MEMORANDUM

BRISTOL LABORATORIES

UNIT OF BRISTOL-MYERS COMPANY

 

 

 

 

FROM.” _bederbere DATE___ Sevtember 22, 1955

J, Lein , Wew Antibiotics Screening Program <
TO SUBJECT conguttantehty AFrangeseat ——
CL.

lear Joe:

T am sorry we did not get to see you at Lansing. We had originally tentatively

Planned to go as is indieated by the titles we submitted but ve found thet we had
relatively little time after our Celerade trip and furthermore that we would have to
drive around Chicaco on Labor Day in order te make the meeting. The airplane schedules
Ore limited on account of the holiday and even the sore erowded and we veren't able to
eet satisfactory reservations. IY an hoping that there will be an opportunity for a triv
and visit during the tine thet you indleated. However, I cannot make any definite plans
jast yet beeanse of the uncertainty about when the remodeling of our laboratory will
begin and any traveling that I de hinges upon that. I will let you know what
possibilities, if any, should materialize,

I wan interested in your comments about the cancer meeting and mY Own general impressions
are consiatent with yours altheugh perhaps not so extreme. I think there are a number
of workere who are making useful applications of genetic concepts in the field, for
example, Ted Hod@$ra. It's hardly their fault if the transplented tuser celle don't
always display completely familiar behavior and it is indeed rather remarkable to see
how plastic the chromosome content of tumor cells can be and remain consistent with the
viability of these eslla. This is something that was quite a shock to me when I

listened to those meetings on the Ascites tumors in Hew York. If you are going into the
matter of tumor agent screening in any large way, | would certainly recommend that you
get in touch with Ted Hoshtra who ia now not very far away from you in Buffele at the
Roswell Park Memorial Imatitute. I believe you asked me once to sugweet the names of any
other possible consultants who might be able to give you background material that waa
outside the reale of my own competence and I use thet as Justification for bringing ur
hia nane.

T still cannot azree with you that the somatic mutation theory of cancer is completely
out. It seems to me still entirely possible that the firet event, a triezer to the

chein of avente leading to the development of the malignant tumor, is an ordinary somatic
mutation. nce the tissue cell has achieved a certain anatomy and recid growth rate one
might naturally exneet, ae one would in any population of microorganisms, subsequent
changes to occur sporadically and tn the ravidly growing cells of the tumor which were
eelected for antibacterial activity etc. thet is characteritatics of metastatic tumor cells,
that ie to say, the initial event conld still very well be a somatic mutation of a kind
which im » way releases the cell from its interne] and external controls. It ia quite
apparent that there are many deep-seated genetic ehanges taking place later on of which
the amupleid variations in chromosome number are the most obvious and the most dramatic.
Thies may have importonce only insofar as one might be somewhat less discouraged about
preventing the initial step of tumor formation if {t were not a somatic mate tion.

You comaented on the use of transplanted tumors in screening teste for chemotherareutic
agents in cancer. ‘“hatever comment I would add would make very little difference singe it
2

feo: J. Lein September 22, 1955 New Antibiotics Sereening
Frogram - Consultantship
Arrangenent

is not easy te ese at the present time what alternative systens could be used for
sereening purposes. It is quite obvious that a tumor which hae been transplanted for
amy mumber of generations hae been subjected to the most rigorous seleetion fer ite
abllity to survive the shock of transplantation, new environment and what net aad in
turn might be expested to be somewhat mere resistant to interference from chene-
therapeutic agents administered to the animal. On thie dasie it might be advisable to
use for tests of chemotherapeutic activity tumer lines of not very great antiquity.
George Klein hae commented that it is now possible to preserva Ascites tumor suspensions
by freezing and this might be a useful lead in the development of the standerdised
inocula for teats of therapeutic agents. I really don't see how you can conceivably
get away from the use of transplanted tumers and the question will then come about what
is the most sensible type of tumer to use in the sane therapentic sgents. I'm sure
that Ted Hoshtra would be much more knowledgable on ench matters since he's werking on
that kind of material and I omiy read about it.

I was impreased by your report on the adeption of the notion of alkalating agents as

@ common factor in chemotherapeutic and radiomynetic activity. This is a point that
hae become increasingly obvious with the work of the British group at the Chester Kadey
Cancer Lab in London but I was interested to hear that it had been so generally
adopted. Of course I was using the vor alkeiating in a more general cense. I don't
know what other terme to use that would include both substitutions of alkyl and serial
and acetyl groups which for present purposes are eqnivalent.

I agree with you that it would be quite useful to continue to look for radionynetic
agenta vartilowlerly those of natural origin. The very fact that they can be produced

by some kind of living celis would be at least preliminary evidence that they have sone
sort of specificity in their action which probably would be less likely to de true in
reagents like aeetylchloride which in principal have the same kind of pave effect. It
seene to me in fact rather remarkable that an agent with sach potency would be a

netural product and one wonders what mechanism the producing organiem had to protect
iteelf ageinst such » compound as aszaserine., Do you have any ideas about thet since this
question may eventually become of great importance aprime te developing the bases for
differential toxicity by such arente.

It possibly will remain true that none of these reagents will be able to co anything
that i-rays cannot. The one hope that yeu wight have is to develop some other haadle
on these reagents that will give then a certain ameunt of differential activity. With
regard to their relationship between the effects of these compounds on X-radiations, it
seems to me that an entirely plausible case has already been built wo for the roll of
perexyl or similar types of derivatives in the mediation of X-ray metivity and J am a
little bit surprised that there have not been more reports on the syathesis and testing
of various kinds of organic peroxides for this type of behavior. Hew some o¢ theea
peroxides are of course highly unstable and almost explesive bat others are relatively
atadle in aqueous solution. I would think thie ie something your chemistry group might
be interested to think about unless of course I've simply overloeked a large body of
work that may have been done in thie direction. It seome to me there is every reason
to expect that thie type of compound will be aa useful in radiomynetic action as would
any of the ether reagents that have been proposed.

T am sorry that you had such a disappointing result on the comparative resistance of
strains B and B/r. I wonder did you check on these etrains with ultraviolet light to
fe: J. Lein September 22, 1955 New Antibiotics Sereening
Program -
Consultantship Arrangenent

see if they fit the original deseription that Witkin gave of then? I have the
impression but I can't document it that some workers have found that these strains

in their hande are no longer ae divergent in the irradiation survival as they
originally had been. ‘Whether or not it will be feasible te reiselate suitable
irradiation-resistant variants is an open question. I agree with you that the queation
of correlated resistance in these radiation-resistant strains has been a somewhat
muddled one and there has never been a very satisfactory explanation. I agree

equally that {t ie very unlikely that these are ecincidental effeets but after all they
conceivably could be considering that the mtante are always selective in the presence
eof mutagens. To my mind it is much more likely that the straine are simply cumulative,
spontansous mutations in this rather leng time during which they had been maintained
separately from one another. In any event the levels of differential resistance which
were described are, as I reeall, rather slight and iaconstant from one strain to
another and I think can probably be ignered altogether. The more important question ta
precisely what technique to use in differentiating the two. I am not surprised that
inhibition rones would give a very poor differentiation fer the reason that the
diffusion and inhibition zone technique sets up a very sharp concentration gradiant

so that there might be expected to be very little difference in the diameter of the
sone even for rather different effective thresholds of activity, In such a case it
night be more advantageous to compare strains by means of a gradiant plate techni que
which gives you a wach more gradually spread out gradiant concentration of the agent.
After all the use of inhibition sones is based on the very fact that at the boundaries
of the sone there is a very rapid change in the concentration of the reagent that ie
diffusing from the center. Since the two strains would be compared side by side on e@
single gradiant plate it would seem to me that this could be quite a workable procedure
although it would not lend itself to quite the mass scale of testing that = diffusion
sone teehnique would. Im the leng run I would suspect that a differential inhibition
test of this kind would be much more feasible for you than one based for example on the
induction of becteriovhage and I will be very surprised indeed if there turn out to be
any serious discrevamsies in the range of compounds that weuld have the radiomynetic
effects in theae two systems, .

You also brought up the peesibility of useing the induction of petit eolany variants as

a screening procedurc. Fundamentally I think this would be a wholesome and preductive
idea but I am very dubious about the possibility of setting up a sareening procedure
that would be useable on such a large scale basis. One cannot really rely upon celony
sise as a criterion of this petit colony change despite the name that is given to it and
you will note that in all of the pablications on this question, Prusaey always eays that
a sample of colonies was tested by using the knotty reagent or seme euch vrocedure. One
of my students has been actively interested in thia system so we can speak with a certain
amount of experience. I suaepect if you were screeming for the induction of vetit
Colonies it would be necessary to expose your population of wild tyne yeast in liquid
medium to the action of the agent and then to plate thie population out which would mean
using a variety of dilutions on account of the variable amount of killing that might

be expected and then combining both a colony sise inspection with a sampling for tests
of oxidative activity either with knotty reagent or with a simplified phenol red acetate
medium that Lindengren described a few months ago in the Journal of Bacterioclory,
technique which,by the way, we have found to be very useful. Thies sounds rather tee
laverious for » routine sereening in the first event but 1¢ {6 something that night very
well be worthwhile te leek into on a selected group of antibiotics once you had then.

Now I have to tell you that a very wide range of reagents hae this petit-indusing activity.
In the first place they do oceur spontaneously but in most strains at a relatively low
To: J. Lein September 22, 1955 Bew Antibiotics Screening
Frogram - Consultantahin
Arrangement

rate. Acriflavin wae among the first reagents to be tested and almost all the work
that Prussey has done has been with that. However, we have found, akin with «
number of other investigators, that ultraviolet light is also a very active reagent
for produeing petite. Finally there have been seattered reports that such things

as X-rays, higher temperature, triphenyltetrasolium chloride and I think, bat I's
not certain, nitregen mustard, were eleo highly efficient in vrodueing petit
variants. One possible advantage of the petit system is that there ic already some
evidence that the scope of reagents which will have that activity is even wider than
the usual radiomynetic reagents bat for the moment I would be cuite discouraged about
carrying out that kind of a sereening on any large scale for preliminary screening
purposes, It would be worth giving the matter some thought since this would be o new
dimension of Biological activity of chemiesl agents.

Of the screening proposals thet might be apropos to your aims of finding radionynetic
agents, I think the ones that already have been mentioned are the outstanding candidates.
They would be: (1) differential vetween 3 and B/r cr similar radiation-resistant
mutants, (2) differential survival of haploid and dipleid yeast and (3) induction of
lysogenic bactoria. A fourth possibility would be the induction of aasily seored
mytotic or somatic segregens of heterozygous yeast. Mr. Right, my student whe is
working with yeast, will be making some yeast dipleids which are heterozygous for a
recessive red pigment character and these might be particularly useful fer this
latter purpose. If it does appear that these diploids are white and that they cive
rise to appreciable numbers of red segregens with moderate descs of UV light then we
will send them on to you. I will aleo have ready some haploid and diploid yeasts if
you would care to test them to see if they have apvorecsiabdle differentials in their
response to UV light. They are supposed to according to the literature but I don'¢
know if it will be enough to suit your purposes. Again I think » gradiant plate
technique will be almost eesential in assaying any chemical reagent. If the diploid
ia mot eufficiently different from the hapleid it might te very well worthwhile to

try to obtain some tetraploid strains of yeast which,fer exemple, Boman, at the U. of
Washington in Seattle, has been working on and which are reputedly much more radiation~
resistent then the diploid. In any event I think the preliminary trials of these
screening systens should be done both on the baeis of differential survival and on the
basis of some sort of gradiant plate assay. Unless the latter can be effectively
worked up however, I can foresee thet you will heave some difficulty in large-scale
applications.

Ae for the uee of the 1455, i.e., lambda-eensitive SR indicator system, I will Just
have to wait to hear from you how that works out. I would by the way appreciate your
sending me back the 14858R so that we can keep it in cur ewn atock for similar
purposes. We had simply never bothered to prepare that pertiouler strain sinee we did
have some others which however had a mumber of other irrelevant markere.

Hera by the way is a tip for picking up lytic effects. ‘Ye have noticed that becterial
lysis is accompanied by very considerable hemolysis on blood agar. It seems to me just
conceivable that you could mix a fairly amall mumber of washed lysogenic strentomycin-
sensitive bacteria together with 148§38 in excesa and plete these on a blood agar base.
On such a base you might conceivably gst a very dramatic effeet of UV light if this

will induee even a fairly seall proportion of the lysogenio bacteria. In effect this
produces plaques on the lambda-sensitive bacteria in the background but these plaques
can be very greatly accentuated by the use of blood agar and as a matter of fact I might
5

fo: J. Lein September 22, 1955 New Antibiotics Screening
Program - Consultantehip
Arrangement

tell you that W1485 iteelf had been isolated eriginally by a plating of strain ¥12
on bloos agar which had been exposed to UY light. It was noticed that there were
some colonies that were giving hemolysis and when these were picked and replated it
was found that the hemolysia wae due to the reinfection of some sensitive eclonies
which had been induced by the UV light. The blood agar we used was, I believe, the
ordinary 10% or eo of either bovine or equine blood; I assume that human red cells
would do just as well.

Since I aseune that your aim is to avoid having to use a fairly elaborate dilution
technique in order to estimate the number of free virus particles that are produced

it might even be possible to adapt this hemolytic reaction for the assay of phage
perhans something along the following lines although we have never actually tried 1%.
The lysogenic bacteria might be grown in the presence of or subsequendly treated with
an unknown reagent at a certain concentration in liquid medium, After a time is
allowed for the release of phage in thet syetem, the bacteria verhane might be spun
down. ‘The supernate might then simply be mixed with a standard mixture of strentomyein-
resistant, lambda-senaitive bacteria, that is the 14S5SR, plue « suspension of blood
in nutrient broth plus streptomycin and incubated for a standard perted of time, The
amount of phage liberated in the first teet might then be roughly measured by the
amount of hemolysis in the bleed tubes. It may turn out in the long run that thie
would he even more elaborate than some kind or other of plating method. On the other
hand this may suggest te you other ways cf crude preliminary tests for massive amounts
of induction. Next to a B vs B/r resistance differential, I would suspect the
induetion technique would be your beat bet. If anything more practical eecurse to mea
about using yeast in these systems, I will let you know and we will in any event send
you some haploid and diploid etrains.

The effect of Aoriflavin ia probably a pretty general one in yeast, the main trouble

is to find strains whieh don't already have a rather high rate of formation in petite
er atrains in which the steck cultures are not already vetitse themselves. This is

by no means rare. Ido agree that there is at least « hypothetical analogy between
the removal of the resciratory plaemide in the formation of petit yeasts and chene~
therapy of viruses. This analogy is approximately as clomee as that of the
disinfection of chloreplasts in such orzaniams ae Zuglena and here agein is something
if you were really going to go far afield, which 1 suspect you are not, you micht want
te think about. Were you acquainted with this, Joe, that ia, that such azente as
atrectomycin are eapabdle of bleaching Euglena so that you get the alga without its
ehleroplasts? This was described by Provoseole, Hutner and Pitner in the 1951 Cold
Spring Harbor Sympseium. It has not been the subject of much work since. As far as

I know, atrevtomrcin ia the only chemical reagent that has this effect although with
some straine heating to high temperatures is capable of differentially inectivating the
chloroplast. I do not know if there have been any exceriments on mortypically radio-
mynetic agents. If you could get yourself set up for the culture of Zuglena this
probably would not be too diffieult a test to run since it would juat be primrily a
matter of microscopic examination of Suglena cells to see if they had become bleached
under the conditions of treatment. Just on the romote poseibility you haven't run into
thie kind of story I have a discussion of disinfection exveriments of this general kind
in a 1952 review in Physiological Reviews of which I think I have sent you a reprint
but I am not sure.

Of a1] the things we have talked about, I am perhaps most pleased at your interest in
this question of substituting for the enteric flora because I think there is = tremendous
opening for research, maybe toe tremendous, for a limited attack tut I don't know that
Tos J. Lein September 22, 1955 New Antibiotics Screening

Program - Consuitantship
Arrangement

anybody else is really going inte it. This is the kind of thing for which germ-free
animale would be very suitable experimental materials but uafortunately the developers
of that technique seem to heve sat on their hind legs and not done any other
experimenta with it.

I hope I did not give you toe many ais-impressions about this occurrence of Serratia
in the little girl. This infant had not been under any antibiotic therapy at all and
the reason that I peinted out this ease was that thie was a strain thet apparently was
able to implant itself in competition with other enteric organiame even to the exclusion
of E, coli and without any apparent harmful effeet on the child at all. I would think
that euch « atrain having been isolated under conditions that would seem to miniaize
the possibility of tts pathogenicity and carrying the distinctive color marker, the
eulture might indeed be very useful as a test organian in replacement types of
experiments. It probably would be necessary to at least try to make various kinds of
antibiotic-resistant mutants from this strain in order to use them in your sort of
experiment. There would be the advantage of the red color on the Platings. I am not
sure in fact that the pigment would be of any serious consequence even in a practical
application should any justification fer this appear since it will be apparent only

in infants on their diapers and probably will not ive very auch trouble with ordinary
evacuations. It is ebvious that a tremendous amount of preliminary research would have
to be done but I would suggest that this organien is as good as any. we will send you
this strain along with the other materials that I mentioned. Several months later by
the way thie little girl was st111 excreting this particular organiem only ae a very
small vropertion of her total feeal flora.

Jos, I'm sorry that the very last part of the first recerd that you just sent was
completely garbled. I think someone elee must have come into the office at the time,
at any rate nothing much came through the microphone. You had just started to say that
you had read the Simonti article but I missed completely everything you told me about
Felix's interest in the Actinomycetes and in the Fenicillium so I may have to get that
back again from you.

Yours sincerely,

Joshua
dLijlg