- Welcome everyone, to Wednesday Nite at the Lab .
I'm Tom Zinnen.
I work here at the UW-Madison Biotechnology Center.
I also work for UW-Extension Cooperative Extension,
and on behalf of those folks and our other co-organizers,
Wisconsin Public Television,
the Wisconsin Alumni Association,
and the UW-Madison Science Alliance,
thanks again for coming to Wednesday Nite at the Lab .
We do this every Wednesday night, 50 times a year.
Tonight, it's my pleasure to welcome back
to Wednesday Nite at the Lab, Professor William Sugden.
He works with the McArdle Lab for Cancer Research.
This is the fifth in a series of talks
by McArdle researchers.
We're commemorating the 75th anniversary
of the founding of McArdle Lab.
Professor Sugden was born in Queens, New York City,
got his undergraduate degree at Harvard,
got his PhD at Cold Spring Harbor,
then postdoc at the Karolinska Institute in Stockholm,
and then he came here to UW-Madison.
Tonight, he's going to be talking to us about
understanding Burkitt's Lymphoma,
from its discovery to working towards its cure.
This is the second talk that Professor Sugden
has given in this series.
Please join me in welcoming Bill Sugden back
to Wednesday Nite at the Lab .
(applause)
- Thank you, Tom.
What I'd like to do this evening is to point out
that Burkitt's Lymphoma is caused by a virus,
Epstein-Barr virus, and in particular,
tell you how we know that.
And I want to do that because it's just an amazing story
particularly in its early phase of people dedicated
to cancer research and what a few people were able to do.
It's a lovely story.
Along the way, I want to address what is cancer?
How does a series of questions lead to
understanding a particular cancer?
And how can we use this understanding to develop
therapies for a specific cancer?
The story of Burkitt's lymphoma actually begins
a long time ago, far away, in a different continent,
in eastern African, about 60 years ago.
And fittingly, it begins with Denis Burkitt.
So Burkitt was trained as a surgeon in Ireland
and in England, just before and during World War II.
And by the mid-1950s, as a mission doctor,
he was in Kampala, Uganda.
I met Denis Burkitt.
I didn't know him well but I read a lot of his papers
and I read a lot of about him.
And from all of that, I have the impression that he was
an amazingly perceptive person whose perceptions
allowed him to see things that a lot of us
wouldn't be able to see.
So I'm going to describe some of that to you too.
In 1957, in a hospital in Kampala,
a colleague introduced him to a child who had
massive growths on both sides of his jaw,
upper and lower jaws.
He had never seen anything like this nor had his colleague.
He examined the youngster, recognized
there was nothing he could do to help that child,
and went along about his surgeries.
So in general, he had more than 600 surgeries each year
in Kampala and in rural clinics surrounding Kampala.
And that could've been the end of the story,
but several weeks later, at one of those rural clinics,
he looked outside and saw another child
that presented exactly the same way.
Massive growths on both sides of the jaw,
upper and lower jaw too.
And this was more than a curiosity, this was something
he wanted to understand.
He examined the youngster.
He brought the youngster to Kampala to examine
him with her his mother and recognized that
this was something that he had not seen before,
and he wanted to address two kinds of questions.
In particular, what kind of disease did it represent,
this new growth that he had found?
For example, could it be the response to a parasitic
or even a bacterial infection?
And did this disease represent a new entity,
one that had just entered the population
so it would've been seen before?
To address the first question, he set about just examining
youngsters that he found around Kampala,
and it's amazing because quite rapidly, he found 36 cases
that presented the same way,
large growths both sides of the jaw, upper and lower jaws.
But he also very rapidly realized that for most
of these children, there were additional large growths
elsewhere in their body, in internal organs,
stomach, kidney, ovaries for example.
And he reasoned it was quite unlikely that
a response to a bacterial infection, a parasitic infection,
would give rise to these multiple growths
at distant sites in the body.
He asked two colleagues who were pathologists
to examine the cells in biopsies from the same patient,
from the jaw and from internal organs.
And when they did, they concluded that the tissue
was the same, the cells in each of those distant sites
was similar that it was likely to represent a malignant
outgrowth of blood cells to be a lymphoma.
To address that second question, Burkitt went to a nearby
hospital that was the oldest hospital in east Africa.
It had been formed by missionaries in 1897.
And he went back and he read their records,
just to find out if there were examples prior to that time,
prior to the 1957, in which children presented with
this kind of, what they thought now as a lymphoma.
And there were, there were many examples.
There were pictures drawn by hand of children
from 1910, 1920, that presented the same way.
What those earlier physicians had failed to see is
that in these children who had these massive growths
around their jaws, they also had growths on internal organs,
and they had misdiagnosed this lymphoma
that clearly existed long before 1957.
So,
Denis Burkitt recognized that this growth was a cancer
because it shared these defining characteristic.
Cancer is a disease in which a single cell type proliferates
inappropriately and can migrate to abnormal sites
in the body, colonize them and continue to proliferate.
And if you think back now what Burkitt had found,
growth in the jaws but growth in internal organs too,
the same kind of cell at each of those sites.
That's characteristic of a cancer.
Now, let me just ask you to think for a moment.
The year is 1957, you've identified this new form of cancer,
newly identified form of cancer, this lymphoma.
You have very limited resources but you'd like to be able
to help the kids who have this cancer.
What would you do?
Can you imagine what he did?
I couldn't.
I would not have done what he did but what he did
turned out to be extremely influential in driving forward
our understanding of Burkitt's lymphoma.
What he did is to make the decision he was going to map,
carefully within Africa, where children
were found that had this tumor.
Where did the tumor occur?
And he did so in two ways.
First, he wrote a grant back to people in England,
for 25 pounds, so 1957, 25 pounds was about $75.
And with those 25 pounds,
he had printed a thousand leaflets.
On those leaflets, he described and depicted children
with Burkitt's lymphoma and he sent those leaflets
to a thousand hospitals, rural dispensaries, rural clinics
throughout Africa, and asked physicians in them,
if you've even encountered a youngster
that presents this way, please tell me.
Tell me everything you can about that patient.
And from that initial survey which you've got to admit
is an incredibly small amount of,
funded by a small amount of money, he learned
that this was the most common cancer in all of Africa,
that it was particularly common from 10 degrees
north of the equator to 10 degrees south of the equator,
and then ran further south along the eastern seaboard
of the continent and he referred to this
as the lymphoma belt.
The second thing he did is to write a second grant.
This time for 750 pounds.
Again, it was funded fortunately and with that funding,
he decided to map the boundaries explicitly
of where you found this tumor and I'm going to call it
Burkitt's lymphoma from this time on,
where you found patients with Burkitt's lymphoma
and where you didn't.
So with that 750 pounds, he bought a secondhand
Ford station wagon, got two colleagues to go with him,
and they drove 10,000 miles largely on
non-existent roads, visited eight countries, 57 hospitals
and interviewed physicians in them all,
and interviewed physicians at rural clinics
along the way too, inquiring had they encountered youngsters
with what we are calling today Burkitt's lymphoma.
And from this long 10,000 mile safari,
Burkitt and his colleagues learned two more things.
This lymphoma was common to regions in which the minimum
average temperature exceeded 60 degrees Fahrenheit.
It was common in regions in which the annual rainfall
exceeded 20 inches per year.
Now, I challenge you, with that information,
what more would you know?
I wouldn't know something, but he was surrounded
by people who were experts in tropical medicine.
And what they immediately can see, which I couldn't see,
is that these characteristics are shared by, in fact,
the number of infectious diseases in Africa,
and what brings those infectious diseases together,
what they have in common is that they're all caused
by agents that are transmitted by mosquitoes.
So for example, Anopheles mosquitoes
which carry the protozoan malaria, live with these
characteristics in regions of these characteristics.
And in fact, in the gray is shown where
Denis Burkitt had identified Burkitt lymphoma patients,
and in the black lines running across that gray area,
is where malaria was endemic throughout Africa.
They overlap almost perfectly.
So from this survey, from these insights,
Denis Burkitt made two hypothesis.
The first was that perhaps Burkitt's lymphoma is caused
by something transmitted by mosquitoes, for example a virus.
That was his first hypothesis.
The second hypothesis was that perhaps malaria which
overlaps so well with the incidence of Burkitt's lymphoma
contributes to the cause of Burkitt's lymphoma.
The first hypothesis that there was an insect-born agent
really turned out largely to be wrong.
But that hypothesis was so influential in driving forward
our understanding of Burkitt's lymphoma
that it was really quite important.
And the second hypothesis, that malaria might be linked
causally to Burkitt's lymphoma,
turns out largely to be true,
and I'll come back to that at the end of the talk.
The story of understanding Burkitt's lymphoma now
shifts to England, to London in particular.
But before we go there, I want to give you one more example
of Denis Burkitt's contributions to Burkitt's lymphoma.
He wanted to treat those children.
What he knew at that time, so this is 1961-1962,
is that in the states, companies were making compounds,
molecules that were used as chemotherapeutic agents.
They were being tested as such.
He also knew that he couldn't conceivably afford to buy
those compounds to treat his children,
but he knew that in this country, in the states,
the standard of care for almost everybody who had cancer
at that time, was radiation therapy.
So that people would first receive radiation therapy
and then be tested with these new compounds.
Burkitt not only lacked the funds to buy the compounds,
he lacked the funds to buy any equipment
for radiation therapy.
So none of his children could be treated that way.
So he wrote the manufacturers of these compounds
and said, "Look, if you give them to me,
"I'll test them on my children, and they'll no problems
"with confounding effects of radiation therapy.
"They don't have it."
And the company said fine, we'll give them to you.
He was the most successful chemotherapist
in the world at that time.
20% of his kids survived and the reason that happened
is Burkitt's lymphoma, then and now,
is the fastest growing tumor in people
and as such, it's incredibly sensitive to chemotherapy.
So not only was he instrumental in identifying this new
or newly identified tumor, he was also instrumental
in developing treatments for it.
So now, let me take you back from Africa to London,
when in 1961, Denis Burkitt who is on vacation,
gave a talk and the title of that talk in London
was "The Commonest Children's Cancer in Tropical Africa:
"A Hitherto Unrecognized Syndrome."
In the audience at that talk was Tony Epstein.
Epstein is a pathologist who is trained in part
in New York City at the Rockefeller University with
George Palade to use a new kind of experimental approach
in biology and that was to use an electron microscope.
When Esptein returned to London, he decided to use
the electron microscope to study viruses.
Viruses, as you know, cause lots of diseases in people
but they're way too small
to be seen in the light microscope.
They're about 1/10 to 1/100 of the size of the smallest
thing you can see in a light microscope.
So the electron microscope which can resolve much
smaller structures was ideal to study viruses.
And in particular, Epstein wanted to study viruses
that cause cancers in animals because it was quite
controversial then whether or not a virus could cause
a cancer in an animal and surely, there was no evidence
that it can cause a cancer in us.
So after he heard that Burkitt hypothesize
that Burkitt's lymphoma might be caused
by a viral infection, he decided
to work on Burkitt's lymphoma and search for such a virus.
At the end of the talk,
he went up to Denis Burkitt and said,
"Can you send me live biopsies from your patients?"
And Burkitt said, "Of course."
And that was now possible because there was
a plane connection between Kampala and London.
What Epstein had to do then was to take cells
from these biopsies and grow them so that one cell
would give two, would give four, would give eight,
propagate those cells and culture
and then examine them in the electron microscope
and that's what he set out to do.
And for three years, with two colleagues of Yvonne Barr
and Bert Achong, he tried to do so.
And for 22 biopsies that were sent by this plane, he failed.
They failed.
They couldn't get the cells to grow, nobody had gotten
this kind of lymphoma to grow at all in culture.
The 23rd shipment was delayed by fog.
The plane couldn't land in London,
it landed somewhere further north.
Several days later, the sample made it to the lab in London.
Those samples were solid blocks of tissue
suspended in some sort of liquid.
And when the 23rd sample came, they looked at the liquid,
it was all cloudy.
So they looked in the light microscope to see
what the cloud was composed of and it was composed of cells.
The cells were growing.
We now know, that Burkitt lymphoma cells grow best
in suspension but prior to that chance encounter,
the tissue had always been grown as a block,
and that way Burkitt lymphoma cells just don't grow.
So now Epstein could propagate these cells
and eventually examine them in a light microscope,
and let me show you what he saw.
That's what he saw.
So this micrograph is a testament to Epstein's
tenacity for three years of failure and sticking it out,
and continuing to try to grow those cells.
But it's also an example of really luck preparing
someone to take advantage of chance because in fact,
what you're looking at are virus particles seen
in the electron microscope, Epstein-Barr Virus particles,
but the cells that Epstein was growing very rarely
ever allowed these to be formed.
In fact, about 1% of some cultures would exhibit
these particles, and he was really lucky.
Of course, he worked like mad to find it,
but he is really lucky to observe them.
Now, let me show you what he was seeing.
If I can get this to be, well,
if you look at the lower signal, it looks like a hexagon,
six-sided, and what you're looking through
is a cut across a virus particle and that particle
is actually icosahedral in three dimensions.
But when you cut across it, it looks like a hexagon.
And if you look at again at the lower left,
it's dense in the middle and that density reflects DNA.
That's electron dense.
That's how it's observed in the electron microscope
and if you look at some of the upper images,
what you'll see is there is a faint ring around them,
that's a membrane that encapsulates those virus particles.
And for people who are experienced at looking at these
sort of images, they'll say, "Ah, that's a herpes virus."
So he knew what it was.
At least he knew in what family of viruses it was.
And in those days, we knew that herpes simplex
causes cold sores.
We knew that varicella zoster virus, another herpes virus
causes chickenpox and so the question arose,
is this a known herpes virus?
Or was there an unknown herpes virus associated
with a small fraction of these Burkitt lymphoma cells.
To answer that, Epstein set out to collaborate
with a number of people and in particular,
he collaborated with the Henles in Philadelphia.
So Werner and Gertrude Henle were quite well-known
for a kind of studies called serology,
where they studied viruses in infected cells.
And in particular, they had studied flu and mumps.
When Epstein approached them and told them
about his identification of a virus, a herpes virus
that might be in Burkitt's lymphomas,
they were keen to help him.
So let me explain what they would do.
Some of you may know, maybe not all of you know,
that in our bloodstream, we make proteins, antibodies,
as part of our immune response, and these antibodies
will bind specifically to virus particles and to the protein
constituents of a virus particle
and ideally, they inactivate it.
That's part of our immune response
when we get infected with a virus
And in fact, in that antibody response can be thought of as
a kind of immunologic memory because if I was infected
with Epstein-Barr Virus, let me say, 55 years ago,
which I was, I probably still have, I know I do,
have antibodies that detect Epstein-Barr virus in my blood.
That's the virus that he found.
So, what the Henles could do is to take antibodies
from many patients, bind them to a fluorescent dye,
take these colored antibodies now and bind them
to cells in culture to ask if those cells
were making the virus particles.
That's the kind of experiment they did.
They isolated antibodies from 17 children
with Burkitt's lymphoma, couple them with a dye,
then added those antibodies to the cells that Epstein
had given them in which 1% of them
exhibited virus particles
and this is what they saw.
Most of those cells are reddish, they have no antibodies
associated with them.
They're stained so you can see the cells in the first place.
A few of them are green-yellow.
That's the color of the fluorescent dye.
That told the Henles that the 1% of those cells
were being recognized by the antibodies from the patients.
In fact, that children with Burkitt's lymphoma mount
an immune response to this virus.
The Henles had antibodies from lots of other sources,
ones that were characterized to identify
herpes simplex or varicella zoster.
Those antibodies did not react with 1% of these cells
from which they could conclude that this was a new virus,
Epstein-Barr virus we now we call it.
A new member of the herpes virus family that was found
in 1% of the Burkitt lymphoma cells.
That was very exciting too as you might imagine.
The Henles were very thorough scientists.
They didn't just look at antibodies
from Burkitt lymphoma patients,
they took antibodies from many donors, healthy people,
people with other kinds of diseases
and they tried them in these sort of asset.
And here comes the surprise.
When they did that, they found that many people
had antibodies to this 1% of the cells,
to the virus particles in 1% of these cells.
How could that be?
That was truly puzzling.
A solution to that puzzle came serendipitously
in their own lab when one of the people who had been
carrying out these experiments, who had used her own
antibodies as a negative control,
they did not stain these virus particles in 1% of the cells,
came down with infectious mononucleosis.
She stayed home for a few weeks.
When she was better, she came back to the lab.
She drew her blood.
She used her own antibodies again as a negative control
but it wasn't negative.
Now, she had antibodies that reacted with virus particles
in 1% of these cells.
From which, the Henles inferred and we now know
that Esptein-Barr virus causes infectious mononucleosis.
We know also that a lot of us are infected with this virus
without getting any disease.
In fact, probably almost everybody in the audience,
at least 90% of you and me are already infected
with EBV and we already have it in us,
and we have it in us all our lives.
So, by the early 1970s, we knew that Burkitt's lymphoma
is a common childhood lymphoma whose distribution
overlaps that of malaria in Africa.
We know that Burkitt's lymphoma cells contain
Epstein-Barr virus in some form,
and that Epstein-Barr virus infects most people
and probably causes infectious mononucleosis.
That was what we understood in 1970s.
What I want to do now is to narrow the focus of this talk
and describe work that we've done in Madison
in the last 40 years.
I want to do that so I can describe to you the logic
behind the experiments we've used
that lead us to look for therapies for Burkitt's lymphoma
in a very particular way.
But I want to admit immediately to you that
with this narrow focus, I'm ignoring the vast majority
of all the research that has been done both
on Burkitt's lymphoma and on Epstein-Barr virus
during these last four years.
But I'll come back to a tiny part of that research
at the end of the talk.
In 1973, I went to the Karolinska Institutet
in Stockholm, Sweden to work with George and Eva Klein.
George and Eva Klein collaborated with Tony Epstein
and with the Henles.
And in fact, they were instrumental in demonstrating
that Epstein-Barr virus is also causally associated
with another human cancer, nasopharyngeal carcinoma
which is common in southern China.
I went there to learn about Epstein-Barr virus
and by 1975, I learned pretty much what I could,
and I came to Madison and took a faculty position
at McArdle Laboratories.
I wanted to answer lots of questions about
Epstein-Barr virus but I'm going to describe two in detail
that we've pursued.
One is what does EBV do to the cells it infects?
And the other is EBV maintained in these infected cells?
Recall that in Burkitt's experiments, in the Henle studies,
really only a small fraction of Burkitt lymphoma
biopsy cells displayed any evidence of being associated
with this new virus, Epstein-Barr virus.
What was the virus doing?
How could it, did it, in fact contribute
to Burkitt's lymphoma?
What does it do to infected cells?
To address this question,
we have to know what kind of cell it infects.
And by 1975, when I came here, it was evident
that the kind of cell that makes up Burkitt lymphoma cells
is a B lymphocyte.
Those are the cells in our bloodstream that make antibodies.
It was also possible by 1975 that the infected cell
in infectious mononucleosis could be a B cell.
That really wasn't shown until 1982 but it was possible.
And probably more importantly, John Pope,
a researcher in Australia had shown that he could
infect B lymphocytes,
I'm going to call them B cells for a moment,
it's easier, B cells with Epstein-Barr virus.
So we set out to generate a quantitative assay
to study the infection of B cells drawn from your blood
or my blood with Epstein-Barr virus.
Let me describe the assay.
It's actually quite simple and it's quite lovely.
So on the left, you see a tissue culture dish
that's actually filled with agarose.
And agarose, you can just think of a sloppy Jell-O
and in that sloppy Jell-O, we could embed B lymphocytes,
B cells that we take from somebody's blood
and then we could just watch them in that sloppy Jell-O.
Or, we could expose those cells to Epstein-Barr virus...
...and then plate them in a sloppy Jell-O
and follow them overtime.
These experiments would take on the order of
five or six weeks and in this left dish, you saw nothing.
The B-cells in the absence of EBV did nothing but die.
They didn't divide, they just sat there and then died.
And they died with a half-life of about,
half would die every 24 to 36 hours.
In the right culture dish, for those cells exposed to EBV
after four or five weeks, what you would begin to see is macroscopic colonies.
You could hold it up to one of these lights
and you'd see cloudy areas.
And you could pick those colonies and put them
into cell culture and they would grow,
and they look like something like the little bunch of cells
shown below of that Petri dish.
And so then, we could characterize the infection
and ask what was happening.
We could show that one particle of EBV is sufficient
to induce and maintain proliferation of the infected
B cells and that those infected B cells would divide.
One would go to two, two to four, four to eight,
eight to 16 et cetera for 40 or 50 generations.
And sometimes, they would go on and divide
ad infinitum, they were immortalized.
And all I mean by that is if you fed nutrients
to these cells, they would continue to divide and divide.
To illustrate that, let me give you a number.
If you take one B cell and infect it with EBV,
and allow it to propagate for 50 generations,
and if you could harvest all the cells at the end
of that 50 generations and if you weighed them,
they would weigh about one ton from one cell.
And what that should illustrate for you is the ability
of this virus to induce and maintain proliferation
could be a hallmark of its contributions to tumor formation.
And that's a thought that clearly has driven us
and you'll see it's likely to be correct.
Is EBV in those proliferating cells?
Is a small fragment in them?
Is none in them?
You can surely imagine that the virus would go in
and have some sort of genetic switch,
turn the cell on to divide and then be lost.
So we wanted to know whether EBV DNA was maintained
In these proliferating cells,
whether all of it was maintained,
whether a small fragment of it was maintained,
and if it was maintained, how?
To describe for you how we've answered these questions,
I've got to introduce you to a technique that was just
generated about 1976-1977,
when we were doing these experiments.
It's a little technical but bear with me.
It's called Southern blotting and it's named after
Ed Southern who developed that technique.
And in the center of this image,
you can see a representation of DNA,
a double-stranded helical structure.
And you can think of each strand of that DNA as being
Watson and Crick.
And those two strands are held together,
Watson holds on to Crick, Crick holds on to Watson
by virtue of base pairing.
So in the left, you can see that the left strand
has an A, T, C and G and the right strand
has a C, G, A, T as I read up.
A hybridizes or binds to T, T binds to A, C to G, G to C.
That's how the double helix is held together.
And then if you read from the top down, T, A, G, C,
that's the genetic code that underlies us.
You take a thousand or maybe 10,000 of those nucleotides
and you get a sequence of one gene,
and all our DNA is made up of is a bunch of genes
which can be translated to make proteins,
and we're a bunch of proteins roughly speaking.
What we wanted to know, we couldn't sequence the DNA.
We wanted to know whether all of the EBV genome,
all of its DNA is in these cells, these tumor cells or not.
How could we answer that?
Again, we couldn't sequence it.
EBV DNA is very big but its tiny relative to the DNA
in one of your cells.
If you take the DNA out of a nucleus and you stretch it out,
it's about two yards long.
If you take the DNA out of those cells rather than
EBV virus particle, and you measure its length,
it's about as long as your hair, a single hair as wide.
That makes a big DNA but it's tiny relative
to the DNA in our cells.
How can we characterize it?
Well, their enzymes, proteins that will cleave DNA
and they were available then at a specific sequence.
So that enzyme Bam cuts every time it sees the sequence
G-G, A-T, C-C and it cuts both strands because you can see
its G-G, A-T, C-C in one direction on one strand, and in the
opposite direction the other strand, it is G-G, A-T, C-C.
Every time those six nucleotides are encountered
by that enzyme, it cuts the DNA.
So in the diagram shown below, you can see it cuts
the DNA three times, generating a large and a small piece.
And you can separate those pieces in a gel,
electrophoretically.
The big pieces run more slowly, so A is on the top,
B is on the bottom.
We could do the same thing now with DNA
that we isolate from Epstein-Barr virus particles,
with cells that we infected with purified EBV particles
in cell culture and most importantly,
we could take Burkitt lymphoma biopsies.
Isolate DNA from those biopsies that were frozen.
We've never done anything with them other than
they were sent to us, its frozen samples.
Purify the DNAs, digest with that enzyme Bam,
separate all the fragments in the gel as show on the right
and then detect what's pure EBV DNA, no cell DNA,
because again, Watson binds Crick.
So if we purified DNA from an EBV particle,
made it radioactive then Watson, from that labeled DNA
would bind Crick on that filter,
and we could detect that radio activity with x-ray film.
And that's the Southern blot you're seeing.
And if you look at that Southern blot, you're not
going to be able easily to say whether lane one
is different from lane, is different from lane three.
Their intensities, it's not a perfect blot
but that's a long time ago but the patterns are the same.
And in particular, if you look,
there's a heavy band labeled 2.1.
It's heavy because that represents a repeated sequence
cut by Bam eight or 10 times in the EBV genome,
and you'll see they all have it.
It's all intense.
So from these experiments and the similarity of the patterns
separated from purified viral DNA, from cells infected
in vitro, from Burkitt lymphoma biopsy cells, we could
conclude that all of EBV DNA is present in those tumors.
How is it maintained?
Those tumor cells under the 10, 20, 30, 50 generations
doublings, how's the virus distributed
to those daughter cells?
It has to be synthesized.
How is synthesized?
That's the question we focused on, and if you'll
bear with me, I think I can tell you why we focused on it.
So what do we do?
Well, Chris Kintner, a graduate student, set out
to begin understanding how EBV DNA was maintained
in these proliferating cells with the following experiment.
And this would have been about 1980-1981.
Again, across the center of this slide, you can see
all of the fragments generated when you cut
linear DNA with the Bam enzyme.
EBV DNA in that virus particle
is a linear double-stranded DNA.
Herpes simplex DNA in a herpes simplex virus particle
is a linear double-stranded DNA.
All herpes viruses maintained the DNA
in the virus particle as a linear molecule.
What Chris set out to do is to ask, could he identify
the ends of this linear molecule?
And he was able to do so.
And you can see up there, they're in green.
They're labeled the end fragment.
They are the ends of that linear DNA.
Then you could ask what happens to them?
You know, if there's only a fragment of EBV DNA in a cell,
then one end might be gone.
But what he found is not only were the end fragment's there,
they were coupled together and in fact,
EBV DNA was maintained intact in these cells as a circle.
We call that circle a plasmid and human cells don't normally
have plasmid circular DNAs in their nucleus.
This was really quite surprising.
This then led to the question, how does this circle
which you don't normally find plasmids in human cells,
how is this plasmid DNA maintained
in these proliferating cells?
And again, by this time, you might say,
why is he talking about all of this minutia.
So let me say why I'm doing it.
The hope behind the narrow focus
of our research is this.
If we know how EBV DNA is maintained in tumors,
and if we know EBV helps to sustain these tumors.
Then we can hope to treat the tumors,
by forcing the loss of EBV DNA from those tumor cells.
That's our hope.
And that's what's driven the rest of our research
in the past and, in fact, in some ways to this day.
So let me tell you how we have at least answered the first
two "if clauses" and what we're doing for the last hope.
John Yates was a postdoc.
He set out to understand mechanistically how these
plasmids derived from EBV were maintained in cells,
and he had two hypothesis.
The first was that somewhere in this circle,
there had to be a sequence of DNA that allowed
its synthesis to begin.
This DNA had to be synthesized before cells divide
and so it could get passed onto daughter cells.
So there should be a sequence somewhere in that DNA
that allows it to start synthesis, sequence in the DNA.
He wanted to find that sequence.
The second is since you don't normally find plasmids
in human cell nuclei, probably EBV was encoding
one or more genes that allowed this origin, this site
at which DNA synthesis initiates to function.
So he wanted to find viral genes that acted
at this hypothetical origin, and he did both.
Let me show you how he did it.
Again, you can see EBV DNA now arrayed as a circle,
as you would find it inside a Burkitt lymphoma cell.
When you cut it with Bam enzyme,
you get all of those fragments.
What John did is take each fragment and make a mini plasmid
out of it and then introduce it in the cell,
which had EBV intact plasmids in them already.
We reasoned that if a cell had an intact plasmid,
it had to make all of the functions necessary
for that plasmid to replicate, to be given
or passed on to daughter cells.
So John put each one of these small fragments alone
in the cells that already had EBV plasmids and asked
could any one of them now behave itself as a mini plasmid?
One and only one did.
The Bam C fragment, from which he concluded
that somewhere in the Bam C fragment is a site
at which DNA synthesis initiated.
To ask or answer the question, could there be genes
encoded by EBV that allow that mini plasmid
to be synthesized, he took each one
of those fragments again and then put one at a time
in the cells that had no other EBV in it.
And you can think of this as taking EBV which is a bunch
of about a hundred genes and then breaking them up
one gene at a time and putting one gene at a time
into a cell that had no other viral genes.
Then into that cell he could introduce the mini plasmid,
the Bam C fragment and asked
could it replicate as a plasmid?
And after hundreds of attempts, John found that the Bam K
fragment provided the only functions EBV provides
for Bam C to replicate as a plasmid.
Everything else comes from the cell.
For us, this was a great step forward, it was after
three years of work where four of us worked together
but John was the person who spearheaded this research.
Let me illustrate schematically what he had found.
There are two genetic elements required
for the plasmid replication of EBV.
In the middle, in that line with those bars is the sequence
drawn out schematically that represents the Bam C fragment
and its origin of synthesis we call oriP
for origin of plasmid replication.
What we now know is the Bam K fragment encodes
the only gene product EBV needs for that Bam C
to replicate as a plasmid.
It's a protein we call the Epstein-Barr virus
nuclear antigen one or EBNA1,
and it binds to all those white bars in oriP.
When it binds to the origin, it allows DNA synthesis
to initiate by recruiting lots of machinery from the cell.
When it binds to the white bars, and the black
and white pattern, what it's doing is holding the plasmid
next to adjacent, to sites in human chromosomes,
and that helps to bring it to daughter cells
when the chromosomes go to daughter cells.
We've studied oriP and EBNA1 a lot,
and in those studies, one thing that we really wanted
to be able to do was to see oriP in a live cell
and watch it be duplicated when it was synthesized
and asked how is it distributed to daughter cells.
That would be a nice experiment.
And we actually did that experiment.
Really Asuka Nanbo did that experiment
and she learned something that we did not expect
we would learn and that piece of knowledge has
allowed us to say we know that EBV provides
Burkitt lymphomas a selective advantage in order
to be maintained as a plasmid.
So I want to show you Asuka's experiments.
So on the left, you can see one cell with one dot,
I hope.
And that's before the cells makes any DNA.
So it's just at what we call
the beginning of the cell cycle
and below in a cartoon, you can see the intensity
that Asuka measured of that one dot,
and it's 143 arbitrary units.
Cells synthesize DNA at a given time,
we call that the synthetic phase or S phase.
And after S phase, you can see that that signal becomes
more intense and you can read the intensity down below.
It goes from 143 to 250 arbitrary units.
What's happened is that the EBV plasmid has been duplicated,
but it doesn't separate so far from itself
that we can resolve that in a fluorescent microscope.
So what are you looking at?
What you're looking at is Asuka took oriP plasmids
and introduced additional sequences to them.
These sequences bind an irrelevant protein
that's fluorescent.
So you can tag the viral DNA with this fluorescent signal
and observe it in a light, excuse me,
in a fluorescence microscope,
and now that's what you're seeing.
The dot on the left represents one plasmid and because
you can measure the intensity of that fluorescent signal,
the second dot is twice as intense approximately.
We know it got duplicated.
This is all because again, the plasmid has added to it
additional sequences that bind the fluorescent protein.
After the synthetic phase, cells divide.
Now look what happens to the plasmid when they divide.
Each daughter cell gets one and you can see
that the intensity goes down by half.
And that's exactly what we would've guess would happen
and it did, 26 out of 31 times.
The five times on the right is what we did not guess,
did not expect and they turned out to be really informative.
Again, in G1 before the synthetic phase,
you can see a cell with one signal,
and its intensity in this cartoon is about 177.
After the synthetic phase, the intensity does not go up.
If anything, it goes down.
You can follow that one cell when it gives rise
to two daughter cells and look at the signals,
and the two daughter cells and only one cell has a signal.
And its intensity has dropped slightly from which
we have to conclude that in five of these examples,
the EBV DNA was not synthesized.
Now, you might ask, so what?
Well, what this really tells us and we've confirmed this
in many different cell types that about 16% of the time,
EBV DNA fails to be synthesized in proliferating cells.
Again, so what?
Well, just think about it.
Start with any number of molecules you would like in a cell
and most of these cells usually, they're about 10.
Asuka had to look for cells that had only one
so she could follow one signal at a time,
but most of the time, there are about 10.
If the cell divides, for there to remain 10,
each daughter cell has to get on average 10.
But if you fail to synthesize some of the DNAs,
maybe you're only going to get nine and nine
and eventually eight and eight, et cetera.
If there are defects of this magnitude and synthesis,
what will happen is as the cells divide,
they're going to lose EBV DNA and eventually,
those cells will have no EBV DNA.
Okay, except when you take out Burkitt lymphoma biopsies,
and you look in those cells,
every cell has EBV plasmids in it.
How can that be?
And that was insights that Asuka experiments
allowed us to draw.
So some EBV plasmid DNAs failed to be synthesized
each synthetic phase, leading to their decrease,
and average number per cell each cell cycle.
The implication of this finding is that EBV
will be lost from any population of proliferating cells,
unless it afford cells a growth or survival advantage,
so that those that have it outgrow those cells that lose it.
All Burkitt lymphoma cells have EBV DNA in them,
they're all plasmids.
We conclude those plasmids must be providing
those tumor cells, one or more selective advantages,
so that the cells that actually have lost EBV,
and they're going to be there just to pass away.
They die, they're outgrown, we don't see them.
We want to know what the selective advantage is
that EBV affords these Burkitt lymphoma tumor cells,
and Dave Vereide, a graduate student set out to answer that.
What is the selective advantage?
The way he did it is really based on something
you've already heard.
I've said that EBNA1, the one protein encoded by EBV,
is essential for EBV plasmids to be synthesized
and distributed to daughter cells.
They've made a derivative of this protein
that could block the function of the normal protein.
I'll call it an inhibitor.
He could then introduce that inhibitor into a wide variety
of Burkitt lymphoma cells and ask what happens?
What happens is they all lose EBV DNA.
They all lose the EBV plasmids.
Then what happens?
So in this plot on the Y axis, you just see the
number of doublings of the cells he's following.
Zero, one, two, three, four, five, six,
seven, eight, nine, 10, et cetera.
And by 10 doublings, one cell will give rise
to about a thousand.
And on the X axis, you see the number of days
that he followed these cells.
And the black line shows the cells in which this inhibitor
is not expressed and they're growing perfectly.
And the red line that's dotted shows what happens
when the inhibitor is expressed.
Those cells lose EBV plasmids.
They cease to proliferate and they die.
EBV is providing, if you think about it, a massive
selective advantage to Burkitt lymphoma cells.
It allows them to survive.
It also helps them grow we know now,
but it allows them to survive.
That was a very marvelous day we understood, too.
So we now know, EBV induces and maintains
proliferation of infected B cells.
EBV DNA is maintained as a plasmid in these cells.
EBV must afford Burkitt's lymphoma cells advantages
to be maintained in them.
And when EBV plasmids are lost from them,
Burkitt's lymphoma cells die.
It would be incredibly nice if we could take Dave Vereide's
approach and take a derivative of EBNA1, and introduce it
in the Burkitt lymphoma cells in Burkitt lymphoma patients,
and treat them successfully but we can't.
That's an experiment you can do
with isolated cells in a lab.
You cannot do it in people.
Surely not in a population in rural Africa.
But we would love to build on the finding that if you
inhibit EBNA1, you can cure cells of EBV DNA plasmids
and that forces the cells in general to die.
So how can we do that?
Let me describe the efforts of Ngan Lam and Mitch Hayes
in the lab who are now screening for small molecules
that will do the same thing and can be used in patients
that Dave Vereide's derivative of EBNA1 did.
Let me explain how they're doing this.
We know from the research of a number of groups
that this EBNA1 protein has to come together
as a dimer to function.
It can only bind to DNA in the Bam C fragment
when it's a dimer.
It can only carry out any of its known functions
when it's a dimer.
So what Ngan and Mitch have done is to take half
of firefly luciferase, that's the molecule in fireflies that
emits light when it encounters substrate, luciferin in ATP.
But they took a half which has no enzymatic activity
and fused it to EBNA1.
They took the other half and fused it
to another molecule of EBNA1.
No lights emitted unless EBNA1 forms a dimer
and brings those two halves of luciferase together.
And if you inhibit that dimerization,
you inhibit light emission.
This is a very rapid assay that you can now look
for inhibitors of the dimerization of EBNA1.
You can introduce these fusions into cells,
then introduce a compound you want to test and ask,
have you inhibited light emission?
So with funding from the Leukemia and Lymphoma Society,
with help from Mike Hoffman, who has really begun
and directs the Small Molecule Screening facility
in the comprehensive cancer center here,
Ngan and Mitch have screened more than 400,000 compounds.
They screened them in multiple assays.
From those 400,000 compounds, they've reduced their workload
to about 90 which are candidates
for inhibiting EBNA1's dimerization.
If any of these molecules actually inhibit
the dimer formation specifically, it will serve as a lead
to develop a therapy for Burkitt's lymphoma,
and that would be marvelous.
So I want to end with two additional thoughts.
One is I want to bring you all back now to this large body
of work that has been done on Burkitt's lymphoma,
and on Epstein-Barr virus for the last 40 years
that I haven't touched on at all.
And I want to do so just to briefly address a question
which I imagine some of you have been thinking about.
And that question is,
why do many of us get infectious mononucleosis?
I had it.
Anybody else had infectious mono on the group?
Several of us have.
Why do many of us get infectious mononucleosis
while few of us develop Burkitt's lymphoma?
The answer to this question in part,
comes from Denis Burkitt, but in comes from a number
of people who has addressed these question in England,
in Australia and in Sweden, other places too,
but the main groups have been England, Australia,
and Sweden that I know of.
What they've found is that chronic malaria
and that's what you find in central Africa,
suppresses the patient's immune response.
The suppression allows EBV infected cells to escape
the immune response.
They continue to proliferate in these children for example,
and when they do proliferate, they acquire mutations.
Those are just mistakes in DNA synthesis of their own genes,
not EBV's genes but their own genes.
And that those mutations can promote tumorigenesis.
So, Denis Burkitt's hypothesis turns out
really to be correct, the second one.
EBV and malaria are co-factors in the cause
of Burkitt's lymphoma and that really explains
why in this country, something on the order
of one in 200,00 children might develop Burkitt's lymphoma
and in central Africa, it's more like one in 10,000.
The last point I want to leave you with is a picture
of an extended family, our extended family.
And in the left, in the back, you see Mitch and in the front,
you see, Ngan.
Those are the people who are carrying forward the screen
of now more than 400,000 compounds.
And we surely hope, we're going to find a specific one
that we can then use to develop better,
more potent inhibitors of EBNA1's dimerization.
When you look at this picture, you'll be able to tell
probably that we come from at least five countries,
three continents, and if you count me,
we spend much more than 50 years of age differential.
If we don't succeed in finding that inhibitor
that we're looking for, what I can promise you is that
this younger generation will.
Thank you.
(applause)