Thank you very much.
It's nice once again to be here.
I want to say that it was impressive that Eric Betzig did some of his crucial work in a living room, somebody's living room.
This story starts with chickens.
I don't think that would work.
(laughter)
So late in...
There we go.
Late in the year 1909, a farmer on Long Island in New York noticed a tumour on the breast
of one of his Plymouth Rock hens.
He watched this tumour grow for several months, and then took it to the Rockefeller Institute
in New York City to see what might be done.
The farmer was referred to Peyton Rous, a young pathologist with an interest in cancer,
although not much of a publication record in the field.
He's said to have been a very forceful, even fiery individual.
So perhaps by virtue of his personality, he was able to convince the farmer
to donate his chicken to medical research.
So this serendipitous encounter launched a sequence of discoveries
that spanned almost three quarters of a century, and culminated in the realization
that the seeds of cancer reside in our own genomes.
And my purpose today is to explore how all that happened, in an effort to illustrate
how discoveries are made.
And then in the following lecture, my colleague Harold Varmus will explain
where these discoveries have taken us in the struggle to understand and control cancer.
Now Rous began with a very simple experiment, by attempting to pass the tumours with grafts
from one chicken to another.
The experiment was not exactly a resounding success.
In this example, with each passage...
there you go... Finky taught me that... (laughs)
only a single engraftment succeeded.
That's the black squares there.
Moreover, the transplantation succeeded only when Rous used chickens from the inbred flock of the farmer.
The tumour would not grow in Plymouth Rock hens from other sources, nor in other breeds of birds.
Rous explained this by saying that the tumours were obeying what he called the laws of tissue biology.
We call it histoincompatibility.
Now mundane as this experiment may seem to us, it's widely believed that the passaging of the tumour
was serendipitously essential to the next step, which took Peyton Rous to a new dimension and to lasting fame.
Beginning with the sixth passage of tumours, Rous began to make cell-free extracts of the tumours,
and found that injection of these into chickens from the same flock
could elicit the identical tumour, a spindle cell sarcoma.
He announced this discovery with a two page article
in the Journal of the American Medical Association.
You will not find much about chickens in that journal in this day and age.
Now, Rous realized that he had made, in his words, a unique and significant finding,
but he was very careful about what that finding might connote.
And I quote from his first paper:
"The first tendency will be to regard the self-perpetuating agent
active in this sarcoma of the fowl as a minute parasitic organism.
But an agency of another sort is not out of the question.
For the moment, we have not adopted either hypothesis."
That sort of language would not get you into many journals these days either.
Now within a year, however, Rous had identified a second filterable agent
that caused a different kind of chicken tumour, an osteochondrosarcoma, and at that point,
he succumbed to using the recently coined term "virus,"
the very concept of which was just about a decade old.
In his writings, Rous was never particularly illuminating about his inspiration for his work.
He explained his effort to transplant the tumour by pointing out that transplantation of tumours
had succeeded in mammals, so why not try it in chickens?
He justified his landmark experiments with filterable extracts by pointing out that such work
had never succeeded with mammals, so why not try it in chickens?
(laughter)
That's literally the case.
Perhaps his motive was best captured by Nobel laureate Renato Dulbecco
in his biographical sketch of Rous, who wrote that Rous was a medical man
who wished to learn about cancer as a disease, and a biologist who did not want
to follow the beaten path, willing to hunt for new clues in well-designed but slow experiments.
The work with chicken viruses was greeted with what Rous described in his Nobel lecture as downright disbelief.
Nobody really took it seriously.
And Rous himself eventually became disillusioned when he failed to identify causative viruses
in transplantable tumours of mammals, particularly rodents.
It would be fifty-five years before his discovery of the chicken tumour virus
earned him the Nobel Prize, and Finky, if you're in the audience, that's the real record.
Okay.
Rous certainly deserved the Nobel Prize, because he had fashioned two momentous legacies.
The first, the eventual recognition that viruses are major causes of human cancer,
something that Rous had toyed with but abandoned after his failure with rodent tumours.
That's a story that Professor zur Hausen could tell you.
The second legacy represents an unbroken chain of research
that would eventually link wayward genes to cancer.
For several decades after Rous, his sarcoma virus lay fallow.
Beginning in the 1960s however, the pace quickened, and it became apparent that Rous's virus
was an archetype for a vast and seemingly ubiquitous family
of what came to be called RNA tumour viruses,
signalling the nature of their newly discovered genome, RNA.
The discovery of Peyton Rous now blossomed from a two page report
in the Journal of the American Medical Association to a 1500 page monograph,
and I doubt that many have ever read it cover to cover.
I first encountered these viruses when I arrived at UCSF, University of California San Francisco,
in 1968, to take up what proved to be my only academic job.
As a newly minted assistant professor whose research concerned the replication of polio viruses,
I really was not very much aware of RNA tumour viruses at the time.
And there I was introduced to a fellow newcomer to the faculty, Warren Levinson,
who was schooled in the biological aspects of Rous sarcoma virus.
And all of us pretty much looked like that in those days.
(laughter)
Under Warren's tutelage, it quickly became apparent to me
that this virus represents a potentially powerful tool
with which to probe the secrets of the cancer cell.
It was a simple and tractable experimental system whose biological properties were actually pretty well characterized.
It could transform cells in vitro, in a petri dish, to neoplastic phenotype, literally overnight.
There was a well-established and highly reproducible bioassay for the virus in vitro,
which among other things made the virus amenable to genetic analysis.
The virus could be propagated and purified in very large quantities,
sufficient for biochemical and structural analysis.
And its inability to replicate in human cells meant it posed little if any threat
to those of us who were working with it, and so it could be worked with under
relatively unconstrained conditions.
At the time, no other RNA tumour virus offered such a powerful suite of advantages.
So we bashed ahead with the chicken virus.
Warren and I joined to study Rous sarcoma virus,
and two great puzzles defined the field at the time.
First, it appeared that the viral genome could be established as a heritable property of the host cell.
How could this happen with a virus whose genome was RNA rather than DNA?
And second, there was that powerful tumorigenic potential to be explained.
Warren and I resolved to take on the replication of Rous sarcoma virus, just how does it propagate,
about which virtually nothing was known at the molecular level.
And in doing so, we implicitly took on the puzzle of how the virus could establish itself
as a heritable property of the host cell.
And I can dramatize that puzzle by reviewing what happens
when Rous sarcoma virus is applied to rodent cells.
At first it appears that nothing has happened, because the virus cannot replicate in those cells.
But in due course, clones of neoplastic cells emerge, cells that will form a tumour
in the same species of animal.
And although these cells produce no virus, their morphological phenotype is determined
by the particular strain of virus that was used.
And mirabile dictu, if at any subsequent point the transformed cells are fused
with normal chicken cells, the virus re-emerges, phenotype intact.
Where had it been hiding?
Now looming over that puzzle was the central dogma,
enunciated with special authority by Francis Crick,
but generally ingrained in most biologists at the time.
The transfer of genetic information was thought to be unidirectional from DNA to RNA to protein.
It turns out that future events about, which I'll talk in a moment,
would prompt Francis Crick in a bit of hindsight to point out that chemistry may preclude
direct transfer of information from protein to RNA, but there had been no inherent reason
to discredit transfer from RNA to DNA.
But that bias was existent and powerful.
In any event, two individuals paid little heed to the central dogma:
Howard Temin and David Baltimore.
The two first crossed paths in 1955 at a science camp for high school students
put on by the Jackson Laboratories in Bar Harbor, Maine.
At the time, Temin was a student at Swarthmore College who was serving as the guru for the camp,
the one adult presence.
Baltimore was a high school student from New York City who attended the camp.
Temin is on the right, Baltimore is on the left.
Look at that.
I've never had one of those before.
Now, although Baltimore went on to attend Swarthmore as well,
Temin apparently had nothing to do with that, the two parted ways until the spring of 1970,
when they independently wreaked havoc with the central dogma.
Now, Temin and Baltimore came at the puzzle of Rous sarcoma virus
from very different vantage points.
Temin began work with the virus as a graduate student at Caltech, where he first of all
helped develop the quantitative bioassay that we all then used thereafter.
And at Caltech, he became familiar with the phenomenon of lysogenic conversion of bacteria
by viruses that we call bacteriophage.
And during the course of infection, the phage can go into hiding by inserting its genome into the genome of the host cell
and from that position can bestow new properties on the host cell.
The phage can be brought out of hiding by irradiating the cell.
Temin thought this might just well represent how Rous sarcoma viruses might replicate,
stabilize its genome in the host, and initiate neoplastic transformation of the host cell.
There was only one problem with this of course.
Lysogenic phage had double stranded DNA genomes, whereas Rous sarcoma virus has
a single stranded RNA genome.
How could that create a lysogenic phenomenon in the host cell?
By 1964, Temin had solved this problem in his own mind by proposing what he called
the DNA provirus hypothesis.
The RNA genome of Rous sarcoma virus must be transcribed into DNA,
which then serves as template for the synthesis of both viral messenger and genome RNA.
Temin ruefully remarked in his Nobel lecture that for the next six years
this hypothesis was essentially ignored.
He might well have said soundly denigrated.
I heard that done on many occasions.
Most observers absolutely loathed the idea.
Undeterred by criticism, Temin produced a variety of suggestive data to support his idea.
Using chemical inhibitors, he demonstrated that infection by Rous sarcoma virus
requires both new DNA synthesis and DNA dependent RNA synthesis.
And using molecular hybridization, he claimed to have detected viral DNA in Rous sarcoma virus infected cells,
although in all honesty the data were quite frail.
In 1969 however, Satoshi Mizutani joined Temin's laboratory and in his first experiments,
literally his very first experiments, he demonstrated that new protein synthesis
was necessary for the early stages of Rous sarcoma virus replication.
That result was actually never published, but it allowed Mizutani and Temin
to conclude that the RNA directed DNA polymerase required by the provirus hypothesis
must pre-exist infection.
And at the time there was no precedent for such an enzyme in normal cells.
We all know better now.
So in that case ignorance was indeed bliss, because it directed Mizutani and Temin
to look for a DNA polymerase in the virions of Rous sarcoma virus itself.
David Baltimore came at the problem from a completely different perspective:
the replication of animal viral genomes, which I have portrayed here,
in terms of how the viral messenger RNA is generated in order to get replication underway.
Baltimore had cut his research teeth on picornaviruses such as polio virus,
whose single stranded RNA genomes serve directly as messenger RNA
once they have gained access to the cell, so that's... yes.
Consequently, we define the polarity of the genome as positive.
That's a convention in the field.
The messenger in turn produces all the necessary viral proteins including an RNA polymerase
that replicates the viral genome.
Baltimore discovered this RNA replicase while a graduate student.
He completed, incidentally, he completed his thesis work in 18 months.
The replicase was the first enzyme of its kind to be uncovered,
and it was consistent with the idea, the conventional view that virus particles themselves
contain nothing but the viral genome and the requisite structural proteins.
That view was changed by the discovery that a variety of viruses
carry within their virions an enzyme encoded by the viral genome and required to initiate infection
by producing viral messenger RNA.
And in chronological order of those discoveries:
An RNA polymerase was discovered within the virions of the large pox viruses
that transcribes the double stranded DNA genome of these viruses into messenger RNA.
An RNA polymerase that transcribes the double stranded RNA genome
of reoviruses into viral messenger RNA.
And an RNA polymerase that transcribes RNA genomes of negative polarity into messenger RNA.
And it was the last of these that carried the greatest weight for Baltimore
because he and Alice Huang discovered the first such example in vesicular stomatitis virus
and followed that by finding a similar polymerase in Newcastle disease virus.
Like Rous sarcoma virus, these are envelope viruses with single stranded RNA genomes.
But they have a negative polarity, remember.
And although the genome of Rous sarcoma virus has a positive polarity,
Baltimore was struck by the possibility that it might utilize a virion polymerase for its replication.
So even before publishing the results on the negative stranded RNA genome viruses,
Baltimore went looking for both RNA and DNA polymerases in the virions of RNA tumour viruses.
And he had to get the virus from other labs, he had never laid a pipette
on an RNA tumour virus before.
He literally leaped into the field unannounced.
He was undeterred by the reigning scepticism about Temin's DNA provirus hypothesis,
because as he explained in his Nobel lecture, "I had no experience in the field
and no axe to grind."
And that too can be a state of bliss for the right scientist.
In the late spring of 1970, the paths of Temin and Baltimore finally converged again
when they simultaneously reported their respective discoveries of an RNA directed DNA polymerase
within the virions of RNA tumour viruses, which soon acquired the name reverse transcriptase.
And in turn the RNA tumour viruses were reffered to as retroviruses.
Now the discovery of reverse transcriptase of course was a transformative event intellectually.
But it also provided a powerfully enabling technique:
the ability to copy any single stranded RNA into DNA,
which made it invaluable to both fundamental research and the burgeoning biotechnology industry.
For my colleagues and me it was a godsend.
Now we could make probes so that we could examine infected cells for viral nucleic acids at will,
and we quickly set up assays to do so.
So, the means by which the virus could produce proviral DNA were at hand,
but was there really a stable provirus in the infected cell, and if so, where was it hiding?
Was infection by Rous sarcoma virus truly an analogue of lysogeny?
The first credible sighting of proviral DNA was actually biological in nature.
Introduction of DNA taken from Rous sarcoma virus infected cells
into uninfected cells by transfection gave rise to the original virus.
The entire viral genome must have been lurking in the DNA of infected cells.
But exactly where was the provirus and what did it look like?
Those questions were taken up by Harold Varmus, who joined me in San Francisco in 1970,
and we were to work together for the next sixteen years.
Here we are in 1978, with me as usual one step behind Harold.
Well, his legs were considerably longer than mine.
And using molecular hybridization with radioactive probe copied from the Rous sarcoma's genome,
we and others were able to demonstrate where and when the provirus was synthesized,
what molecular forms it took within the infected cell, its eventual integration into
the chromosomal DNA of the host cell, where it is perpetuated and expressed
as an unwelcome addition to the cellular genome.
Now all of this adhered to what Howard Temin had posited a decade before.
And contemplating this intimate interaction between viral and cellular genomes
prepared us for what was soon to follow.
Meanwhile progress was made towards solving a second great puzzle:
How does Rous sarcoma virus elicit malignant growth?
The first step was the demonstration by genetic analysis that the virus possesses an oncogene
that is responsible for malignant transformation of host cells.
This is a beautiful story which time does not permit me to tell,
but it was fundamental to the field.
And this oncogene was dubbed src because it causes sarcomas in chickens.
Now src proved to be one of the only four genes, simply put, in the Rous sarcoma virus.
And remarkably this gene plays no role in viral replication.
That's the job of the other three genes in this exquisitely compact and efficient genome.
Now src gene itself raised two puzzles of its own.
First, what is the biochemical mechanism by which the gene elicits cancerous growth,
and second, why does the virus have such a gene, given that it is not required for replication?
The mechanism puzzle was solved with a discovery by Art Levinson in our lab
and Mark Collett and Ray Erickson's lab in Denver that src encodes a protein kinase.
Two years later, Tony Hunter at the Salk Institute surprised himself and everyone else
with the discovery that the src kinase carries out a heretofore unknown reaction,
phosphorylation of tyrosine.
And there is a direct line from this line of experimentation
to modern therapeutic practices, but that's also a story for another time.
The ways in which these discoveries emerged are illuminating.
In Denver, it was an inspired guess, based on the pleiotropism of src.
Protein phosphorylation ranks among the most versatile agents of change known to biochemists.
What better way to evoke the myriad changes that give rise to the neoplastic cell?
In San Francisco, shrewd enzymology by Art Levinson led to the recognition
that the src protein was actually phosphorylating itself,
and thus might well phosphorylate other proteins as well.
And at the Salk Institute, Tony Hunter's fortuitous use of an outdated buffer that's pH had gone off
led to the separation of phosphotyrosine from phosphothreonine for the first time in recorded history.
And here is Art Levinson, in creative costume at a lab Halloween party, many years before he became
CEO of Genentech, chair of the Apple board of directors...
he doesn't dress like that for those meetings...
and most recently the founder of his own company, Calico.
Now what about the second puzzle, the origin of the src gene in Rous sarcoma virus?
Many years, two opposing lines of thought directed us to the genome of normal cells.
First, there was the so-called virogene oncogene hypothesis,
formulated by Robert Huebner and George Todaro.
It was sort of an effort to create a universal theory of cancer.
I for one didn't take it very seriously, but nevertheless it was out there.
The thought was that retroviruses had deposited themselves with their oncogenes
in the germ lines of ancient ancestors of modern species.
And these genes were normally repressed by the cell, but they could be activated
by various cancer-causing agents.
By this account, we might possibly find src in normal cells, but it would be in the form
of a retroviral oncogene, and its origin would remain unexplained.
The opposing thought was Darwinian in nature.
Since src is irrelevant to the viral life cycle, it is not likely to have arisen in concert
with the remainder of the viral genome.
There was no apparent selective pressure to accomplish that.
Instead the intermittent interaction between
viral and cellular genomes during the course of infection might have created an accident
in which src was acquired from the cell, in essence reversing the hypothesis of Huebner and Todaro.
And this thought also favoured a search for src in normal DNA.
The search required a radioactive probe that could be used in molecular hybridization
with the exquisite specificity to detect the src gene with great sensitivity.
Now in his previous work as a postdoctoral fellow, Harold had used deletion mutants of bacteriophage
to detect the expression of individual genes by molecular hybridization.
And we were able to adapt this technique to our purposes.
Because our collaborator Peter Vogt had isolated spontaneous deletion mutants of Rous sarcoma virus
that appeared to remove the bulk of src.
So as illustrated here, we could use the RNA of one of these deletion mutants
to perform what later came to called subtractive hybridization.
First transcribe the genome, the entire genome, both the replication genes and the src gene,
into complementary radioactive cDNA.
Then second, hybridize that cDNA to RNA from the deletion mutant which would capture all the DNA
except that representing src.
And finally remove the double stranded hybrids, leaving the single stranded cDNA for src,
the probe we needed.
The preparation and use of the src probe was laborious, given that the work far antedated
the advent of recombinant DNA.
And the requisite experiments occupied more than three years.
The fact that they were done at all was a tribute to the valiant efforts
of two postdoctoral fellows, Ramareddy Guntaka, who demonstrated that we could probably make the probe we needed.
And Dominique Stehelin, shown here, who carried the work to its decisive conclusion.
Today, with the assistance of recombinant DNA and other contemporary technologies
you could probably have it done in a matter of weeks or months,
but I for one am very glad we didn't wait.
By 1974 we knew that our probe could detect sequences homologous
to the oncogene of Rous sarcoma virus in the DNA of various avians.
The top panel here demonstrates the specificity of the probe.
It reacted with the RNA from which it had been transcribed,
but not with RNA from the deletion mutant.
The middle panel shows that the src probe reacted with normal chicken DNA,
but so did a probe for the deletion mutant.
And that reflects the presence of what we call
endogenous retroviruses, known to pollute the genomes of many species, ourselves included.
The bottom panel is the crucial one.
Only the src probe reacted with DNA from other avians, and this was our first indication
that we were picking up something that was not linked to a retrovirus,
and unlike the genomes of endogenous retroviruses, was conserved across substantial phylogenetic distances.
Note the results with emu DNA, among the most primitive of surviving avians.
We published these findings in 1976 as a brief note in Nature, two figures and two tables.
The simplicity belied the extraordinary difficulty of the underlying experiments,
and the remarkable paradigm shift that they represented.
We were pleased and so apparently was the Nobel committee.
But oh how times have changed.
A week ago, a colleague told me that he had asked a class of graduate students to read that paper.
Their reaction?
They got the Nobel Prize for that?
(laughter)
Yeah, we did.
(laughter)
Gradually, we built the case that our probe was detecting a normal cellular gene.
We found that it was conserved across vast phylogenetic distances,
suggesting that it serves a vital function.
We found that normal cells contained a protein with the same biochemical properties,
the same size as the viral src protein, and that this was expressed in numerous tissues.
And when at long last we could use recombinant DNA to clone cellular src, we sealed the deal.
Cellular src had the structural hallmarks of a normal cellular gene,
the viral oncogene src had indeed been pirated from the host cell
as a fully spliced version of the progenitor, and at some point the cellular gene
had sustained a mutation which converted it to an oncogene.
And it soon became apparent that src was more than an isolated curiosity.
The inventory of retroviral oncogenes mounted steadily, and each of these had been derived from
the genome of a normal cell.
Thus for each viral oncogene, there was a cognate proto-oncogene in the cell.
Accidents of nature had uncovered a battery of potential cancer genes in normal cells.
The cellular src proto-oncogene is a well behaved and vital switch
in the signalling network of normal cells, we know a great deal about that now.
The viral src oncogene, however, is a mutant malefactor,
whose gene product has been constitutively activated,
a genetic gain of function that creates a cancer gene.
Gain of function, by one means or another, proved true for all the other
retroviral oncogenes as well.
It was easy to imagine that the battery of cellular proto-oncogenes could be a keyboard
on which all manner of carcinogens could play, independent of viruses, creating cellular oncogenes
without the intervention of a retrovirus, for example.
Such has proven to be the case.
Proto-oncogenes that have suffered gain of function are a virtually inevitable feature
of all human tumours.
And as such they are major drivers for tumour genesis, and consequently,
candidates as targets for new therapeutics, and that is a thriving industry now.
With the assistance of modern genomic tools, the number of these genes has been expanded well beyond two hundred.
The gain of function that creates oncogenes can be affected by various means.
First, gain of chromosomes, or focal gene amplification, both of which increase gene dosage.
Chromosomal translocations, which can either disturb the control of gene expression,
or create mongrel gene products that malfunction.
Point mutations, which can disrupt the control of gene expression, or create malfunctioning gene products.
And defective epigenetic control of gene expression.
All of these have been found in human tumours.
But the end result is always the same:
The equivalent of a jammed accelerator with the capacity to drive tumour genesis.
The transduction of normal cellular genes into oncogenes by retroviruses, an accident of nature,
had brought genetic seeds of cancer to view for the very first time.
What are the factors that drove this line of discovery?
Well here are a few, for the students.
First of all, an eye for the main chance.
David Baltimore discovered a reverse transcriptase with his very first experiment
with an RNA tumour virus.
He was not thinking about it before, but he saw an opportunity and seized it.
Judicious disregard for received wisdom.
Consider Temin and Baltimore's benign neglect of the central dogma.
A willingness to gamble.
Consider the years invested in the pursuit of cellular src, in what could easily have been a fool's errand.
Faith in the universality of nature.
The example?
Our conviction that there was something to be learned about human cancer from studying
a chicken virus, albeit not in anybody's living room.
The choice of experimental system.
The attractions that drew me to Rous sarcoma virus.
Technical innovation.
The assay that found cellular src.
And self-confidence.
Howard Temin's steadfast pursuit of his ideas during more than a decade in intellectual exile
while he promulgated the provirus hypothesis to disbelieving ears.
So I conclude with a shout out to Rous sarcoma virus,
which has been a fecund friend of cancer research.
Consider its offspring.
Demonstration that viruses can cause cancer.
Reverse transcriptase, a truly disruptive and generative discovery.
The first example of a gene that can directly cause cancer, src.
Nothing like it had been seen before.
The role of protein kinases in tumorigenesis.
Phosphorylation of tyrosine, now a prime player in cell signalling
and a prime target in cancer therapeutics.
Proto-oncogenes, the first glimpse of a genetic keyboard for carcinogens.
And to date, for what it's worth, five Nobel laureates.
Not bad for a chicken.
Thank you.
(applause)