Thank you Professor Fuchs, Ladies and Gentlemen, the title of the discussion as it was given in the program
is "Carcinogenesis: Chemical, Physical and Biological".
The concern for this subject is one of very broad character.
Yesterday you heard a call for the scientific community to be concerned with matters of public interest.
Well, this one is such a one.
In our country, well, first of all we are all surrounded by what seemed to be a variety of agents in our environment,
which may, and in fact in some cases we know, do have serious consequences for human health.
These include many of the natural environmental agents such as ultraviolet radiation from the sun or ionising radiation
either from outer space or from the earth surface or created by man.
And of course a wide variety of chemicals.
Some created by the needs of mankind, some existing in nature in ordinary food stuffs.
Now, as Professor Fuchs said a moment ago, it seems a far cry from the work
which he described on photosynthesis to the subject upon which I chose to talk today.
In a sense it is, in another sense it isn't.
After all the kinds of questions which we sought answers to when we undertook to determine or to try and find out
how a green plant uses sunshine to manufacture food and material, involved the use of all sorts,
of all branches, of all disciplines of science.
Organic chemistry, physical chemistry, physics, biochemistry and biology.
And later, as I have found in the last few years, even such matters as human behaviour and economics.
This subject is a similar one in that sense, in that it involves all kinds of human knowledge.
And the specific way in which I came to be involved in this is something that perhaps at least some of you will appreciate.
About 4 years ago there came into my laboratory in Berkley a young man, an Italian who had had a personal, he's an organic chemist,
and he had had a personal experience with the disease, a cancer.
As a result of that he applied for and received a postdoctoral fellowship from one of the private agencies
that exist all over the world, particularly dedicated to the problems of cancer.
He also was much interested in working in our Berkley laboratory
because some of his friends had been there before him and had told him a little bit about the place.
So when he arrived his fellowship specified that he had to work on cancer.
But we had nothing going in the laboratory on this subject specifically.
And so we had a conference, Ercole and I sat down, he came into my office,
we discussed what could be done that would fulfil the requirement of his fellowship and my interests.
And after some discussion we agreed that the photochemistry of some carcinogenic agents in the environment would do this.
And that is how in detail, specific detail we became involved in the problem.
That was the beginning of it.
And you see that's the nature of our lives, moving from thing to thing, from adjustment to adjustment.
And expanding, always expanding our interests by virtue of the flux of young people which goes through our laboratory.
It is the thing which I hopefully believe is so, that keeps us alive in that particular group of workers.
Well now, without following that line, without trying to give you the details of what Ercole did
and how it expanded into what I'm going to discuss today, I'll point out what he did as we go along.
Now, why is this subject relevant, as I said earlier there are carcinogens, various kinds of them,
physical, chemical and biological surrounding us all the time.
The chemicals which we add to our food, which we use as drugs and with which we are in contact every day.
Either in our clothing or in our furniture or in our houses.
Contained materials some of which might be and in fact are known to induce the appearance of tumours in man.
The only way in which this kind of relationship is established is by an epidemiological study,
we don't do experiments with men usually.
And it's interesting to remind you that the earliest known such connection was discovered by a British physician
almost 200 years ago in the case of the chimney sweeps who cleaned out the chimneys of British homes.
You know, each year they collect a lot of soot from the coal that they were burning
and these chimney sweeps would have to go through those chimneys and sweep them out.
It turned out that there was a particular kind of cancer, cancer of the scrotum
which was very prevalent amongst these people.
And eventually that cancer was traced to a chemical contained in the soot which collected in the chimneys.
And this is the one in fact or one of them with which I will spend most of my time.
There are many other such materials now that we know about.
That occur in our food or in our environments.
The other end of the story, in other words not only do we have a wide variety of carcinogens,
that is chemicals, radiation and biological materials.
The most recent of which has, and you've heard about it here before, are the viruses.
But the consequences of their action, the way in which the carcinogenesis is expressed
varies a great deal depending upon that part of the body in which the cancer appears.
It looks very different from one to another.
So we have a wide variety of agents, very different in character.
And we have what looks like a wide variety of consequences also very different in overt appearance.
However it is my belief and it is going to be the thrust of everything I say this morning,
that this wide variety of agents act through a single kind of mechanism.
Not one mechanism but a single kind of mechanism.
And that the wide variety of expressions of the consequences, of the biological consequences are all of the same general sort.
Involving a change in the genetic content of cells.
And the expression of that change of genetic content may be in a variety of forms, but have one thing in common.
And it is this commonality between the inducing agents on the one hand
and the consequences on the other that I would like to bring into one story if I can.
Now, as an example, yesterday at lunch I was reminded of a story or a bit of advice
which I frequently find myself induced to give to some students.
And what I am going to do today and I'm going to say today is really the embodiment of that advice.
Very often a student will arrive or work in the laboratory and spend his entire time gathering data
without really trying to integrate it.
Saying that well, he doesn't have all of the information and he's going to wait until he has collected it all before he begins to think about it.
Now, this isn't really the way science grows.
It is true that without data science can't exist or can't continue, can't develop.
But it isn't really the way in which a new generation of thought is created.
At least not in my experience.
And I have to tell them a little story and I do it in this way.
I say to them it is not, in order to be creative in science one must, occasionally, not always,
in fact very seldom, get the right answer.
But occasionally you must get the right answer about how nature is working when you have only half of the data in your hand.
And half of what you have is wrong and you don't know which half is wrong.
And then you get the right answer, you are doing something creative.
And that's what I'm going to do today.
(laughter).
I haven't got all the data, some of what I have is wrong but I don't know which parts of it are wrong,
but I'm going to try and put it together.
And eventually there will emerge from this construction certain kinds of questions
which one can put to the, experimental questions which one can put to the test.
And as we put them to the test we will modify our concepts to fit.
So that the new concept changed by the new information grows eventually into a correct one.
Well, now let us go on with the nature of chemical carcinogenesis as we understand it today.
Of course it might change but today there is a basic unity, at least there appears to be a basic unity in the nature,
in the chemical nature of the substances which are known to be carcinogenic.
Produce cancer either in man or in experimental animals or more recently in even simpler systems
such as cultures of living cells in a bottle, in a flask from various sorts of animals.
And still simpler systems in which we test the chemicals on certain relatively purified enzyme systems
which are molecules themselves.
And so we have a range of test systems from the whole animal on the one hand,
well mankind on the one hand, to animals, to tissue cultures, to molecules.
And we do all of them.
When we look at the collection of molecules which we know to be carcinogenic in one system or another,
we see that there is some commonality in their construction.
And the first slide is simply a catalogue of such chemicals.
All of them have one thing in common.
They all produce electrophilic bits.
This carbonium ion, this carbonium ion when the carbon to oxygen bond is broken.
The negative charge remains on the oxygen, the positive on the carbon
and this carbonium ion then is an electrophilic agent seeking a high density of electrons.
And this is true of all of these first four that I have put on the slide machine here.
All of them are alkylating agents, electrophilic reagents giving a positive carbon atom.
Even without any enzyme interference.
And the kinds of electron dense, molecules with which they react are listed over here in this column.
And for those molecules which are common in cells, the electrophile, I should say the nucleophiles
are mostly in the nucleic acid basis either in DNA or in RNA or in proteins.
There may be others as well, these are the principle ones that seem to be involved in the change in the genetic composition of the cell.
There are a number of other molecules listed in the lower half which by themselves are not electrophiles,
at least not from their first look.
However after they are changed enzymatically this one by oxidation of this NH bond to an NOH bond
which then can be esterified with acetate or sulphate and this now drops off as a negative ion
leaving behind a positive nitrogen ion, which in turn can be and is a powerful electrophile.
Just as these carbonium ions are.
The same sort of system occurs, the same sort of change occurs on this carcinogen,
which used to be used as a food colouring many, many years go, it's no longer so used.
It was found to be a carcinogen of the liver.
And the mechanism of its transformation from this yellow die into a carcinogen is probably very much the same as this one here.
By an oxidation of the nitrogen to the NOH and then eventually getting into a nitronium ion
so that it can attack the cellular nucleophiles.
The same thing can be said of these nitrogen compounds.
Now, down here there is a very simple symbol, PAH meaning polycyclic aromatic hydrocarbons.
And these require enzyme activation and when they are so activated they also appear to be nucleophiles.
And it is this group of compounds about which I will speak mostly in terms of the chemical consequences.
That group of compounds, the particular example of which is shown in the next slide.
All are produced always when any combustion of any organic material occurs.
For example when you burn tobacco in a cigarette.
Some micrograms of benzopyrene, the one on the left, is produced.
When you burn gasoline in the internal combustion engine some milligrams of benzopyrene are produced
and come out the back end of the exhaust pipe.
When you burn coal in a power plant or in a heating system in a home,
a good bit of this polycyclic aromatic hydrocarbon is part of the combustion products.
And so that particular group of compounds finds its way into our environment in many different ways.
Even when you burn wood or I should say agricultural refuse
as a result of the harvesting or the pruning of trees.
This also is one of the products that comes out in the smoke.
And so it is a common environmental component.
It is perhaps most effective when it is inhaled into the lungs as a result of smoking a cigarette
because then it gets right to the sensitive tissues.
And so the very few micrograms from each cigarette are deposited right in the proper place if you like.
Well now, the basic notion about how this material which is a relatively inert,
it seems to be a relatively inert chemical, these polycyclic aromatic hydrocarbons.
How can they become, how can they be made to fit into the notion of electrophilic activity which we mentioned a moment ago.
In the last decade a great deal of work has been done implicating an enzyme system
which is present in all living cells to some extent, but is induced to a higher level in cells, in most cells,
particularly in liver when they are exposed to such materials as this.
Not only this one but other materials of the same kind will do the similar thing.
These are called, for obvious reasons, the aerial hydrocarbon hydroxylases.
A group of mixed function oxidases, that is enzymes which will use molecular oxygen
to add one of the oxygen atoms to the aromatic hydrocarbon.
And use two hydrogen atoms from a reduced coenzyme to take care of the other oxygen atom.
This is what is meant by mixed function oxidase.
They are present in most cells and tissues but they are raised to a higher level
when such cells and tissues are exposed to these aromatic hydrocarbons.
Well now, the next slide shows a way in which that happens.
Aromatic hydrocarbon hydroxylase and molecular oxygen
and I didn't put on here the reduced pyridine nucleotide which takes the other oxygen atom.
And it was proposed that one of these oxygen atoms is used to hook on to the aromatic hydrocarbon
in the number three and four position making this epoxide.
Now, that's a very unstable compound which with a little bit of acid opens to give a carbonium ion at one of these positions,
either that one or that one, which then will react to give a hydroxyl and the remaining carbonium ion
will then react with the nucleophile giving the carbon to carbon bond which I mentioned at the beginning.
Thus this is proposed as an obligatory intermediate to make this compound into an electrophile
which can then be carcinogenic if this reaction occurs.
A side reaction which destroys this is simple hydrolysis of this epoxide linkage to give a diol.
There are many lines of evidence which indicate that this hydroxylase
is indeed involved in making this compound into a carcinogen if one blocks this enzyme in some way.
Either by a chemical agent which blocks it or by a mutation which prevents it from being made.
This material then does not become carcinogenic in such cells.
So the participation of the hydroxylase seems to be well established.
However the isolation of such a product has never been achieved.
And so we don't really know whether this is indeed the way in which the hydroxylase produces its effect.
And it was at this point that the student whose story I told you earlier on, entered the picture.
He undertook to examine the reactions of these aromatic hydrocarbons with components of nucleic acids.
And the one he chose was N-Methylcytosine.
The next slide shows what he found.
Instead of finding the hydroxylated compound on the three or four position,
he found that the reaction took place on the number six position, I should say on the four or five position.
The reaction took place not here but on the number six position to give this product.
Now this is a photochemical induced coupling.
However the same reaction can be induced with the aryl hydrocarbon hydroxylase and oxygen.
Exactly the same reaction in which one gets this kind of a coupling,
rather than on the sixths position or on the one and the three.
One, three or six and not on the four and five.
And that was his contribution then to this business.
Well now, we have thus examined the chemical behaviour of a number or carcinogens, this one in particular
but as yet we have no clue as to what the biological consequences of such an event might be.
Why, if this happens, even if it happens, why should that produce the biological consequences
that we know it produces, namely cancer.
Now, here I have to leave chemical carcinogenesis, I'll say one more word about it.
But we're going to leave chemical carcinogenesis for a moment and examine what we know about biological carcinogenesis.
And then after I describe to you what I think is the nature of biological carcinogenesis
we will see if we can't put the two things together and make a single unitary concept of the whole thing.
Before we leave this chemical reaction let us see if we can imagine
what the molecular consequences at least of such a change would be.
Now I'm going to, I don't have pictures of it and I don't have a model, it's much too big to carry from Berkley to Lindau.
So I'm going to ask you to imagine this thing.
Most of you have heard of the structure, of what the structure of the genetic material of living things is,
the nucleic acids, either the DNA or the RNA, most all mammalian, well all animals
have DNA as their principle storage material for the genetic information.
And that this DNA is primarily first constructed as a sequence of basis
of which that N-Methylcytosine is one or that cytosine is one instead of the methyl group.
There's a ribose sugar there.
And it is a long string of such basis of which there are four different kinds,
the cytosine is only one of them and there are three others.
But they all have similar kinds of electron dense materials in them.
They wind up in the double helical structure which you've all heard about.
In which the basis are stacked like playing cards one on top of the other, 3 1/2 angstroms, roughly 3 1/2 angstroms apart.
Now, if this aromatic hydrocarbon were to slip in between some of these basis
and form that kind of a covalent carbon to carbon bond as it is described here.
This is a carbon atom and that's a carbon atom.
If this were to happen in the double helix, the normal helical structure would be distorted.
In fact one could not maintain it if that reaction occurs.
And the normal helical structure would be distorted
so that at least one of the linear molecules would be looped out, it could not fit in the double helix.
It would have to be looped out as a sort of a loop outside of the double helix.
At least that, perhaps more.
That is a molecular consequence of such a reaction.
And at the end of this conversation I will try to deduce or introduce that again
and go from that to the biological consequences of such an event.
But before I can do that we have to examine what we know or what we think we know
about the way in which a virus can induce cancer.
Can change the character of cells to make them into malignant cells.
Now, most of the work we have done in Berkley, at least in my laboratory that is, has been not with whole animals
although we've done some of that and I will show you in the very last slide some of that.
But mostly it has been done either with enzymes, isolated enzymes
and more recently with cells of animals growing in the test tube, in a tissue culture.
And we've examined the behaviour of those cells under various influences, under viral influence,
under chemical influence and under both.
In order for you to get some idea of how this is done I want to show you a picture of a tissue culture of mouse cells
which have been infected with a virus and wherever the virus particles have been introduced into the cells
and have transformed them by whatever mechanism - and we'll come to that in a moment -
those cells which are transformed show as little piles of cells.
And the next slide shows that tissue culture in which a few of the cells had been transformed.
On the left we have only a very weak magnification, only a tenfold magnification and the dividing line is right here.
This background is a background of normal cells, cells which have not been infected with virus
and which grow to confluence and then stop growing.
That is when they touch each other, when all the cells are touching each other they signal each other.
They send signals to each other in such a way that the growth of the cell stops.
However those cells which had been infected and transformed by the virus,
the sarcoma virus, no longer are subject to that control mechanism.
And even though they come to confluence, to touching each other, they keep on growing.
And pile up on top of each other.
And these little piles are called foci.
And there are visible in this picture, one large focus and another smaller one and still two more smaller ones.
There are I think four foci here, one large one and three small ones
in which the piles of cells are visible even to the naked eye, one hardly needs a microscope.
This is the same picture blown up 40 times instead of ten times, this focus plus that one.
You can see, there it is.
And each one of these little bright spots is a rounded up cancer cell piling up on top of some more.
And here you see the big focus which is that one.
And then at the edge of the smaller one here.
This kind of system is one which has been widely used in the last decade
to explore chemical, viral and radiation carcinogenesis.
Rather than using whole animals.
One of the reasons for this is one can grow a million cells on a dish only three centimetres in diameter.
If one had to grow, do similar statistics with a million animals, even mice,
you can imagine what kind of a housing problem that would be.
Well, this has facilitated the work enormously.
And it is this kind of tissue culture work which is dominant in our laboratory at the moment, it's really the next stage,
most of our early work, some of you may remember, was done with single cells of plants, algae.
And we kept looking for single cell cultures, we've got them now.
And we can grow them both on plates and in deep cultures and this is the way we go about it.
Now, what is the nature of the process of the change?
Or what do we believe the nature of the change is which has transformed a normal cell into a malignant cell
which keeps growing, which is not regulated.
The next slide is a diagrammatic representation of that process of transformation.
If we can lower it just a little bit, the top number one here represents, this is a DNA containing virus,
this is a monkey virus, Simian virus 40.
Which contains DNA and is represented here by this oval with the three black dots containing the DNA of the virus.
When the virus infect in the cell here, is represented by the larger circle where the X is, representing the DNA of the cell.
The chromosome material of the cell.
The virus when it infects the cell, the virus injects or allows the DNA of the virus to enter the cytoplasm of the cell.
Now, normally, normally, it's hardly normal but an infected cell may reproduce the DNA of the virus in the cytoplasm
and eventually that cell membrane will rupture and more virus particles will be liberated.
That's the normal course of events of a viral infection.
However on a statistical basis a few cases will occur in which the viral DNA may be inserted into the cellular DNA in some way.
And we're going to come back to how that happens.
If that happens and this is the beginning of that process.
Here is the end of it, if that happens, then the cellular nucleus contains not only cellular DNA
but may contain all or some part of the viral DNA.
And when it divides and grows it now has new information in it, some of it obtained from the virus, here's a pair of them.
If that information, if that DNA of the virus has instructions for the manufacture of certain kinds of proteins
which will change the membrane of the cell, so that when it comes in contact with another one,
the message to stop growing is somehow blocked, it doesn't happen then the cell will continue to grow
and we will speak of the cell as having been transformed.
This will make itself evident in other ways as well.
New proteins will have been, will be made by this cell which are coded for by the viral DNA.
And we will see it in the membrane proteins of the cell.
In the new antigens which the cell contains.
As well as in terms of the nature of the growth mechanism of the cell.
Both of these things have been shown.
And the fact that the cell contains viral DNA has also been demonstrated by hybridisation experiments
in which you measure the way in which the cellular DNA after transformation interacts with virus DNA.
And you find that there is a homology between them.
There is a certain amount of the viral DNA which has indeed been incorporated into the nuclear DNA as well.
Well now, this is how we conceive the mechanism of transformation by a DNA containing virus, it's a monkey virus.
But the fact is that the first slide I showed you,
not the first one but the one ahead of this in which I showed you the transformation of mouse cells,
my sarcoma virus, is not that kind of virus at all.
Mouse sarcoma virus, Moloney sarcoma virus is not the DNA containing virus.
It's a virus whose genetic information is RNA, not DNA.
And the question then is if this is the nature of the transformation process, how can an RNA containing virus,
a virus whose genetic information is entirely RNA, how can that achieve this result.
Well, this was known six or eight years ago or thereabouts and Temin, a Wisconsin biochemist suggested
that in order for this to happen there must be in the virus, or induced by it, an enzyme which could copy the RNA sequence into DNA.
Now you should realise that this was at the time almost heresy because the gospel said
that you could only go from DNA to RNA and then to protein, that you couldn't go the other way.
And here was a young man suggesting that you could go the other way, in fact that you had to go the other way.
Well, it took about five years before that enzyme was actually demonstrated
in the mouse sarcoma virus and other oncogenic, as cancer producing RNA viruses.
And thus the idea of the integration of viral information, genetic information into the chromosomes of the cell
as the act of transformation from normal to cancer cell could be maintained.
If such an enzyme copied the RNA virus into DNA.
And the next slide shows that schematically.
The copying of RNA virus, the normal behaviour of the cell, here's the DNA of the cell
is copied by DNA dependent RNA polymerase to RNA which then makes proteins and a new cell
and this is as shown on both sides, both to protein new cell and to replicate the DNA of the normal cell.
However if we infect the cell with a viral RNA of an oncogenic virus and if it carries with it
this RNA dependent DNA polymerase, RNA instructed DNA polymerase, that's what RDP is supposed to stand for,
RNA dependent DNA polymerase, we can copy this piece of RNA of the virus into a piece of DNA which now represents the information.
The genetic information of the virus in DNA.
And then if there's a lot of it present it stands a chance of being inserted into the ordinary synthetic sequence
of the cell replicating its own DNA and we could get a piece of this,
some part of it at least added into the nuclear DNA of the cell.
And we have then a transformed cell.
If we have cellular DNA plus viral DNA, we thus get a transformed cell.
Now, if this is the case, then if we were to find a chemical which could inhibit that particular enzyme, that particular enzyme.
We should not only be able to inhibit the replication of the virus
which has to go through this step through DNA and then back again into RNA.
That's the normal replication, there's a line missing from here to there to indicate viral replication.
We should not only be able to inhibit the replication of the virus, but because we prevent this copy from being made,
we also inhibit the transformation.
Well about the time that virus and its enzyme was discovered, I should say the enzyme was discovered.
It was also found that a class of drugs which had been used to inhibit RNA polymerase in bacteria
and was a very useful drug in controlling a variety of bacterial diseases, particularly tuberculosis,
that one of its derivatives did indeed have specific inhibitory capacities for that enzyme.
Now the next slide shows the picture of that drug and some of the derivatives which we have made.
And all it is therefore is for you to get an idea of what they are.
I'm not going to use them in any detail or discuss their synthesis.
This is the nucleus of rifampicin, a class of antibiotics.
And on this position are hooked these various side chains.
The R group represents this entire rifampicin nucleus and these are a few of the several hundred of rifampicin derivatives
that have been synthesised in the search for anti TB drugs.
The one which has turned out to be the most useful for tuberculosis, for inhibiting the RNA polymerase of bacteria is this one.
But it turns out that this one will not inhibit the RDP, the RNA dependent DNA polymerase
or reverse transcriptase as it is called.
The one which does that is the one below it, this one.
This one does indeed inhibit that RNA dependent DNA polymerase.
And in addition to this some of these others have been made
and of these I'm going to speak only of this one which is a very potent inhibitor,
even more potent inhibitor of RNA dependent DNA polymerase than is that one.
And these are the two drugs, this one called DMB which I will speak of later and this one,
well I call it, with a chain open here with two 8's, I call it R82.
So later on you will see that.
Those two are the only two I will speak about again.
Well now, the first thing we did was to use the drugs that we had available, and the one we had available was that one,
and I call it DMB, to see if it would inhibit, prevent the cancer producing virus from transforming or making a cancer.
And the next slide shows the result of that experiment.
Here we have, we're counting now the number of foci, that is the number of cancers which the virus produces.
Without any drug we will produce 140 foci for 100,000 cells.
With three micrograms of the drug in the medium we have reduced the number of foci to 90.
With six we have reduced it still more, with nine we have reduced it still more.
Thus the basic idea that the chemical which inhibits
that particular enzyme can prevent that virus from transforming the cells is confirmed.
But something else is in this slide which caused us some concern.
Well, not only concern but excitement.
These two columns, XC plaques, are simply devices for counting the number of virus particles
that are liberated from the cells.
And here you can see that in both of these experiments
the drug has not reduced the number of virus particles at all.
If anything, it's increased them a little bit.
And so there is something wrong with the theory.
Now, we don't like to throw it all away so we only threw part of it away or really we added something to it.
We suggested that this same drug, these two that I mentioned earlier, too,
they would inhibit the reverse transcriptase which is required for copying the viral RNA into DNA.
But also there are other enzymes involved later on in the integration of that piece of DNA into the cell.
And we make the suggestion which is illustrated in the next slide, that not only does the drug inhibit this enzyme
but it may inhibit one or more of these which are required to insert that piece of DNA into the cellular DNA.
In fact we can use a small enough amount of drug so that this stuff will not be inhibited.
And we will make more virus but one of these other enzymes.
And I've listed three here, is more sensitive to the drug than is this one.
And at low enough levels this will operate but one of these, one or more of these will not.
And thus we can separate the integration phenomenon, that is the transformation from the virus replication step.
And that is what I think has happened.
Now, if that's the case, then we have to find out which of these enzymes is involved in the integration, they seem all to be.
And there may be others which I haven't listed here, which I don't know about, so I have to look, I have to find them.
And then this has another consequence which we can test.
This says, this idea suggests that if we were to activate these enzymes,
if we could find a way of activating this group of enzymes here
especially highly in the presence of a large amount of this stuff,
we should enhance greatly the rate at which this stuff is integrated into the cellular chromosome
and at which transformation occurs.
Now, this is a synergism experiment.
This is an experiment in which we use something to activate that excision repair system on the left here.
And at the same time we will have present a large amount of viral misinformation if you like.
And if that situation is true we should enhance the rate at which that misinformation is integrated into the cellular chromosome.
Now, that can be done and this is now where we come to the marriage between chemical carcinogenesis and biological carcinogenesis.
It turns out that one of the things that the chemicals do, as I mentioned at the beginning is to deform the cellular DNA.
Loop it out, react with it and change its normal shape into an abnormal shape.
Now, apparently when that happens, enzymes for cutting out the mistake that loop out are induced
and the whole, these enzymes are called the excision repair system, cutting out and repairing.
So if we put the chemical in which does the reaction which I described earlier
and if we have at that time present a large amount of viral DNA, which is carcinogenic,
the probability of inserting it is greater, should be greater, should be greater, it's a test.
And I found in the literature a number of such tests.
We are doing them ourselves with the hydrocarbon.
And I found in the literature a number of such tests with other chemical carcinogens.
And I'm going to show you one of them because it does show exactly that effect.
This is some work of Stitch, a Canadian biochemist who reported it a couple of years ago.
And the next slide shows his result.
In which he uses a chemical carcinogen and a viral carcinogen together.
The viral carcinogen in this case is an adenoma virus, adenoma virus 7.
It's a DNA virus.
And if he puts the adenoma virus on at the same time he puts on the chemical treatment,
in this case the chemical is 4-nitroquinoline and oxide which is a very potent liver carcinogen.
And this is a Syrian hamster cell tissue culture.
If he puts the SA 7 on, that is the adenoma virus on alone he gets 17 foci.
This is the control experiment.
The virus alone introduces 17 foci per two million cells, you see here it is per two million cells,
17 foci per 2 million cells.
If however he puts on the chemical and then within an hour and a half he puts on the virus,
you see that instead of 17 transformations he got over 400 transformations.
I should tell you that the chemical alone in this particular hamster cell system produces no transformations.
The chemical alone will not do it.
But here the virus alone does 17, the chemical alone zero, the chemical with the virus 400 or more.
A very potent synergism.
A confirmation at least of the concept I gave you on the previous picture.
That the chemical triggers the introduction of the misinformation which must be there at the time the triggering occurs.
There is another statement here, the whole excision repair system
which is induced by the nitroquinoline and oxide is over in about eight to ten hours.
So 24 hours later it doesn't work.
The whole excision repair system has stopped and then adding the adenoma virus doesn't do any,
there is no synergism at all.
In fact it seems to be suppressed a little bit.
This is the sort of confirmation that the chemicals are triggering the introduction of already present strips of misinformation.
And those strips could be derived from a variety of sources.
In this case that piece of information is derived from the adenoma virus.
But it could come from other sources.
It could be in extra nuclear DNA which might be in the cell.
It could be in inactive DNA in the cell which is then moved around into another part of the chromosome by this whole system.
All of these are different possible processes by which an inactive piece of DNA is made to express itself in the cell.
And the chemical doesn't create the information, it integrates it.
It triggers the integration and this may be the unitary process of the chemical,
of the ultraviolet radiation and of the viral or other plasmid information.
The way in which they work together.
The chemical alone could not on this theory produce the transformation
without the presence of some other source of misinformation.
And that the integration phenomenon with all the enzymes involved that are involved, play a role.
Well now, if that's the case we want to see if those other enzymes, those ligases particularly,
the ones that hook together the loose ends of the DNA, play a role in the continuing reproduction of transformed cells.
And it turns out that much to our surprise they seem to do so, although the interpretation of this result
that I'm going to give you now is very, very questionable.
We began to explore other, the second drug that I mentioned, the one that has two C8 groups attached to the hydrazine.
It seemed to be a very special drug, had all sorts of properties for penetrating cell membranes that we liked.
And we went on, it had very good enzyme inhibiting properties.
And so that was the drug of choice.
And in order to use it we had to test its toxicity on cells, before you can use it for any experiment.
You have to see is the drug itself toxic to the cells.
And the next experiment is the result that we found when we studied the toxicity of the R82.
Can you focus that a little better, it looks blurred to me, that's much better, thank you.
I'm now speaking of another drug of that same class.
Instead of that 16 membered ring, which I showed you on the whole catalogue of drugs, I have opened the ring,
it's very much the same but it has, instead of having a big 16 membered ring, it has a tail of two 8's.
And that's why it's called, has this name.
And now on normal cells, we are counting cells and we're determining what happens to normal cells
when we add this drug to the medium in which they are growing.
And look at this picture on the left, this is the number of days in which the cells are growing.
We inoculate roughly 100,000 cells and then we wait and we count them.
And the zero here means no drug at all and you see that in three days there is over one million cells
and in four days there are about two million cells.
The same thing happens when we put three gammas of this drug in it, this R82.
So the three gammas of the drug, three micrograms, three millions of a gram of the drug don't do anything, doesn't do anything to the cells.
Even five micrograms of the drug per millilitre doesn't do anything to the cells.
We have to go up to ten before we begin to see any effect on the normal cells.
And by the time we get to 15 or 20 we are really killing off the cells.
So the drug is toxic at high enough levels but at low levels it isn't.
Now when we look at T-cells, these are transformed cells, cells which have been transformed by the sarcoma virus.
These are cells which are in effect cancer cells.
And here again is no drug, counting the transformed cells, which grow even a little better
when there is no drug than the normal cells, they grow a little faster as they should.
But even as little as three micrograms of the drug suppress the growth of the transformed cells.
Five micrograms suppress the growth of the transformed cells and ten of course stop it entirely.
And if you now take a section here at the second day and count the number of normal and transformed cells
at the second day, 48 hours, as a function of how much drug is present.
You see that the normal cells resist three, five and even ten micrograms,
one has to go to 15 before one suppresses the growth of the normal cells.
But the transformed cells are suppressed even at the lowest level.
Now, that was a surprise to us because the transformed cells, the cancer cells are in general much more vigorous than the normal cells.
And here is a case in which the thing seems to be reversed.
Now let me show you just quickly the last three slides in which you see what happens to the cells themselves.
That was just a count and the next slide shows some pictures of the cells themselves and you can see what happens to them.
This one is the normal cell without drug.
This is the normal cells with drug.
Here are the transformed cells.
You see the difference between transformed cells and normal cells.
Normal cells are elongated with their nuclei in them and growing in stringy close packed.
The transformed cells are rounded up and pile up one on top of the other.
The transformed cells however in the presence of the drug have disappeared.
You see there are very few left.
Normal cells grow normally even in the presence of five milligrams of drug.
I guess this is five or six milligrams of drug.
And here the transformed cells have sort of disappeared.
At least they haven't grown.
Looks as though they've died.
Now we've done another experiment of the same sort.
We've taken a tissue culture and the next slide shows it.
We've taken a tissue culture and we have infected it with Rous sarcoma virus.
And this is normal cells in the periphery and this is one of the foci that you saw on the very first slide of tissue culture.
And the two white arrows are intended to mark the boundaries of the focus.
Here is another one just like it, two of them.
This one we have simply put fresh medium on and let it grow for 82 hours.
And it grows enormously.
It grows into a great big focus.
In fact it grows into a very large, such a large one that the centre no longer gets nutrient and is necrotic,
the cells are dying in the centre but they keep growing around the outside.
This is a sort of a model of a solid tumour if you like.
Here is an identical one here but after the focus has been formed, we have added the drug to it.
And 82 hours later you see the focus has not grown at all.
If anything it seems to be fading away.
So as a model of the treatment of a tumour after it appears, by this drug, this tissue culture seems to be a positive experiment.
And finally looking at the cells themselves we have taken a mixture of the cells.
Half of them normal and half transformed.
Next slide, half normal and half transformed in the top of the slide.
And you see the transformed cells are the rounded up ones and the normal ones are the long ones.
You see the transformed ones and the normal ones.
This is the beginning of the experiment.
We add the drug and let it go for two days and it looks as though there are very few transform cells left,
one, two, three, a few of them.
Most of the cells are normal.
We don't know whether that normality, normal appearance of the tissue culture is due to the overgrowth of the normal cells
over the transformed or whether it is due to the toxicity of the drug on the transformed cells simply eliminating them.
And that's a very fundamental difference.
In other words does the drug reverse the transformation or does it simply kill the transformed cells.
It looks as though it simply kills them but we don't know for a fact that that is the case.
And that's about where the experiments are.
Now as a final picture let me tell you that we have gone a little further than tissue cultures.
This is chicken cells in tissue culture and you have to keep that in mind, it's a long way from a human being, very long way.
But we have gone one step further, we have used not this drug but the first one, DMB drug on rats
which have been induced to have mammary tumours by a chemical, a condensed ring system hydrocarbon.
I'm now returning to where this talk began.
If this is all, if all the ideas hang together.
These chemicals should not only prevent the transformation but conceivably could have a therapeutic effect
even after the transformation has become visible.
That wasn't the case, that idea wasn't extent when this last experiment was done.
We used a strain of animals which has in it the necessarily genetic information
so that when the animal is given one injection of a condensed ring hydrocarbon, dimethyl benzanthracene
or trimethyl benzanthracene, let's say dimethyl benzanthracene.
It's a condensed ring hydrocarbon which is in all these combustion smokes.
All the animals developed mammary tumours.
They have the genetic composition to do it.
And the next slide, this is the last slide, shows, look at the upper part only.
Here is the control experiment, N equal 17, that is 17 animals got an injection
of dimethyl benzanthracene and they were all dead at eight weeks, zero survived.
Percent unaffected zero, none unaffected, all dead in fact.
It's a very potent titration.
You know it's a hard titration to do because it takes such a long time.
And of course it's a hard titration to do with very much statistical validity
because it takes a lot of animals.
I mean 17 animals is not a very great number.
Of course if we had a million it would be better, but we don't.
I mean we don't have a barn for a million rats.
So we have to do it in this way.
Well now if we give, we have here 12 animals which got dimethyl benzanthracene.
You see they all die after eight weeks.
These 12 got ten milligrams of the drug every seven days.
And the course of the disease was at least altered, it was slowed down.
Eventually they all died.
If however we give ten milligrams every four days, in other words more drug,
the course of the disease is altered still further and some of them even survive.
The significance of a whole animal experiment of this kind is very difficult to be sure of.
You know it's bad enough, may I have the lights please, it's all done now the slides,
it's bad enough to try and interpret an experiment with tissue cultures, with whole cells.
We want to keep pushing it back to the enzyme and to the substrate as far as we can get.
So I've shown you experiments at all three levels, enzyme inhibition, cell inhibition and animal inhibition.
The least certain of these is the whole animal work.
Next in confidence is the tissue culture or cell work.
And finally the one that is most, we have most confidence in is the enzyme inhibition.
But for that one we have only the one enzyme, the reverse transcriptase.
The only one we know.
We know that there are three or four others involved in the transformation and we are just now in the process of trying to find out
which ones they are, fish them out and do the experiments on the isolated enzymes.
And we'll know then what is going on.
We will have to find out also in detail what the chemical change is which induces the formation of those enzymes.
We only have an indication of that.
And that's where we are today.
Perhaps if some of you students up there three years from now won't be here I'm afraid,
I don't know where you'll be, you'll be someplace else.
But if things go one tenth as well as, if one tenth of the guesses I have made today are correct
there will be something to tell you about another time.
Thank you very much.
Applause.