed for transformation
a) E1A immortalizes cells
b) E1B caused tumors
Polyomavirus
1. Contains 2 oncogenes, both of which are needed for transformation
a) Large T immortalizes cells
b) Middle T caused tumors
Recombinant viruses
1. E1A and Large T caused cells to be immortal, but did not cause cancer
2. E1A and Middle T caused cancer
3. Large T and E1B caused cancer
4. E1B and Middle T caused no change
Immortalizing oncogenes and Transforming oncogenes
1. Immortalizing oncogenes make cells immortal
2. Transforming oncogenes cause immortal cells to divide
Human oncogenes
1. ras oncogene
a) E1A and ras caused tumor
b) E1B and ras caused no change
c) Large T and ras caused tumor
d) Middle and ras caused no change
2. myc oncogene
a) E1A and myc caused cells to be immortal
b) E1B and myc caused tumor
c) Large T and myc caused cells to be immortal
d) Middle and myc caused tumor
3. myc is an immortalizing gene and ras a transforming gene
Astrocytomes
1. Most common form of brain cancer
2. Steps
a) Loss of both p53s
b) Loss of some of chromosome 9
(1) Including a tumor suppressor gene
c) Oncogene on chromosome 7 over expresses a growth hormone receptor
d) Loss of chromosome 10
ENVIRONMENTAL FACTORS
A. Benzopyrene immortalized cells
1. If exposed, only ras oncogene needs to be activated
2. Twice as likely to get cancer since only 1 gene needs to be turned on
B. 3-methylcholanthrene immortalizes cells and turns on the ras gene
XI.ANTI-ONCOGENES
A. Genes whose products suppress tumors
1. Potential use in gene therapy
2. Must be knocked out for tumor to occur
3. Recessive
a) Mutation of one has no effect
b) Oncogenes are dominant
(1) Only one needs to be present
4. Cell proliferation is under both positive and negative control
a) Positive control needs to be amplified
b) Negative control needs to be repressed
B. Retinoblastoma
1. Predisposition to cancer can be inherited
2. In some families, the Rb gene is missing, hence only one needs to be knocked
out
a) People with acquired form of retinoblastoma usually have only 1 tumor
b) People with the inherited form have many tumors
3. E1A and Large T gene products bind to Rb gene product
a) Ties up all free Rb
XII.OTHER
FACTORS
A. Angiogenesis factors cause tumor to be infiltrated with blood vessels
B. Loss of adhesion molecules
C. Hormones that cause lactation immortalizes cells
1. These hormones cause expression of myc gene
D. Epidemiology of cancer show 4 to 6 hits are necessary
XIII.MULTISTEP
PROGRESSION
A. Fully malignant cancer cells do not usually develop from a single cause
B. Causes
1. Radiation
2. Chemicals
3. Retrovirus integration
4. Random point mutation
5. Random DNA amplification
6. Random deletion
C. Initial changes may be inherited
1. Familial retinoblastoma
2. Familial adenomatous polyps of the colon
3. Multiple endocrine neoplasia
a) Cancer of the thyroid, adrenal medulla, and parathyroid
4. About 5% of colon cancers involve inheritance
5. Other cancers, such as breast, colon, and ovarian also run in families
D. Colorectal cancer
1. Deletion of a gene on chromosome 5
a) May inherit one chromosome 5 already missing this gene
b) Limited proliferation
c) Benign tumor
(1) Adenoma class I
(2) Grows as a polyp
2. Mutation of ras proto-oncogene to an oncogene
a) Still benign, but faster growing
(1) Adenoma class II
(2) ras codes for protein kinase
3. Gene encoding cell adhesion molecule on chromosome 18 is deleted
a) Adenoma class II
(1) Still benign
4. Alteration or mutation of tumor suppressor gene on chromosome 17 (p53)
a) Malignant carcinoma
5. 8 - 10 more mutations for metastasis to occur