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FUN2: 9:00-10:00
Scribe: Patricia Fulmer
Tuesday, November 18, 2008
Proof: Sally Hamissou
Dr. Engler
Microbiology: DNA Viruses 2
Page 1 of 5
SQ- Student Question HPV- Human Papillomavirus SRC- sarcoma forming genetic material
Rb- Retinoblastoma gene Myc & Ras- Oncogenes
Slides are numbered based on the updated power point version
How RNA and DNA viruses help us understand oncogenes and tumor suppressors
I. Introduction [S1]:
a. Viruses are important ways to understand how cells work.
b. Tumor viruses are an example of how viruses have helped us understand cancer.
c. This lecture will be half about viruses and half about cancer and how the viruses have helped us understand
cancer.
d. [These slides not in our posted presentation] He had us partner up and discuss what kind of cancers cause
the most deaths each year and whether or not these are different for men and women. (Question posed at 00:45
in the audio)
e. For men: lung, colon, prostate with pancreatic cancer being mentioned.
f. For women: lung, breast, colon were ranked first through third, with cervical and pancreatic being mentioned
also.
g. Cancer is an important problem for doctors, especially dentists because of oral cancer and optometrists
because of ocular cancer.
h. He will be using cancer and viruses, specifically RNA and DNA tumor viruses, to help us understand how cells
work.
II. Characteristics of Cancer cells [S2]
a. Cancer cells undergo unregulated growth- grow when they aren’t supposed to.
i. Normal cells don’t grow and remain at a quiescent state
b. Cancer cells become immortal (active growth when they should be quiescent)- our normal cells do not grow
under everyday conditions
c. Cancer cells have increased nutrient uptake
d. Cancer cells in tissue culture become anchorage independent- they don’t have to be stuck on any substrate to
grow
e. The cell cycle (regulation of growth) in cancer cells becomes active- full mitosis and meiosis cycles
i. Growth signaling pathways activated by oncogenes and tell cell to start growing when it doesn’t need to by
oncogenes (many RNA tumor viruses have oncogenes in them)
ii. Pathways to prevent cell proliferation are disrupted- tumor suppressors normally keep the cell from growing
(DNA tumor viruses disrupt tumor suppressors so that the brakes come off of the growth pathways)
III. Cancers develop in many steps [S3]
a. Cancers don’t happen overnight
b. Takes a whole series of mistakes/mutations to cause cancer by making the normal cell become a cancer cell
and then reach metastasizing cell state.
c. All of these are genetic mutations or act that causes the cell to lose its ability to turn its growth off.
d. Build up of these mutations causes a normal cell to undergo a shift toward metastasis/tumor formation
e. Viruses help us understand many of these steps.
f. This slide shows a series of oncogenes, mutations of tumor suppressors, and other causes a normal cell to
progress to become cancerous.
IV. Some types of cancer associated with tumor viruses [S4]
a. Many kinds of cancer
b. Leukemias- blood borne; derived from lymphoid cells
c. Carcinomas- derived from epithelial or endothelial cells
d. Sarcomas- derived from connective tissue cells
e. Rouse sarcoma virus (chicken virus) causes tumors in connective tissues in chickens- granddaddy tumor virus
studied
V. Definitions [S5]
a. Need to keep the two pathways separate in our minds: one adds gas to the system (tells the cells to grow when
it’s not supposed to) and the other pathway is a tumor suppressor that has been inactivated so the brakes come
off
b. Oncogene adds gas to system
c. Oncogenes are found in acute transforming RNA tumor viruses- called “gain of function” mutations. If either
copy of this gene is activated, you have gain of function and cell starts to become a tumor. It is an altered gene
whose product can act as a dominant factor to help make a cell into a tumor.
FUN2: 9:00-10:00
Scribe: Patricia Fulmer
Tuesday, November 18, 2008
Proof: Sally Hamissou
Dr. Engler
Microbiology: DNA Viruses 2
Page 2 of 5
d. Proto-oncogenes are normal cells that are almost always involved in some way in control of cell growth—normal
form of oncogene
e. In some way the cell is getting a signal to grow when it shouldn’t.
f. The converse is a loss of function mutation on a tumor suppressor gene, loss of function. It’s like taking the
brakes off your car.
i. Normal activity prevents tumor formation, therefore suppressing any growth of a tumor, but because of a
mutation that normal activity does not occur.
ii. Loss of function by mutation increases chances of cancer.
g. You have to knock out both copies of the tumor suppressor genes on the chromosome for the mutation to take
effect.
h. DNA tumor viruses knock out the protein product of these tumor suppressor genes.
VI. Oncogenes can increase tumor susceptibility in transgenic mice [S6]
a. Oncogenes help send bogus signals to tell the cells to grow
b. Myc is oncogene- expression will cause the mouse to die over time
c. Ras causes mouse to die sooner
d. A combination of the two will do the most damage (steep slope on graph). They act synergistically and have a
dramatic incidence of tumor.
e. Mutations accelerate each other.
VII. How do oncogenes and tumor suppressors work? [S7]
a. Idea of gain of function ad loss of function
b. Mutation will cause the oncogene to activate- only takes a single mutation; causes cell to proliferate
c. Must have both copies of tumor suppressor gene mutated for that mutation to be expressed. It is the loss of
growth brakes.
d. [S8] Don’t memorize this slide! Point is we have lots of places with oncogenes and lots of places with tumor
suppressor genes that will keep cells from growing when they shouldn’t. As you begin to mutate the cell loses its
control over growth.
VIII. General overview of DNA and RNA transforming viruses [S9]
a. Lots of viruses that are classified in 2 ways: DNA tumor viruses and RNA tumor viruses
b. A lot of natural tumor formation occurs.
c. There is Rous sarcoma virus (RSV) that was discovered by Peyton Rous and is a RNA tumor virus.
d. It was a chicken virus discovered in 1911. The chickens formed tumors at a high rate. If you ground up the
tumor, mixed it with saline, and put it into another chicken, that chicken got tumors too. That’s how he knew it
was a virus. This was the 1st example of a virus and 1st RNA tumor virus to be found.
e. There are other RNA tumor viruses like HTLV1 (involved in T-cell leukemia, prevalent in Japan; about 1% of
people with this virus will develop a cancer of some sort) and Hepatitis C (associated with hepatocellular
carcinoma)
f. DNA tumor viruses
i. HPV causes warts and has some kinds associated with cervical cancer (HPV16, 18, 31, and others)
1. The vaccine against cervical cancer consists of the protein shell of HPV from types 16, 18, and 31
(the three most common forms)
2. The HPV vaccine consists of the protein shells for type 16, 18, 31.
ii. Epstein Barr virus (EBV) causes mononucleosis-in certain situations, EBV can cause a nasopharyngeal
carcinoma called Burkitt’s lymphoma; this is more common in third world countries. It’s an immunological
defect that allows the cancer to occur
g. Hepatitis B viruses are also associated with hepatocellular carcinomas and are DNA viruses
h. All of these viruses help us to understand cancer
IX. Retrovirus life cycle requires integration into the chromosome [S10-11]
a. In retroviruses, the viral RNA gets into the host cell, you make a DNA copy that gets integrated into the host and
makes new genomes and new proteins to make new virus. This is important to remember for understanding
RNA tumor virus.
b. When we talk about tumor viruses, we are mainly talking about retroviruses (viruses that have reverse
transcriptase and perform the RNADNARNA and proteins method)
c. 2 classes of these:
i. Acute transforming viruses – carry oncogenes in the virus RNA genome.
1. Can transform cells in culture
2. May be replication competent (can replicate by themselves-example is Rous virus) or replication
defective (cannot replicate by itself)
ii. Non acute (chronic) transforming viruses- don’t carry oncogenes
FUN2: 9:00-10:00
Scribe: Patricia Fulmer
Tuesday, November 18, 2008
Proof: Sally Hamissou
Dr. Engler
Microbiology: DNA Viruses 2
Page 3 of 5
1. Cause tumors more slowly by inserting their DNA copy somewhere close to a proto-oncogene in the
chromosome
X. Chronology of understanding oncogenes [S12-14]
a. History lesson- one of the 1st things what Rous realized was that sometimes his experiment didn’t grow a virus.
He discovered that there are temperature sensitive mutants of RSV. When you grow them at 37 degrees C, they
make virus and tumors. When grown at 41 degrees, they only make virus—no tumor.
b. At 41 degrees C, there’s some protein denaturing that is important for the tumor to form. Therefore, the thing
causing the tumor is a gene product.
c. 40 years later- it was found that defective mutants that couldn’t form a tumor at any temperature. The genomes
were shorter in these viruses. Things that could make a tumor were longer and things that could not make a
tumor were shorter. So these mutants were missing some piece of information that caused the tumor to form.
That means the tumor making component is found in the RNA genome and is present on the wild type but
missing on the mutant.
d. Piece of information missing on the mutant is called SRC—sarcoma forming. Nobel Prize to Harold Varmus and
Mike Bishop for figuring out the next step.
e. Reverse transcriptase takes RNA and makes DNA in retroviruses. They took the wild type RNA from a Rous
virus that could make tumors and used reverse transcriptase to make small pieces of DNA that were
radioactive. Did hybridization experiment with this DNA and RNA from a mutant virus that was without a SRC
(hybridization experiments anneal the DNA and RNA), and the stuff left over was from the wild type DNA that
would have matched up with the SRC that was missing from the mutant. Call this extra DNA the SRC-specific
probe. It is specific for the SRC gene. They then took this probe and hybridized it to RNA from a normal virus,
and it annealed 100% to the SRC gene. Now had a probe that could show them an oncogene.
f. Discovered there are different kinds of oncogenes on viruses that can’t grow by themselves. They need a helper
to grow.
g. These have to be grown in mixed cultures with wild type and viruses with the oncogene in them for growth and
tumor formation
h. Varmus and Bishop did another experiment to see if the probe would bind to chicken DNA
i. 65% of the probe bound to a region of chicken chromosome. This means that there is a copy of the SRC gene
inside the chicken DNA and many other mammals as well, including humans.
j. This raised the idea of a proto-oncogene. The viruses have captured bits of a DNA copy of mRNA and
incorporated it into the virus. This bit causes the tumor and the virus transports the bits from cell to cell.
k. Studying these oncogenes will tell us what proteins/genes are involved in cell growth control.
l. Each retroviral oncogene has a cellular proto-oncogene counterpart that is captured by the virus and carried
along with it.
XI. Genetic changes convert a proto-oncogene into an oncogene [S15-16]
a. Genetic changes activate the oncogenes from a proto-oncogene. Many different ways to do this.
b. One way is in the ErbB oncogene- normally is the epidermal growth factor (EGF) receptor, but when ErbB loses
the extracellular domain of EGF, it sends a signal to the cell that makes the cell think EGF is present always and
it is time to grow.
c. Another kind of oncogene is Neu. This is a point mutation where Val in plasma membrane gets changed to Gln,
and this means that no matter what the new oncogene recognizes, it tells the cell it is there and tells cell to grow
when it shouldn’t.
XII. Four classes of oncogenes [S17-18]
a. There are a lot of places that have oncogenes. (He said this often during lecture)
b. Have to know all four classes of oncogenes for the TEST!!! Know an example of each one!!! Just need
one example.
c. The first class are things that look like growth factors. The virus makes a protein that is secreted out of the cell
and binds to its receptor on the outside of the cell, pretending to be a growth factor when it is not one.
d. Only two are known, very rare.
e. Sis mimics Platelet Derived Growth Factor (PDGF)- pretends to be a growth factor when it’s not
f. Phony growth factors = Class one!
XIII. Class Two: Mutated Receptors [S19-20]
a. 2nd Class consists of mutated cell receptors.
b. When you mutate a receptor, the signal is always on.
c. ErbB and Neu are both mutated receptors that send false signals for the cell to grow. There’s also fms, ros and
sea
d. ErbB is a deletion of EGF receptor extracellular domain and neu is a point mutation that causes the receptor to
always send the signal to grow.
FUN2: 9:00-10:00
Scribe: Patricia Fulmer
Tuesday, November 18, 2008
Proof: Sally Hamissou
Dr. Engler
Microbiology: DNA Viruses 2
Page 4 of 5
XIV.
Class Three: Intracellular transducers [S21-22]
a. The 3rd kind of oncogenes are intracellular transducers- shuttle signal from receptor to nucleus; there are a
lot of these
b. There are 4 kinds: tyrosine kinases, serine/threonine kinases, Ras (or G) proteins, and Phospholipase C (PKC)
i. These are the proteins that are targeted in cancer drug research currently- trying to block the bogus signal
these are transporting
c. These mutated transducer molecules send incorrect ON signals
XV. Class Four: transcription factor oncogenes [S23]
a. These are mutated transcription factors that turn on genes for growth at the wrong time
b. And there are a lot of examples of this
c. Transcription factor AP1 which controls the transcription of a lot of genes is composed of 2 proteins (Jun and
Fos). Each one can be an oncogene. A mutation of either one can cause transcription factors to turn on when
they shouldn’t.
d. There’s Myc and Myb and many others that are oncogenes and transcription factors that do their job at the
wrong time for the cell.
XVI.
The same oncogenes can be found in more than one virus isolate [S24]
a. And these themes, oncogenes, come up again and again and again. No matter where you look you’ll find
examples of oncogenes and different kinds of acute transforming retroviruses. You’ll find them in different
animals as well. They’re in many places.
XVII. Non acute transforming viruses [S25-27]
a. Acute is a genetic mutation that affects the growth signaling pathway in one of four ways:
i. Acts like a phony growth factor
ii. Mutated receptor
iii. Mutated transducer- sends signal at inappropriate times
iv. Transcription factor
b. Non acute viruses don’t carry an oncogene. They activate the proto-oncogene copy in the chromosome by
inserting.
i. Very slow process (1-2 years tumor formation because not efficient)
ii. Chronic viruses are transformation by insertion
iii. Viruses insert near an oncogene- they turn on the gene by rearranging chromosome in some places or
amplifying the gene in that local place
XVIII. Insertional activation of proto-oncogenes [S29]
a. Insertion near a proto-oncogene can cause effects by:
i. The viral enhancer acting on the nearby proto-oncogene
ii. The viral promoter transcribing a nearby oncogene (he said proto-oncogene, but the slide says oncogene)
iii. Sometimes transcription is altered causing the proto-oncogene and viral RNA to fuse
iv. Rarely the gene is inactivated
b. This doesn’t occur to every cell, so the process is very inefficient
c. Summary: acute RNA transforming viruses carry oncogenes, chronic don’t carry oncogene but activate the
proto-oncogene in the chromosome.
XIX.
Some DNA tumor viruses block tumor suppressor pathways [S30-31]
a. The acute pathway has oncogenes, the non acute does not. It activates/alters the proto-oncogene in some way.
b. DNA tumor viruses do the alternative pathway, which takes off the brakes
c. Lots of DNA tumor viruses
i. Adenoviruses- we’ve all been infected by these at some point
ii. Papillomaviruses- what the cervical cancer vaccine is for
iii. Papovaviruses
1. Simian Virus 40 (Monkey virus)
2. JC and BK viruses (human)
d. These all have proteins that function to take the brakes off
e. Adenovirus’ protein is E1A- there’s a number of tumor suppressor gene products that bind to E1A so they can’t
do their normal binding on the cell and suppress growth
f. The proteins for Papillomavirus (E7) and Papovavirus are the same way. They do this because the viruses want
the cell in S phase because that’s when all the material to make DNA copies and protein are most available.
The cell wants to remain in G0 (quiescent).
g. The virus must initiate cell cycle by making these proteins and sequestering the suppressors.
XX. Retinoblastoma [S32-35]
a. A rare form of ocular tumor
b. Occurs in childhood
FUN2: 9:00-10:00
Scribe: Patricia Fulmer
Tuesday, November 18, 2008
Proof: Sally Hamissou
Dr. Engler
Microbiology: DNA Viruses 2
Page 5 of 5
c. Tumors develop from neural precursor cells in the immature retina
d. About one child in 20,000 is afflicted.
e. Two forms of the disease:
i. hereditary (multiple tumors affecting both eyes) - germline mutation in one copy of gene predisposes
individual to retinoblastoma
ii. non-hereditary (single tumor in one eye)
f. Deletion in chromosome 13 of a tumor suppressor gene(recessive mutation)
g. Gene product of retinoblastoma gene keeps cell shutdown
h. If you mutate both copies (must be both because recessive), all brakes are off and start to form a tumor
i. Mutations in retinoblastoma gene (Rb) has often been implicated in breast and small cell lung cancer and others
as well
j. Retinoblastoma usually binds to another transcription factor E2F, which controls transcription and gene
expression of most genes a cell needs to copy its DNA; it makes all the things a cell needs to be ready to copy
DNA in S phase
k. Rb works normally by inhibiting E2F from doing its job so that the cell does not enter S phase and grow
l. A mutation of Rb means that E2F can start working, and the cell will move toward S phase
m. Normally Rb is bound to E2F and it keeps it from doing its job to make all the genes that will make DNA. At a
specific point of G1 phase of cell cycle, Rb is phosphorylated and releases E2F, allowing it to perform its job and
the cell to continue the growth process. Mutating Rb is the equivalent of phosphorylating it, and it stops working,
E2F is released, and the cell shifts toward S phase and making DNA.
n. Adenoviruses and those like it function to bind to Rb and cause the cell to move toward S phase.
XXI.
How do oncogenes and tumor suppressors work? [S37]
a. DNA tumor viruses work by taking off the brakes
b. They bind to the Rb protein, which causes E2F to become free and the cell to shift toward S phase (which the
virus wants).
c. Also can shift the cell toward unregulated growth, not unlike when Rb is mutated in a tumor.
d. Acute transforming RNA tumor viruses show us that there are oncogenes and that oncogenes are dominant,
that there are proto-oncogenes in the cell chromosome that can be activated by inserting a virus near them and
changed to an oncogene, and that the cell gets a signal to start growing
e. There are also tumor suppressors- loss of function mutations that have to have both copies inactivated; release
things like E2F that will shift the cell toward S phase—equivalent of making them into a tumor
XXII. Summary [S38]
a. Viruses have shown us a lot about oncogenes and tumor suppressors
b. Acute tumor viruses exert their effect through growth signaling—send signals that say the cell needs to grow
unregulated; leads to tumor
c. DNA tumor viruses want to shift cell to S phase so they can copy their DNA; sequester away tumor suppressor
proteins (Rb, p53 for example) that normally keep the cell shut down so that they can speed up cell’s entry into
S phase
d. This shows us a lot about how the cell normally works to keeps itself regulated and shut down
e. If you mutate Rb, get Retinoblastoma. You have to mutate both copies-that’s why we have hereditary form (both
genes have been mutated so you have tumors in both eyes). The non-hereditary form has only knocked out the
genes in one cell, not the whole genome, so you get tumor in one eye only.
SQ: Are RNA viruses acute and DNA viruses non acute? No, there are two types of RNA viruses. There are the acute
kinds that carry oncogenes. The non acute RNA tumor viruses do not carry oncogenes but instead they insert their DNA
copy into the chromosome somewhere close to a proto-oncogene and turn the proto-oncogene on when it shouldn’t be.
DNA tumor viruses generally don’t cause cancer unless something bad happens. You can think of them as chronic.
You’ve all been infected by human Adenovirus, but probably none of you will get a tumor directly due to that infection.
That’s just not what it normally does effectively, unless you put it into a situation where it can’t grow. Papillomavirus can
lead to a tumor in some cases, but you can be protected by taking the vaccine to keep it from growing.
[End 49 min]