Download Differences between Cancers inTerms of

Differences between Cancers in Terms of
Evolution of Drug Resistance*
L. W. LAW
(National
Cancer institute,
National
An increase in resistance to drugs is only one of
several kinds of variation that may be detected in
populations of neoplastic cells by change of the
environment.
Heterogeneity
of the neoplastic cell
population gains support from many facts. The
attainment
of autonomy,
loss of histocompatibili
ty genes, fluctuations in cell size, chromosome
numbers, and nuclear abnormalities,
increase or
decrease in specificity of certain neoplasms as de
termined by transplantation,
and changes in be
havior of neoplasms through microisolation of
clonal sublines
all suggest
the action
of natural
or
applied selection on a population of cells exhibiting
a multiplicity of variants.
The problem of resistance in neoplastic cells, as
in microorganisms, will remain a most important,
perpetual threat to the successful use of therapeu
tic agents. The mechanisms of drug resistance, as
may be seen, are for the most part unknown or
controversial.
This is not a sign of neglect
but a
reflection of the difficulty of the problems encoun
tered. This will become apparent in the discussion
which follows.
The development of lines of neoplastic cells
showing resistance to and, on rare occasions, de
pendence
upon certain
carcinostatic
drugs is now
established (8, 39, 45, 46, 50, 51, 54). The informa
tion available concerning the adaptive response,
mechanisms concerned, etc., is, however, only of a
preliminnry nature. The use of microorganisms has
been helpful in attempts to understand the man
ncr of origin of resistant variants of neoplastic
cells, and inferences may be drawn concerning bio
chemical and physicochemical
mechanisms from
these studies. The development of populations of
neoplastic cells, especially of lymphomas,
in as
citic form, gives encouragement
to the use of
methods which have been helpful in the study of
microorganisms. The analogy between populations
of neoplastic cells and microorganisms is obvious,
and there appears much to be gained in pursuing
S Pr@ted
at
the
meeting
of
the
Scientific
Review
Corn
mittee of the American CancerSociety, held at the Westchester
Country Club, Rye, N.Y., March 93-95. 1956.
institulsi
of Health, Bethesda, Md.)
this analogy in method and in concept. It will be
appreciated,
however,
that
only a beginning
has
been made in such an endeavor.
Encouraging
results, particularly
in lympho
cytic neoplasms, have been obtained with com
pounds which interfere with intermediary metabo
lites involved in the biosynthesis of nucleic acids.
Some of these compounds have been designed es
pecially to interfere with specific reactions. The
backlog of information relating to the biochemical
pathways involved in nucleic acid synthesis has
now reached a stage where some degree of success
may be anticipated in determining mechanisms
through which certain antimetabolites
exert their
inhibitory effects. The specific modifications
of
metabolic pathways provided by resistant mu
tants
of neoplastic
cells should
prove
useful.
The
identification and characterization
of specific cc
enzymes F (6,
@,
44, 95) concerned with the utili
zation of PGA and related compounds, studies on
the site of action of azasenine (%5,8@), 6-mercapto
purine (18, 79), and @,6-diaminopurine (10) indeed
give much hope for an early solution of the in
hibitory effects of these compounds in terms of
specific biochemical events. Consequently,
it ap
peared wise to concentrate our efforts at the
moment on resistant populations of neoplastic
cells developed through the use of antimetabolites
which interfere with nucleic acid synthesis, in the
hope of defining specific mechanisms and exploit
ing these to the advantage of the host.
Much of the discussion presented here relates
to studies on the common morphologic form of
lymphoma in the mouse, acute lymphocytic leu
kernia. Our continuing efforts are directed also to
other forms of lymphomas available as ascitic nec
ple.sms, such as type A and type B reticular nec
plasma (17), plasmacytomas,
and granulocytic
leukemias.
In addition to the antifolic and antipurine corn
pounds discussed here, more recent efforts have
been directed also to antagonists of pyrimidinea,
such as uracil antagonists
and orotic acid ana
logs(98).
698
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L&w—Evolution of Drug Resistance
The failure of initially effective compounds to
maintain remissions in leukemic patients and to
inhibit completely populations of leukemic cells in
the animal should not deter those interested in the
chemotherapy
of neoplasms. The striking initial
effectiveness on restraint of growth is worthy of
exploration. Failure of drug action is encountered
in bacterial chemotherapy and is the result of the
development of resistance or dependence. The
possibilities of exploiting the biologic variables of
resistance and dependence, once the specific bio
chemical or physicochemical mechanisms are un
derstood,
appear to be extremely
good.
699
Tun Dnvni.opurn@r&@m
Cniaacrimiwrice
OF RI@8ISTANT
Since reference will be made throughout the
text to specific variant sublines of the lymphocytic
neoplasm
L1@10 (5@), it appears worth while to re
view briefly the unique characteristics of these
lines. The original line of this lymphocytic nec
plasm (L1@10-sensitive) is now in the 860th trans.'
fer. An ascitic sublime was established and has now
been carried through 51 transfers. It is interesting
to note that no detectable changes in response to
drug s have been observed in the sensitive subline
during this long period of transfer, and the re'
TABLE 1
NONSENSIT1VITY IN S@v@R.&iNEOPLASMS DEvELoPED
THROUGH THE USE OF C&RCINOSTATIC AGENm
@R)Species
Refer.Neeplasm
Resistance
and
rnceLymphocytic
Designation
leukemia
C
86C
strain
Composnd
or
need
Dependence (D)
AK-4
AK mice
A-methopterin
R
I
C58
A-methopterin
R
L1210
DBA/2
mice
mice
Various
4-amino
PGA
8
R
and
D
and
D
(4
lines)
4@,51
antagonists
C
L1210
DBA/9
mice
8-Azaguanine
R
C
L1210
DBA/2
mice
6-Mercaptopurine
R
DBA/2
mice
Thioguanine
DBA/9
mice
Thioguanine
4'
L1210
C
L1210
(ascites)
and
A-
46,
4@7
50
R
or
D
54
R
to
both
54
R
(2
lines)
(8
lines)
methopterinI
CRC
Plasma-call leukemia
Fibrosarcoma
58(ascites)Fibrosarcorna
Carcinoma
L5178
(ascites)
DBA/2
mice
6-Mercaptopurine
L5178
(ascites)
DBA/2
mice
A-rnethopterin
I@4946
(ascites)
AKR
A-methopterin
R
70429 (ascites)
Sarcoma 180
Ehrlich carcinoma
@SRmice
Swiss mice
Swiss mice
mice
Azaserine
6-Mercaptopurine
N-methyl colcliicamide
R and D (6 lines)
R
R
79
18
Walker rat sarcoma
Rat
N-acetyl derivative of
R
14
R (9 lines)
41
mustardCarcinosarcoma
N
Walker
Fibrosarcorna
Wistar rat
YOshidasarcoma
Rat
ethyl)-amine-N-oxideMany(ascites)
TEM
Methyl-bis (i9chioro-
R
98
lines of trsnaplantable Iymphomu are in exiatencewhich abowa natural realatenceto the many agmta discussedhere. In fact, two I@'niphocyticass
plums,
respectively.S
L8054 (C68 mice)and P.488 (DBA/5 mice)appsrently
Unpublished
observations
from
this
grow optimally in the presence of 8-aesgusnine and 5-mercaptopurme,
laboratory.
In general, the failure of neoplastic cells to re
spond to a drug after initial success may be the re
sult of variables either in the host or in the cells.
Burchenal first described the phenomenon of re
sistance to A-methopterin,
which arose in a popu
lation ofleukemic cells derived from a lymphocytic
neoplasm (Ak-4) of the mouse (8). In the same
year we were able to select resistant and dependent
leukemic cells with various 4-amino substituted
folic analogs, using the lymphocytic neoplasin
L1@10in DBA/@ mice (45, 51). Since then, nu
merous resistant
and dependent
variants
have
been recorded as being developed, in the lympho
m.as particularly, but also with such solid tumors
as Sarcoma 180, the Ehrlich carcinoma, etc., to
several different types of agents (see Table 1).
sponse of the ascitic population has been essential
ly that of cells carried by transfer to the subcuta
neous connective tissues. This indicates that the
endogenous environmental
variables within the
host are relatively nonselective as regards drug
sensitivity, for this neoplasm.
In contrast, selective pressures applied through
the use of certain antimetabolites to the popula
tion of sensitive cells have resulted in the develop
ment of variant sublines differing from the control
sensitive line in various physiologic and biochemi
cal aspects. Consecutive serial transfers of the leu
kemic lymphoblasts in mice receiving either near@
maximum tolerable levels, or periodic increases in
the level of a drug, result in the establishment of
populations of cells showing differing levels of re
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700
Cancer Research
sistance to, or dependence on, the drug used. The
several sublines now in existence have been de
scribed (4o, 46, 50, 51, 54). Each of these sublines
has been maintained for more than 100 serial pas
sages in mice given the drug originally used to de
velop the new population of cells, to minimize re
version
to a sensitive
population.
In several
sub
lines, however, notably AM-D, A-methopterin
dependent; 8-AG-D, 8-azaguanine-dependent; and
TH-R, 6-thioguanine-resistant, the characteristic
resistance or dependency has been maintained for
80, 55, and twenty transfers, respectively, in the
absence of drug. These characters are thus shown
to be stable, irreversible,1 and heritable.
Resistance,
a
condition
in
which
leukemic
lymphoblasts of leukemia L1@1Ogrow optimally
either in the presence or absence of a drug, and de
pendence,
wherein
these cells grow optimally
only
in the presence of a drug, have been observed fol
lowing the use of two classes of antimetabolites:
folic acid antagonists and purine (or pyrimidine)
antagonists. Variant cell lines have been obtained
using the following compounds : 4-amino-PGA
(aminopterin),
4-amino-N'°-methyl-PGA
(A-me
thopterin),
4-amino-9-methyl-PGA
(A-ninopte
rin), 4-amino-9,N'°-methyl-PGA
(A-denopterin),
8-azaguanine,
6-mercaptopurine,
and 6-thiogua
nine.
In addition to L1@1O,resistant lines have been
developed in two other lymphocytic neoplasms,
L4946 (A@KR mice) and L5178 (DBA/@ mice)
(50),
and
in the
plasma-cell
neoplasm,
704@9 (CSH
mice) (7@)(see Table 1).
Partial dependence upon the drugs used in the
selection of variant lines has been observed in two
L1@1Olines, resembling, in this respect, a strain of
Neurospora requiring sulfanilamide
(19) and the
streptomycin-requiring
strain of Meningococcus.
The emergence of resistance in certain nec
plasma has been determined
by various means,
such as volume of ascites (58), tumor cell volume
(@8), mitotic index (@9), or weight of the localized
tumor mass (41, 47). Caution should be exercised
in assuming the development of the character of
resistance without additional collateral evidence.
Transplantable
lymphomas,
which grow as a local
ized mass of tissue in the subcutaneousconnective
tissues, are easily converted to the ascitic form,
and transformations from one form to the other
1Irreversibility is used here in a relative senseto distinguish
genetic from physiologic adaptation in which populations of
cells revert readily and quickly to sensitivity in the absence of
the drug. This is discussed later. One characteristic of a muta
tine is its ability to revert, and such reversions have been
studied and the frequencies determined. The difficulties in de
tecting such reversions in neoplastic cells in the absence of
adequate selection or isolation technics are apparent.
are then accomplished with ease. Thus, many cri
teria may be used for a comparison of the original
and derived populations of neoplastic cells. For ex.
ample, in the A-methopterin-dependent
(L1@10/
AM-D) line, optimal growth of leukemic lympho.
blasts leads to earlier leukemic death in mice given
4-amino-PGA analogs. This difference is also re
flected in lymphomatous growth in the subcutane
ous connective tissues, in infiltrations of lympho
blasts into liver, spleen, and lymph nodes, in
escape of lymphoblasts into the blood stream, and
in hemoglobin levels. A florid leukemia develops
in the presence of the drug (45). (See also the be
havior of other resistant and dependent variants
[471.)Recentlythetechnicof measuring
total vol
ume of ascitic cells, coupled with the influence of
tumor growth on liver glutathione levels, has been
shown to be a simple, yet precise, criterion for
studying the action of certain chemotherapeutic
agents on populations of leukemic cells, and, addi
tionally, for distinguishing
resistant and depend
ent variant lines from sensitive populations (75).
Certain characteristics
of the resistant and de
pendent variants of L1@1Ohave been determined:
1. Cross-resistance
(or
dependence)
to
all
4-
amino substituted PGA antagonists has been
shown for the L1@1O variants developed through
the use of these compounds. The so-called weak
PGA antagonists, such as N'°-methyl-PGA and
9-methyl-PGA, are not inhibitory, nor do they
provide for optimal growth of the dependent lines.
Neither PGA nor CF influences the growth char
acteristic of the sensitive or dependent lines of
L1@1O, but
both
compounds
block
the
antileu
kemic action of A-methopterin on sensitive leu
kemic cells and the growth-promoting capacity of
this compound on the dependent cells.
Sensitivity of the variant lines to other in
hibitory
compounds
such as TEM,
alpha-peltatin,
the various available antipurines, and azaserine is
similar to that of the sensitive line.
%.
Cross-resistance
(or
dependence)
shown to all purmne antagonists
has
studied
been
for the
L1@10variants developed through the use of 8azaguanine, 6-mercaptopurine,
and 6-thioguanine.
These compounds, plus @,6-diaminopurine, chloro
purine, and purine, show no inhibitory effects on
the resistant lines and support growth in the de.
pendent lines, while remaining inhibitory to
L1@1O-sensitive leukemic cells. Whereas sensitivi
ty of the variant lines to TEM, alpha-peltatin,
and
azaserine has remained similar to the sensitive line,
a pronounced increase in sensitivity has been oh
served for all four antipurine variants (8-AG-R,
8-AG-D, 6-MP-R, and TH-R) to the 4-amino
PGA analogs. This will be discussed in more detail
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Law—Evolution of Drug Resistance
later.
The several variant lines of L1@1Ohave retained
their characteristic cross-resistance and collateral
sensitivities
throughout
more than 100 serial
transfers. Ascitic forms are identical in response
to the neoplasms
carried as lymphomatous
growths.
Although
encountered
morphologic
changes have
among the various sublines
not been
following
the development of resistance, it is quite apparent
that striking differences may be detected in
growth rates and certain metabolic aspects in cer
thin lines (see Table @).The population of ascitic
cells, dependent on 8-azaguanine (8-AG-D) for
optimal growth, still exhibits logarithmic growth
characteristic
of the
sensitive
line
but leads
to
701
almost ubiquitous and on occasion present insur
mountable obstacles to the clinician, too often
failure of therapy is related without supporting
evidence to bacterial resistance at the cellular
level. This may also be the case following drug
failure in neoplastic diseases. Host variables, such
as hepatic detoxication, the development of a more
efficient renal excretory mechanism, the influence
of degradative enzymes, etc., conceivably may ac
count for drug failure. Lack of effectiveness in the
treatment of granulocytic leukemia in the mouse,
for example, with potassium arsenite has been
attributed to host variables (71).
Nonetheless,
considerable evidence is now at
hand from studies of neoplastic cell populations in
the experimental animal to state with certainty
TABLE 2
DIFFERENcEsOBsERvEDIN Two LINEs or THELYMPHOCYTIC
NEOPLASM
L1210
Ascitic fluid
Mean survival following inoculation of 1X1O'
ascitic
cells
8-Asaguanlae
dependent (8-AG-D)
Sensitive (S)
bloody
nonbloody
12.7 days
7.7 days
PGA metolxhsm:
CF content of cells
PGA to CF conversion
A-meth.inhibitionofPGA to CFconversion*
incorporation:
Na C'4formate(CNA)f
0.52 @igm/gm cells
23.5 ,@gm/gm cells
2.87 pgm/gm cells
146.5 14,m/gm cells
x
xX7
47 ic/mole C
2,6-Diaminopurine-2-C'4 (CNA)
8-Azaguanine-2-C'4 (RNA)@
97 sic/mole C
12 ,@c/moleC
91 j@c/moleC
2.86 j@cfmoleC
314 pc/mole C
Drug rezponse:@
+
+
±
A-methopterin
8-Asaguanine
Azaserine
C Unpublished
data
of
Nichol
and
++
Law.
t CNA —Combined nucleic acids.
@
Data of Skipper it at. (see 5. 77).
I — — no increase
in survival
time;
each
+
— 100 percent
death from leukemia much later than the sensitive
line. The slower growth rate is not always char
acteristic of resistant and dependent cells, how
ever, for a 6-thioguanine-resistant
variant in
ascitic form has a mean generation time of 9 hours,
strikingly similar to the sensitive ascitic popula
tion (54).
The differences shown by the two lines (S and
8-AG-D), relating to PGA metabolism and incor
poration of sodium formate,
@,6-diaminopurine,
and 8-azaguanine, are probably the direct result
of changes in pathways of synthesis of nucleic
acids.
MANNER OF Omoiw
OF Acguiw
Dauc
RnsisT&@icn
As in the case of antibacterial agents, the devel
opment of antineoplastic agents has brought to the
foreground new aspects in the evolution of neo
plastic cells. Although drug-resistant bacteria are
inoreaae
over
controls.
that drug failure results from changes within the
cells.
Two general alternative possibilities need to be
considered in the reaction of a population of bac
teria or of neoplastic cells to a drug. These media
nisms need not be mutually exclusive:
1. Physiologie adaptation.—Thedrug induces a
change in the population, for example, as in en
zyme adaptation. The change disappears rapidly
and entirely following removal of the drug. Thus,
there are no genetic changes to be found.
@. Genetic
adaptation.—Individual
cells
of
a
given genotype are unable to cope with the new
environment. Mutation (or other genetic changes)
thus provides a basis for new heritable variation.
If these mutants are able to propagate themselves,
a new population arises from clones better fitted to
survive. Mutants
arising independently
of the
drug are selected by the drug and isolated, and a
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Cancer Research
70@
new population
characterized
results.
Genetic
by the randomness
adaptation
is
of the change
and by stability in the absence of the drug.
The roles of mutation and selection in drug re
sistance in bacteria have been sufficiently docu
mented both by indirect and direct methods to be
convincing that an important mechanism is one of
genetic adaptation. Indirect methods of analysis
which have proved useful are the fluctuation test
(60),
the
replica
plating
method
of Lederberg
(57),
and the method of statistical clones developed by
Newcombe (63). More rigorous proof of the genic
nature of drug resistance has been obtained in
Eackeriehia coli, strain K-1@, in which hybridiza
tion and segregation take place (55, 88). Recom
binations of characters
of the parental strains
occur similar to the Mendelian processes of higher
forms.
All the stable forms of drug resistance, resist
ance to streptomycin,
azide, chloramphenicol,
and
terramycin, studied adequately by bacterial cross
ing technics, with E. ccli, are related to mutations
of genes which almost certainly must be nuclear
and chromosomal. Particularly, in E. cdi exhibit
ing high levels of resistance to streptomycin and
to sodium azide, the changes are known to be due
to gene mutations. They occur in the complete
absence of the drug, and streptomycin is known
not to act specifically on the streptomycin gene
locus to induce mutations (55, 64). In one study,
streptomycin
resistance in E. coli has been shown
to result
from
a single
recessive
gene
mutation
(56). Incontrovertible evidence exists, from studies
with Neurospora, in which the classical procedures
of genetics can be used, that drug resistance can be
associated with a gene mutation (19).
Analysis of transduction
(97) and of transfor
mations (@,34) to resistance in bacteria, as well as
the Mendelian analysis mentioned above, are con
firmatory in indicating that the types of resistance
observed in bacteria represent changes of a com
posite and discrete nature as demanded by genetic
theory.
Alternative theories have been proposed for the
origin of drug resistance. In brief, those which
have received most attention are: (a) direct induc
tion of genetic changes by the drugs employed
(59) ; this
will
be
dealt
with
subsequently;
and
(b)so-called“self-adjustment―
ofenzymebalances
to permit growth under new conditions of the en
vironment without chromosomal change or selec
tion (@7). This theory is, in essence, quite similar
to physiologic adaptation, mentioned previously.
For the most part, proponents of this theory have
dealt with changes in the population of cells as a
whole, using sub-inhibitory
concentrations
of
drugs, and the adaptive changes in populations of
cells have been readily reversible and nonstable in
the absence of the drug. No attempt has been
made to resolve the behavior of individual cells in
the population. These variations are probably of
a nonheritable nature and should be expected in
populations of bacteria or neoplastic cells. The
mechanisms underlying such variations are un
doubtedly dissimilar from heritable variations of
a stable and irreversible character. Attempts
should be made to distinguish the types of varia
tion encountered in neoplastic cell populations,
but this may be quite difficult in certain situations.
Claims of specifically directed, adaptive resist
ant mutants have been made by bacteriologists.
This Lamarckian belief that mutations are induced
specifically by the agent used in selection probably
is the result of the adaptive features
and the fact that bacterial genetic
their infancy.
Studies on the mutagenic effects
neoplastic compounds have been
of the process
studies are in
of certain anti
reported in E.
cdi (@6). Only nitrogen mustard and azaserine
were found to be potent mutagens as determined
by reverse mutations from streptomycin depend
ence to nondependence. None of @6other corn
pounds having some degree of antineoplastic po
tency was effective. Novick and Szilard (70) have
also found dimethylxanthine,
8-azaguanine,
mutagenic
but not
paraxanthine,
@,6-diaminopurine,
for E. coli, expressed
and
to be
as resistance
to
T-5 phage. It is to be noted that these mutations,
as well as those resulting from various treatments,
such as x-rays, ultraviolet, the mustards, etc. , are
really nonspecific changes. From studies of muta
gens in bacteria, Neurospora, and Drosophila, it
may be concluded that many genes are affected
more or less indiscriminately, and these effects
give no reason to assume that specific genes mu
tate in the proper direction
to ensure
drugs or, in the case of neoplastic
cinostatic
survival
to
cells, to car
agents.
An indirect method used to distinguish between
physiologic and genetic adaptation in bacterial
populations exposed to unfavorable environments
was first utilized by Luria and Delbrlick and is
known
as the “fluctuation test― (60). This test was
adapted to a study of the origin of resistance to
A-methopterin
in the L1@1O lymphocytic
nec
plasm (48). It was concluded from these data, in
which resistant sublines were seen to make their
appearance in the absence of the drug, along with
other collateral evidence, that mutation and selec
tion constituted the mechanism by which resist
ance arose. Increase in resistance was seen to occur
in a stepwise fashion. Stability of resistance in the
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L&w—Evolution of Drug Resistance
absence
of antagonist
would appear to strengthen
the assumption of mutation and selection as an
important mechanism.
Resistant
and dependent
variants
obtained
through the use of other antimetabolites, such as
A-ninopterin, A-denopterin,
8-azaguanine, 6-mer
captopurmne, and 6-thioguanine, appear also to
arise in discrete stepwise fashion, resembling also
the penicillin pattern of resistance. The stability
of these lines in the absence of the drug used to
select them has been mentioned previously.
Most microorganisms do show some degree of
adaptation
708
as shown by Novick and Szilard (69, 70). Guano
sine has been shown to prevent completely, in E.
coil, the mutagenic
action of a dimethyixanthine,
theophylline.
Theophyffine,
as well as caffeine,
8-azaguanine, and other compounds, increases the
mutation rate of resistance to T-S phage in this
organism. Guanosine suppresses completely the
mutagenic effects of these compounds. Thus, the
possibility must be considered that antimutagenic
agents might be used in conjunction with chemo
therapeutic agents to prevent emergence of resist
ant variants.
to most of the drugs to which they are
@. Variability
is
great
within
a
population
of
subjected, and this might be expected to occur also
in populations of neoplastic cells. Usually adapta
tion occurs in a gradual manner, but at the end of
a long process an extremely high degree of resist
ance may be attained. The degree of resistance at
tamed by neoplastic cells in man or in experimen
microorganisms following selection for drug resist
ance. Changes in resistance to other drugs, both
increased and decreased, are observed (86). These
changes may be related or unrelated to the selec
tive drug mechanisms. When unrelated, it is ex
pected that each neoplastic population may have
tal animals appears to be relatively much lower,
different
probably because the intensity with which car
cinostatic drugs can be used is seriously limited in
these species.
The emergence of a sudden adaptation, similar
to streptomycin or isoniazid resistance and de
velopment of resistance to one drug may attain
predictable changes in metabolic pathways, result
ing in increased sensitivity to other drugs, remains
to be determined for various neoplasms and van
ous drugs.
pendence
in microorganisms
(one-step
pattern),
drug sensitivities.
Whether
deliberate
de
It has been observed in our laboratory with the
has also been observed in neoplasms. Studies with
use
E. coil, strain K-1@(sexual crossing studies), have
(L1@1Oand L5178) that resistance to, or partial
revealed that resistance in a single step to high
levels of streptomycin results from mutations in
herited as changes in a single gene locus (56, 64),
in contrast to the polygenic system involved in
stepwise (penicillin-like) resistance (11, 1@).In our
laboratory the plasma cell neoplasm, 704@9, has
dependence
upon, purine antagonists
such as
6-mercaptopunine,
8-azaguanine,
and 6-thiogua
nine is invariably accompanied by a striking in
crease in sensitivity to 4-amino-PGA antagonists
been found to be extremely sensitive to an appar
ent glutamine antagonist, azaserine. Several lines,
showing a rapid development of resistance to, and
partial dependence upon, high levels of this com
pound,
have
now
been
isolated
and
are
under
study (7@). The change appears to be stable and
irreversible, but the pattern of resistance, unlike
those observed for antifolic and antipurine corn
pounds, appears to resemble a streptomycin-like
pattern.
The mutation-selection mechanism in the origin
of resistant populations of neoplastic cells would
appear to have fatalistic implications. Spontaneous
mutations
cannot
be prevented
effectively,
and
they cannot be reversed in a directive manner, at
present. Once the biochemical or physicochemical
mechanisms of drug resistance are known, how
ever, it is conceivable that resistance may be ex
ploited to the benefit of the host. There are certain
hopeful signs in this direction:
1. The development of compounds with anti
mutagenic
potentialities
seems now to be a reality,
of
(53).2
On
two
the
different
other
lymphocytic
hand,
increased
neoplasms
resistance
to
A-methopterin in Line I leukemia is accompanied
by increased sensitivity to 6-mercaptopurmne (89),
unlike the behavior of the L1@1Oacute lymphocyt
ic leukemia. Documented evidence concerning the
development of collateral sensitivity in neoplasms
is indeed scanty.
8. Many instances are known in experimental
animals, in which resistance is confined to the nec
plasm. Other tissues of the body do not attain the
same degree of resistance to drugs. The general
toxicity
mustard
elicited
for such compounds
and A-methopterin
as nitrogen
is substantially
un
altered. Thus, if resistance in specific instances
may be related to specific physicochemical changes
such as enzymic constitution and active transfer,
for example, it is argued by Danielli that the phe
nomenon of resistance may be turned to advan
tage for the host. A theoretical model for such ex
ploitation is given (14). If resistance in a neoplastic
population is the result of the formation of an
adaptive enzyme and the drug is, therefore, ren
dered ineffective by the detoxicating action on the
2 Unpublished
observations
for
the
L6178
neoplasm.
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1956 American Association for Cancer Research.
704
Cancer Research
drug, this drug could be replaced by another which
is activated by the adaptive enzyme. For ex
ample, a neoplasm which becomes resistant to ure
than (NHrCO-OEt)
or ethylphenyl carbamate
<.‘.‘)
NH-CO-OEt)
mightbeexpected
todoso
by formation of an adaptive enzyme which will
split the ester or the peptide linkage. A drug such as
phenylurethan
mustard [(ClCHsCHs)sN@(@@
NH-CO-OEt]
could be used which may be acti
vated by the adaptive enzyme. The employment
of such models depends upon characterization of
resistance in terms of specific biochemical or physi
cochemical
variables,
and much needs to be
learned in this direction. Nevertheless, it should be
recorded that such distinct possibilities do exist.
4. The problem of resistance in neoplastic cells
may be of far greater importance than that en
countered in microorganisms.
In bacterial infec
tions the rapid development
of new antibiotics
with different mechanisms of action, the possibili
ty of using massive doses of antibiotics continu
ously, and the employment of associations of drugs
are, for the most part, means of combating the
dangers of resistance. Theoretically, to prevent the
emergence of resistance or dependence in a popu
lation of neoplastic cells, especially that occurring
in discrete, stepwise fashion (L1@1Olyinphocytic
neoplasm and antifolics) , a drug should be given
continuously at high levels. The importance of us
ing drugs, showing no cross-resistance,
simultane
ously rather than in sequence hardly needs to be
stressed.
A striking
example
of genetic
synergism
is found in Bacillus megaterium (87), sensitive to
isoniazid and Na-p-aminosalicylate
(PAS). High
mutation rates of 6 X 1O@ and 1 X 10' per bac
terium per generation were obtained when each
drug was used singly. The mutation rate obtained
following simultaneous
treatment
(mutation
to
double resistance), however, was approximately
the product of the individual frequencies. Syner
gism has been observed in certain neoplasms with
the simultaneous administration of drugs known
not to show cross-resistance; for example, Amethoptenin and 8-azaguanine in L1@1Oleukemia
(49), A-methopterin
and 6-mercaptopurine
in
L5178 leukemia,2 and azaserine and 6-mercapto
purine in Sarcoma 180 (13). Synergism has also
been observed by Skipper, using several combina
tions of drugs (78, 80). Studies of simultaneous ad
ministration
of drugs in childhood leukemias are
now in progress and involve the use of such corn
binations as A-methoptenin and 6-mercaptopu
nine,1 and azasenine and 6-mercaptopunine (7). It
should be appreciated that the success of these
regimens will be determined only with adequate
numbers of patients, since many are refractory to
each of the compounds employed. Inferential evi
dence may be obtained in these trials of the nature
of the resistance in man, especially if the magni
tude of response (remission) is comparable to that
achieved in the experimental animal.
The interaction of drugs at the physiological
level may range from synergism to additive ef
fects, indifference, or even antagonism. On the
basis of present knowledge concerning media
nisms of action of antineoplastic
agents, it would
seem premature to predict what combinations of
drugs would effectively eliminate or effectively
suppress resistant forms.
Tests for cross-resistance (or dependence) and
collateral sensitivity for a number of variants and
drugs
are presently
limited
to leukemia
L1@1O. In
our laboratory, studies are now in progress to de
termine patterns of cross-resistance and collateral
sensitivity in other lymphoma series, e.g., type A
and B neoplasms (17), granulocytic leukemias,
plasmacytomas,
etc. It would be unwise to gen
eralize on the anticipated efficacy of combinations
of drugs even for a given morphologic form of nec
plasm (e.g., lymphocytic leukemia) in a given spe
cies. The observations of Szybalski and Bryson
(86) indicate the significance of bacterial type as
well as of the drug employed in affecting cross
resistance patterns.
The role played by transformation (Pneumo
coccal type) (@, 85) and of transduction
(Salmo
nella type) (97) in the development of resistance
to drugs in a neoplastic cell population is yet to be
determined. It is possible, for example, that
through autolysis of neoplastic cells transforming
materials may be released. It is of interest to note,
however, that resistance in Pneumococcus to peni
cillin or streptomycin can be conferred on sensitive
organisms by purified DNA preparations
of the
resistant cultures. In the case of penicillin resist
ance, transformation occurs stepwise, characteris
tic of the genetic pattern of development of the
resistant organisms contributing the DNA. In the
case of streptomycin
resistance, it is possible to
show a transformation phenomenon characteristic
of a single, high-level resistance (85). It was earlier
reported by Voureka (89) that resistance could be
lost in a bacterial population, as well as gained
through advent of a transforming principle. Trans
formation in the direction of sensitivity is of spe
a Personal communications
from Dr. James Holland, Roe
well Park Memorial Institute. and Dr. Emil Frei, National
Cancer Institute.
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1956 American Association for Cancer Research.
LAw—Evolution of Drug Resistance
cial interest to those interested in the chemothera
py of neoplasms. More recently it was found, how
ever, that several other preparations produced the
same results in the resistant cells (90). Thus, these
changes appear to be nonspecific and nonadaptive.
The possibility of achieving in neoplastic cell pop
ulations transformations
to sensitivity does not
seem hopeful in view of the experiences to date in
bacterial populations.
The frequency of such
genetic transfers is low, and it may be impossible
to achieve a selection or isolation mechanism nec
essary for the change from resistance to sensitivity.
Possrnz.n
Mncaar@zsaes RESPONSIBLE
FOR DRUG RESISTANCE
Relatively
little evidence
is available
concerning
biochemical mechanisms responsible for resistance
to drugs. There have been several unusual oppor
tunities to discriminate
among certain possible
mechanisms in bacterial systems. These will be
discussed briefly in relation to resistance in popu
lations of neoplastic cells where some data are
available to suggest certain mechanisms or to
exclude others.
Model systems in bacteria have been developed,
particularly
with such antimetabolites
as anti
folics, antipurines, and antipynimidines.
Although
specific mechanisms
have not as yet been un
covered, the experimental findings have given
direction to such studies which used mammalian
cells. Conceivably, the following mechanisms may
account for the selective changes brought about
through the use of drugs.
705
activities of the sensitive L1@1Olymphocytic neo
plasm and of two nonsensitive variants, 8-AG-R
(resistant to 8-azaguanine) and 8-AG-D (depend
ent on 8-azaguanine),
there were found to be no
real differences in the deaminase concentration
among these lines.4 These results are not surpris
ing, however, since the two variant lines used show
cross-resistance
and cross-dependence
to such
other compounds as 6-mercaptopunine
and @,6-di
aminopunine
(47). It is difficult
to conceive
of in
activation of these compounds by 8-azaguanine
deaminase. The possibility that different mecha
nisms exist for naturally resistant neoplasms con
trasted with those developed through selection,
however, should be investigated further.
Further,
suggestive evidence exists that de
gradative enzymes might also be of significance in
determining
the distinctive
patterns of nucleic
acid synthesis existing for different normal tissues
(4) and may indeed account for the differing re
sponses
of neoplasms
to the same drug. The dem
onstration of metabolic differences between differ
ent tissues lends optimism to the search for corn
pounds having selective action on certain neo
plastic tissues.
The possibility of organisms acquiring the abili
ty to convert
folic
antagonists
to metabolically
active compounds was first suggested by Hutchi
son and Burchenal (87) in S. fuecalis made resist
ant to A-methopterin,
and by Kidder et at. (48) for
a strain of Tetrahymena found able to utilize PGA
antagonists
for growth.
Resistant
S. faecalis
grew
well on PGA antagonists except on those with a
1. Development ofan enzyme with increased abili
methyl group in the 9-position, suggesting deami
ty to convert or inactivate the drug.—The guanine an
nation of these compounds by the organism and
tagonist, 8-azaguanine, has been shown to be an release of a source of PGA then used as a growth
factor.
inhibitor of neoplastic
growths in mice, rabbits,
The occurrence of growth-promoting contami
and rats, and of the human brain neoplasm, glio
blastoma multiforme, in tissue culture (see @1). nants in the folic acid antagonists, however, pre
Carcinomas and lymphocytic neoplasms appear to sumably accounted for the observed growth in
these two organisms (16, 88, 68). As shown later
be the most sensitive forms. Among lymphocytic
for S. faecalis (68), CF was formed (apparently
neoplasms tested in our laboratory, approximately
from PGA) when commercially available aminop
50 per cent are sensitive
to this analog.
Hirschberg
term was used but not when purified arninopterin
et at. (31) made the interesting observation that
was used. There appears to be no definitive evi
those neoplasms responding to 8-azaguanine have
dence at present to indicate that mammalian cells
a relatively low level of the enzyme guanase, which
or microorganisms can acquire the ability to con
converts guanine to xanthine and also deaminates
vert PGA antagonists
to metabolically
active
the 8-azaguanine analog to inactive 8-azaxanthine.
The nonsensitive neoplasms studied were found, in compounds.
This mechanism of conversion of folic antago
contrast, to have high guanase activity. Glioblas
nists by deamination
to PGA or PGA derivatives,
toma multiforme tissue was devoid of measurable
which are then utilized for growth, has been con
guanase activity,
whereas normal brain tissue
showed extremely high levels of activity (8@). sidered and rejected as it relates to resistance or
Though limited, these are interesting and impor
4 Personal
communication,
Dr. Alfred Gellhorn,
College of
tant observations and should be extended. In a Physicians & Surgeons, and unpublished observations, this
preliminary
survey
of the comparative
guanase
laboratory.
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1956 American Association for Cancer Research.
Cancer Research
706
dependence
in leukemic
not stimulate
cells.
PGA
and
CF do
growth of A-methoptenin-resistant
L1@lOcells, at least under the conditions of paren
teral injection,
and CF is shown to prevent the
growth-promoting
influence
an A-methoptenin-dependent
of A-methoptenin
in
line of L1@l0 (AM
D) (47). Conceivably, deamination or deamina
tion plus demethylation
may account for the de
velopment of other resistant populations of nec
plastic
cells, however.
There
is no reason
for as
suming a priori the existence of identical media
nisms for all folic antagonist-resistant
populations.
Recently, the possibility has received some at
tention that yeast, bacteria, and mammalian cells,
under certain conditions, are able to inactivate the
4-amino analog of PGA (aminoptenin), rendering
such cells insensitive to the inhibiting compound.
This possibility requires further consideration and
investigation as it relates to the type of resistance
known to be stable, irreversible, and heritable in
contrast to a strict physiologic adaptation
of the
population of cells.
Recovery from the inhibitory effects of aminop
term in the yeast, Candida tropicali@, and in
certain bacteria which do not show a nutritional
requirement
for PGA is associated with the ap
pearance of free ptenidines and accumulation of a
diazotizable amino-compound.
The investigations
by Webb (91, 9@) included the nonexacting bac
tenia Aerobacter aerogenes and A. cloacac and
Escheriehia cdi.
The
diazotizable
amino-corn
pound observed in these studies probably is the
p-aminobenzoylglutamic
acid residue resulting
from cleavage of the aminoptenin molecule at the
linkage between C-9 and N-b. This compound
apparently does not undergo further degradation.
Two main components of the free pteridines have
been identified.
In C. tropicalis and the bacteria studied, the
initial inhibition is frequently followed by partial
or complete recovery, and growth in the recovered
cultures (second logarithmic phase) is again sus
ceptible
to inhibition
by aminoptenin.
Thus, there
is no distinction made between susceptible and
nonsusceptible
organisms and no definitive evi
dence that a selected resistant population differs
from the sensitive population
in being able to
cleave the folic antagonist. It would be of interest
to know, for example, whether a naturally resist
ant organism such as Sacch. cerevisiae has the abili
ty to cleave aminoptenin and whether resistant
populations
of bacteria
developed
through
muta
tion and selection have acquired this ability.
In attempting to extend these findings to mam
malian cells we have employed the sensitive
(L1@1O)
and
A-methoptenin-dependent
(L1@1O/
AM-D) and sensitive (L4946) and A-methopterin
resistant (L4946/AM-R)
lymphocytic
neoplasms
in ascitic forms following the Bratton-Marshall
method. No differences have been detected in the
formation of a diazotizable amine among these
groups.6
Among bacteria, specifically A. aerogenes, E.
ccli, and L. easel, PGA in cultures undergoes
cleavage in the same manner as its inhibitory ana
log, aminopterin. In contrast, chicken or rat liver
preparations
are able to cleave PGA but not the
4-amino-antagonist,
aminoptenin (76).
Evidence for inactivation of aminopterin by cer
tammammalian
(andavian)cellshasbeenpre
sented by Jacobson (4@). These interpretations
are
based strictly on the influence of folic analogs, par
ticulanly arninopterin, on mitosis of cells in short
term tissue culture. An arrest in metaphase was oh
served in long-term exposures to aminoptenin in
certain tissues such as intestinal mucous mem
brane, normal bone marrow cells of mouse and
human origin, and normal mouse lymph nodes,
whereas other tissues such as leukemic cells of the
mouse and of man, for example, invariably over
came the initial inhibition in a fashion similar to
Candida and the bacterial systems observed by
Webb. Though it appears that aminoptenin over
comes the initial inhibition as a result of “macti
vation,―there could be found no evidence that the
inhibitor had been broken down or that extensive
chemical changes in the molecular structure had
occurred. “Inactivation―
in these experiments was
based solely on the influence of the recovered
supernatant fluid on hanging drop cultures of
osteoblasts or fibroblasts, and in certain instances
following inoculation into mice. The detection of
activity
or “inactivity― by
more
conventional
methods with microbial assays has not been re
ported. It is of interest to note that the inhibitory
capacity
of the
“inactive―compound
could
be
restored by mild acid hydrolysis.
A most puzzling conclusion drawn from this
work is that no conversion from PGA to CF-like
compounds occurred and that the inhibitor could
not be assumed to block such a conversion. This is
in striking contrast to what is observed in leukemic
cells incubated with glucose, ascorbate, and for-'
mate or senine (40, 68).
These results of Jacobson suggest a possible
“binding―
mechanism for the development, at
least temporarily,
of nonsusceptibility
to a folic
analog. It is reported that leukemic tissues, in con
trast to their normal counterparts,
such as lymph
oid tissue and bone marrow, possess the ability to
a Unpublished
observations
of Dr. Sheldon Resume,
laboratory.
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1956 American Association for Cancer Research.
this
LAw—Evolution of Drug Re@tance
inactivate folic analogs (4@). A survey of lympho
cytic neoplasms in their response to folic analogs
will reveal a wide range of response from natural
resistance to striking sensitivity. To suggest that
all leukemic tissues differ from their normal coun
terparts in this respect is indeed premature.
Although cleavage or “inactivation―may be the
specific mechanisms for resistance in individual
cases, more quantitative
data are necessary to es
tablish this. Woolley, for example, found Endo
ingly heat-labile enzyme, which catalyzes panto
thenate synthesis from @-alanineand pantoate.
Other cases of resistance of strict specificity to
similar agents are known.
In contrast to the specificity of resistance in E.
coli, described above, where the specificity can
hibits the growth of E. cdi by competing both with
p-aminobenzoic acid (PAB) and p-hydroxybenzoic
acid (FOB). Resistant cells, apparently the result
of mutation and selection, were obtained to each
of the two inhibitions, which showed no cross
resistance, and these resistant types were distinct
from E. ccli, showing resistance to the sulfona
mides of PAB and POB. The nature of the experi
ments excluded such mechanisms as increased pro
even be detected among analogs substituted at the
same position in the molecule, is the type of re
sistance encountered among the lymphomas. The
antifolic-resistant
(and dependent)
lymphocytic
neoplasms, L1@1O, L4946, and AK-4, exhibit a
wide range of cross-resistance (and cross-depend
ence) to all 4-amino substituted PGA and pteroic
acid compounds. L1@1Ovariant lines, showing re
sistance to, or dependence on, several purine an
tagonists,
8-azaguaine,
6-mercaptopurmne,
and
6-thioguanine, likewise show a wide range of cross
resistance (or cross-dependence).
Antifolic van
ants, however, remain sensitive to purine analogs,
and punine-antagonist variants remain sensitive to
antifolic compounds. This lack of specificity most
probably indicates only quantitative alterations in
the substrate-enzyme-product
system and greatly
complicates the process of discriminating among
the various mechanisms responsible for resistance.
There is some evidence, although of a prelimi
nary nature, that quantitative alterations, such as
increased biosynthesis and concentration
of the
metabolite antagonizing the inhibitor, or increased
concentration
of an enzyme utilizing the metabo
lite, may account for resistance, in leukemic cells
and in bacteria, to PGA antagonists.
Folic acid (PGA) is known to play an important
role in the metabolism of single carbon units (“for
mate―). Introduction of a one-carbon unit is
known to occur in the and 8 carbons of the punine
bases, the 5-methyl of thymine, the fl-carbon of
serine, the s-carbon of histidine, the methyl carbon
of methionine. Before there was definitive evi
dence for the involvement of the biologically ac
tive form of PGA, CF (foli.nic acid, leucovorin,
citrovorum factor), in formate metabolism, it was
found by Nichol and Welch (66) that 4-amino
duction
PGA antagonists blocked effectively the enzy
myces verno@lig,resistant to pynithiamine, able to
cleave the drug between the pynimidine and pyni
dine rings; however, the amount of cleavage was
insufficient to account for the degree of resistance
existing (94).
A corollary to increased conversion, destruction,
or inactivation of an inhibitor, as a possible change
in resistant mutants, is the possibility of decreased
conversion from an inactive to an active inhibitor.
Danielli
has suggested,
in a preliminary
report
(14),that resistanceina Walkerrat sarcomato an
N-acetyl nitrogen mustard derivative is the result
of elimination of a peptidase thought to activate
the compound. Elimination of those tumor cells in
the population which have a high content of the
activating peptidase would result in a new popu
lation of cells resistant to the compound. It is un
fortunate that more definitive, documented data
are not yet available from this interesting study.
@. Quantitoiive
alierations
in
the
,ubstroie-enzyme
product system.—In certain instances resistance
observed with different competitors of the same
metabolite is strikingly specific. Davis and Maas
(15)
@
707
found
that
or more
p-nitrobenzoic
efficient
acid
utilization
(PNB)
in
of metabc
lites, or even of the emergence of new metabolic
pathways which enable the metabolites to be dis
pensed with. Though stringent proof has not yet
been given, it would appear that resistance here is
due to a qualitative change in the enzyme using
PAB or POB, resulting in a decreased affinity for
the drug compared with the metabolite. These re
salts suggest that such a change in affinity can re
sult from gene mutations. These same investiga
tors (61) have shown also in E. coli, by a direct
demonstration
with extracted enzymes, that a
gene mutation results in the production of a strik
matic formation of CF from PGA.
The metabolic alteration of PGA (or of formyl
PGA, tetrahydro-PGA, anhydroleucovonin A),
when incubated in various tissue preparations, can
be treated as a simple reaction (40, 65, 68), and the
CF (or CF-like compounds) formed can be mess
ured in microbial systems, provided certain re
quirements are met, such as the presence of one
carbon donors and reducing conditions. The effec
tiveness of the 4-amino PGA compounds in block
ing the formation of CF can be shown in such a
system.
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Cancer Research
•708
Comparisons
of the sensitive
and resistant
forms
the incubation experiments inhibited conversion of
of bacterial cells, S. faecalis, and of leukemic cells,
PGA to CF, corresponds closely to the amount
L1@1O and
found in the plasma of man and in the tissues of
mice, after administration
(9, @0).
Attempts to prepare cell-free extracts of leu
kemic cells which give suitable activity for corn
parison with intact cells have been unsuccessful.
However, the strikingly higher activity of ascitic
cells, in contrast to lymphomatous tissue, in con
verting PGA to CF lends encouragement to the
possibility of a direct approach in leukemic cells
Line
I, for the
enzymatic
formation
of
CF have been made (67). The resistant strain of
S.faecalis has developed a greatly increased capac
ity to form CF (100-fold or more), and it is sug
gested that this characteristic is responsible for, or
contributes greatly to, the ability of these cells to
survive high levels of 4-amino-PGA antagonists.
For example, an amount of 4-amino-PGA which
completely inhibits the formation of CF by sensi
tive S. faecalis is without influence on resistant
cells.
This increased efficiency •
in the conversion of
to problems
Hutchison
of permeability
and Burchenal
addition to the enzymatic
and enzyme affinity.
(39) have studied,i@r@
conversion of PGA to
PGA to CF may be the result either of an increase
CF, the liberation of conjugated CF (conjugase
in the concentration of the inhibited enzyme or of
differences in the enzyme having to do with rela
tive affinities for metabolite and antagonist.
activity)
In contrast, the amount of CF formed from
PGA by tissue suspensions of resistant (I/A) and
dependent (L1@10/AM-D) leukemias was some
what less than that formed by the corresponding
sensitive lines. On the other hand, a consistently
greater inhibition of the formation of CF by PGA
antagonists was noted in the sensitive compared
with resistant leukemic cells, and this effect was
consistent
with all 4-arnino-PGA
analogs
studied.
The 10-methyl analog of PGA was ineffective as
an inhibitor, and this has been confirmed in this
laboratory by in vivo studies on growth inhibition
of leukemic cells.
Cell-free “sonicates―of A-methoptenin-resist
ant &faeca& cells, in contrast to intact cells, were
found to be highly sensitive to folic analogs (65).
An amount of aminoptenin, without effect on in
tact resistant cells, was found to inhibit completely
the conversion of PGA to CF in the cell-free sys
tern. Thus, it appears likely that PGA antagonists
do not reach the susceptible enzyme system in in
tact cells to interfere with its function. Recent
work by Anton and Nichol (8) using resting cell
suspensions of sensitive and resistant S. faecali.a
has shown a greater retention of both PGA and
aminoptenin by resistant cells, indicating probably
a “binding―
phenomenon. Interestingly, the abili
ty of a series of variants, differing in degrees of re
sistance to folic analogs, to convert PGA to CF
increased exponentially in a manner related to the
degree of resistance.
It is possible that this limit of accessibility of a
susceptible enzyme system to folic antagonists
may be responsible for the resistance of leukemic
cells, since their sensitivity to antagonists
was
found to be reduced, although to a much smaller
degree. Nonetheless, it is of interest to note that
the concentration of PGA antagonist, which in
I/A
in incubated
Line I (sensitive)
(A-methopterin-i@esistant)
leukemic
and Line
cells oh
tamed from splenic inifitrations, and the effect of
A-methoptenin on these reactions. Sensitive cells
formed somewhat more CF from PGA, but resist
ant cells liberated more free CF from the conju
gate, especially early in the incubation period. The
folic analog was found to be more effective both on
converting PGA to CF and on the conjugase ac
tivity of sensitive than of resistant leukemic cells
(ten-fold differences).
It remains to be determined whether differences
of such degree determine differences between sen
sitivity and resistance to A-methopterin
in leuke
mic cells.
8. Developmentof alternativemetabolicpathways
to inhibited product@.—Someevidence is found to
support this hypothesis of the development of al
ternative
pathways
to nucleic acid synthesis.
These data refer particularly to differences be
tween resistant (to folic antagonists) and sensitive
bacteria and to differences between PGA-antago
nist-resistant
and purmne-antagonist-resistant
lines
of lymphocytic neoplasms.
S. faecalis/A (resistant to A-methopterin)
is oh
served to have a lowered PGA requirement
for
growth compared with sensitive cells in a purine
pynimidine medium (89). This alteration in PGA
response appears related to a decreased de novo
pathway for purine biosynthesis since growth of
S. faecalis/A is more than doubled by addition of
individual
purines to a purine-pyrirnidine-free
medium, whereas no growth stimulation was oh
served under similar conditions with sensitive S.
faecalis. As expected, S. faecalis/A, as well as Line
I/A leukemic cells, were found to have developed
an increased sensitivity to the purmne antagonist,
6-mercaptopurine.
Iii the case of the leukemic
cells there is, however, no definitive evidence of a
change in punine biosynthesis. This observation
may only be a reflection of a change in sensitivity
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1956 American Association for Cancer Research.
LAW—EVOZU&m
correlated with the development of A-methopterin
resistance. A-methopterin-resistant
and -depend
ent L1210 lines, in contrast, do not show an in
creased sensitivity to purmne antagonists.
A metabolic shift of quite a different pattern
was observed by Hakala (@8) in S. faeca1i@ made
resistant to A-methoptenin.
In this mutant the
rate of biosynthesis of thymine derivatives, but
not of purmnederivatives, is limited, indicating dif
ferences in the formation of the co-factors (CoF)
involved in thymine and purmnebiosynthesis.
In our laboratory four separate resistant or de
pendent lines of leukemia L1@10, developed
through selection by 6-mercaptopurine,
8-azagua
nine, and 6-thioguanine, and a line of the lympho
cytic neoplasm L5178 (resistant to 6-mercapto
purine) all show a striking increase in sensitivity
to 4-amino-PGA antagonists, resulting in many
cases in “cures―
if certain conditions are fulfilled
(53, 54).2 The development
of this typical
collat
eral sensitivity has not been reported for other neo
plasms resistant to purmne analogs. If these variant
lines now represent a shift in the metabolic pattern
from utilization of preformed metabolites to one
involving de novo synthesis from precursors within
the cell, then PGA antagonists should be unusually
effective. Likewise, the de novo pathway would be
less influenced by antagonists of preformed purmnes
and would account for the general cross-resistance
to these compounds.
Certain incorporation studies, as reported by
Skipper and colleagues, carried out in vivo under
optimal conditions of growth, with the use of
L1@10 leukemic lines resistant to antifolics and
antipurmnes, give the following support to the
hypothesis
of development
of alternative
path
ways to nucleic acid synthesis:
a) A-methopterin
inhibited the de novo nucleic
acid synthesis in sensitive leukemic cells more than
in resistant cells (81); whereas,
b) This antagonist more than doubled the rate
of de novo synthesis in L1@10/AM-D, A-methop
term-dependent
leukemic cells. Incorporation
of
sodium
@@14
into other normal tissues
in these two groups of tumor-bearing mice, how
ever, was inhibited by the antagonist (81);
c) Incorporation studies of @,6-diaminopurine
@-C'4
into the combined nucleic acids of sensitive,
A-methoptenin-dependent
and 8-azaguanine-de
pendent L1@10 cells in vivo showed interesting dif
ferences. Specific activities (@tc/mole C) in the
combined nucleic acids for sensitive cells was only
half that observed in the folic antagonist-depend
ent cells, while incorporation into 8-azaguanine
dependent cells was approximately
one-third that
of the sensitive and one-tenth that of the folic
of Drug
709
Resistance
antagonist-dependent
variant. There were found
to be no differences in specific activities of certain
normal tissues. Thus, these observations also sug
gest the possibility of leukemic cells differing in
sensitivity to folic and purmne antagonists, utilizing
differing metabolic pathways.
More recent incorporation studies (83) also
show that two additional resistant variants of
L1@10, 8-AG-R
(8-azaguanine-resistant)
and
6-MP-R (6-mercaptopunine-resistant),
do not uti
lize @,6-diaminopurmne as a DNA or RNA source as
well as the L1@10-sensitive cells.
Recent observations6 also point to this differ
ence in metabolic patterns, utilization of exoge
nous metabolites versus de novo synthesis from pre
cursors. Ascitic cells from L1@10 (5), AM-D,
8-AG-R, and 8-AG-D lines, incubated for two
hours at 37°C. under nitrogen, with glucose, as
corbate, and serine reveal (a) real differences in the
content of CF, (b) marked differences in the
capacity to alter PGA to CF, and (c) differences
in sensitivity of these systems to A-methoptenin.
The 8-azaguanine-dependent
(8-AG-D) and -re
sistant (8-AG-R) ascitic cells, particularly
the
former, have a strikingly higher CF content, a
more efficient capacity to alter PGA to CF (nearly
10 times
more efficient
thoptenin-dependent
sensitivity
than sensitive
and A-me
cells) and exhibit a striking
to folic
analogs,
compared
with
the
sensitive and folic antagonist-dependent
lines.
This striking sensitivity to A-methopterin
of the
8-azaguanine
variants has been observed in in
vivo studies mentioned previously.
It is clear from these latter studies that care
must be exercised in attributing
significance to
differences between leukemic and normal lymph
ocytes in CF content and to differences in serum
levels and excretion of CF and antifolic corn
pounds, as a measure of sensitivity to folic an
tagonists, or to acquisition of resistance as due to
differences in the enzymatic formation of CF. For
example, the increased CF content and the in
creased conversion of PGA to CF-like compounds
observed in the 8-azaguanine-dependent
L1@Z1O
ascitic neoplasm strongly suggest that these mech
anisms should result in resistance to PGA (CF)
antagonists, similar to increased PAB production
in certain bacteria accounting for sulfonamide re
sistance. Yet the L1@10/8-AG-D neoplasm is
strikingly sensitive to 4-amino-PGA antagonists.
Thus, in this specific case, an additional mecha
nism suggests itself, a change in the affinity of the
enzyme for the inhibitor.
Some information is available concerning resist
ance of L. casei to the purine antagonist, 6-mer
I Unpublished
work
of
Nichol
and
Law.
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Cancer Research
710
captopunine
(18). This compound
has been shown
to be an effective antileukemic agent, particularly
against lymphocytic
penimental animals
leukemias in man and in ex
(see 6@). Resistance in the
LiflO leukemia to this compound has been de
scribed (50). Though the exact mechanism of ac
tion of 6-mercaptopunine has not been defined, its
interference with certain metabolic pathways is
indicated. L. casei, resistant to this purmne analog,
differs from the sensitive strain in being able to
utilize a pathway for conversion of adenine ribo
tide to guanine ribotide, by-passing the pathway
found in sensitive bacteria utilizing derivatives of
hypoxanthine.
Presumably
6-mercaptopunine
in
terferes by blocking the conversion of hypoxan
thine derivatives to guanine ribotides (18).
This compound has been shown to inhibit
markedly de novo synthesis of nucleic acid punines
as measured by “formate―
utilization in neoplastic
tissues (79). In extensions of this work, to define
more exactly the site at which 6-MP inhibits this
synthesis, with labeled precursors, it was found
(88) that 6-MP inhibits both the utilization of for
mate and hypoxanthine but not of adenine, fitting
the picture described for 6-MP resistance in L.
to a gene mutation. The very nature of their ex
perimental
set-up allowed for the exclusion of
other possible mechanisms of resistance.
An attempt is being made by Skipper (84) to
obtain information concerning such a mechanism
in mammalian
cells with C'4-labeled PGA and
aminoptenin.
A most rigorous test for an enzyme of altered
affinity is through studies of the enzyme in cell
free extracts. Sevag and Gots (74) presented evi
dence for an altered dehydrogenase
system in re
sistant pneumococci, but it has not been shown
that alterations in such a system were related to
resistance.
Recent work by Blakley (6), Kisliuk and Saka
mi (44), and Greenberg (@) at the cell-free enzyme
level of certain individual reactions in which PGA
plays a part, with the possibility of defining the
chemical nature of coenzyrne(s) F, will most cer
tainly allow for elucidation of the precise mecha
nisms of action of PGA antagonists
and of the
mechanisms responsible for the development
of
resistance to these agents.
5. Reduction in the transport of an inhibitor to a
susceptible enzyme.—Evidence has been presented
to indicate a change in @S.
faecalis/A (resistant to
casei. However,
incorporation
of 4-aminoimida
zole-5-carboxamide
was little influenced by 6-MF,
A-methopterin),
rendering the susceptible enzyme
which is difficult to interpret if this compound is a less accessible to the PGA antagonist. The lowered
sensitivity of leukemic cells to this inhibitor may
true precursor of hypoxanthine
ribotide. No data
are available yet on the influence of 6-MP on the also be a reflection of the same mechanism.
Conceivably, such a reduction in transport may
utilization of various labeled precursors of NA
punines in neoplastic cells sensitive to the corn
be the result of reduction in permeability of the
pound in contrast to resistant cells. Several lines of cell, reduction in facilitated diffusion, or may rep
resent a “binding―
mechanism such that an “in
evidence strongly suggest a difference in the pat
tern of purine metabolism between sensitive and active― inhibitor results, similar to that described
6-MP-resistant
leukemic cells, particularly
the
by Jacobson (4@). A good deal of evidence, from
suggests
striking increase in sensitivity of resistant cells to studies with resistant microorganisms,
inhibitors of the de novo biosynthesis of nucleic
changes which prevent the drug from reaching its
acid punines. These studies would be of interest in site of action and which must play a part in the
view of the fact that L. casei resistant to 6-MP
development of resistance (see Abraham [1]).
shows cross-resistance only to 6-thioguanine and
The mechanism leading to inaccessibility of the
not to other purine antagonists, whereas in certain
enzyme system, converting PGA to CF, in S.
neoplastic cells, leukemia L1@10, resistant to faecali1@is not yet clear, although the folic analog
6-MP, cross-resistance is common to many punine
appears to be taken up by resistant cells (98). The
analogs, 8-azaguanine, 6-thioguanine,
@,6-diarnino results of Skipper, studying relative absorption in
purmne, purmne, and chloropurmne.
S. faecalis and S. faecalis/A of @-C―-labeledPGA
4. Development of changes in enzyme affinity for and arninoptenin, suggest that both compounds
the drug compared with metabolite.—Proof is not
enter the cell. A slightly higher activity of aminop
yet available for the existence of a mechanism
tenin-%-C'4 was found in resistant cells (84).
such as structural changes in an enzyme resulting
It is possible that the striking differences ob
in decreased affinity for the inhibitor compared
served in incorporation of 8-azaguanine-@-C'4 into
with its affinity for the metabolite leading to drug
L1@10-sensitive cells and 8-azaguanine-dependent
resistance. Results of Davis and Maas, discussed
cells are the result of differences in transport in the
previously, on resistance in E. cdi to analogs of two populations of cells and could be an explana
PAB and FOB, strongly suggest the existence of tion of resistance, but probably not of depend
this mechanism that most likely is directly related
ence (5).
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Ltw—Evolution
It is clear that many of the mechanisms dis
cussed here may apply to changes leading to re
sistance of bacterial or neoplastic cell populations,
but not to dependence wherein the inhibitory corn
pound is required for optimal growth. A classic ex
ample of a drug-requiring mutant has been given
by Zalokar (96). It has been recorded that in the
sulfonamide-requiring
mutant of Neurospora sul
fanilamide was not functioning as a metabolite but
was restoring growth by partially blocking PAB
metabolism.
The organism was inhibited by
methionine,
a product
of PAB metabolism.
Growth was thus restored by decreasing the rate of
methionine formation by this organism. Strepto
mycin dependence in certain bacteria is a similar
phenomenon, the mechanism of which is not under
stood. The possibility exists here, and in dependent
neoplastic cells, that inhibition of growth by a nor
mal rnetabolite is occurring. There is no informa
tion available at present, however, to establish or
even strongly suggest that such a mechanism exists
in neoplasms showing dependence.
It is conceivable that adaptive enzyme fonxna
tion (that shown to occur against a common genet
ic background) may play an important role in the
development of nonsensitivities in populations of
cells. Such possibilities must be considered as
mechanisms, especially in cases where resistance in
neoplasms appears to be of a reversible and non
hereditable nature, that is, where sensitivity of the
population is reestablished upon withdrawal of the
agent.
Little evidence has been obtained to date con
cerning the role played through possible selection
of peculiar chromosome complexes. It has been
shown (80) that resistance of the Yoshida sarcoma
to the N-oxide of nitrogen mustard is not associat
ed with the selection of a peculiar chromosomal
complex. The chrornosomal features of the sensi
tive neoplasm are left unchanged through the de
velopment of resistance. Most of the neoplasms
studied in our laboratory are known to be diploid
or near-diploid in character. Cytologic studies on
the original and derived population have not been
done as yet.
DISCUSSION AND SUMMARY
Resistance to several carcinostatic drugs has
been established in neoplastic cells. The resistant
variants in the lyxnphocytic neoplasm L1@10, de
veloped in short periods of time through the use of
antifolic and antipurine
compounds,
appear to
arise in a discrete, stepwise fashion, resembling
the penicillin pattern of microorganisms. In sever
al other lymphocytic neoplasms resistance was de
veloped but not always with ease. It is to be expect
of Drug Resistance
711
ed, however, that the ease with which the charac
ter develops will vary from one neoplasm to an
other and from one drug to another. Mutation and
selection appear to constitute the mechanism in
volved. The changes are shown to be stable, ir
reversible, and heritable, and persist in the ab
sence of the drug used in selection. The possibility
should not be ignored that the pattern of resist
ance and the mechanism involved may be quite
different in other neoplasms responding to these
same compounds.
It is indicated that another pattern of resist
ance, developed through the use of azasenine, in a
plasma cell neoplasm (704@9) of C8H mice, occurs
rapidly, probably in a single step, to a high level
of resistance, resembling in this respect the strep
tomycin pattern of microorganisms. Details of this
problem are now being worked out.
The possibility that physiologic adaptation
plays a part in the development of nonsensitivity
to drugs must be considered, though in our limited
experience it has not been encountered. It is evi
dent that these nonheritable, unstable changes in
populations of cells, especially among bacteria, are
more common than was formerly believed (see
Ravin [78]). The mechanisms underlying this form
of adaptation as distinct from the stable, irrever
sible, heritable types are undoubtedly
quite dis
similar. Therefore, at the experimental level, when
nonsensitivity
to a drug arises, it should be deter
mined to what extent the population of neoplastic
cells has responded to the adaptive stimulus and,
if the response is discontinuous, to what extent it
is due to a heritable differentiation among the cells
of that population with respect to ability to adapt.
Successful therapy depends upon this knowledge.
These distinctions can be made in experimental
neoplasms, but it appears impossible, or at least
not feasible, to do so presently in neoplasms of
man.
By analogy, resistance in leukemic children to
the agents known to be effective in the mouse is
assumed to be a result of variables within the cell
rather than in the host. There is no definitive in
formation on this issue, however, and attempts
should be made to obtain an answer.
Although
mutation
and
selection
appear
to con
stitute an important
mechanism through which
resistance arises in neoplastic cells, it is impossible
at the moment to define in genetic terms the pre
cise mechanism(s) concerned, since neoplastic cells
are somatic cells and, as such, are generally
thought not to lend themselves to genetic analy
sis. The possibility, however, of the development
of procedures analogous to sexual processes for a
direct analysis of neoplasms by conventional cross
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71@
Cancer Research
breeding studies is hardly less likely than it was for
E. coli, in which an efficient selective method for
the occurrence of genetic recombination has been
established. Thus, it may be inferred that the
hereditary mechanism in bacteria is similar to that
operating in higher organisms. Fusion of Sarcoma
180 cells in the ascitic
form of this neoplasm
has
been observed, and the likelihood of direct heredi
tary interaction at least may be suggested. Neces
sary suitable markers, which appear most promis
ing at present in lymphocytic neoplasms, are drug
resistance and histocornpatibility
genes. Thus, the
possibility of distinguishing
the process of genic
recombination
in somatic cells may be realized.
The possibility exists that in addition to these con
ventional modes of genetic variation there may
occur among leukemic cells genetic variants
brought about by agencies similar to the trans
forming and transducing factors known to play a
role in the evolution of bacterial types. In bacteria
such agents are known to produce changes in
genetic characteristics analogous to those charac
tens used as markers in leukemic cells.
More information is desirable concerning cross
resistance and collateral sensitivity in an array of
different neoplasms. The experimental situation
necessary is one containing several variant sub
lines developed through selection by several drugs.
This indeed may be most difficult and entails a
fairly large scale screening of neoplasms in their
sensitivities to drugs and the systematic develop
rnent of resistant sublines. Inferences concerning
mechanisms of resistance, the possible use of asso
ciations of drugs, and particularly the use of drugs
in sequence can be gained in this manner. It is
premature to transfer the knowledge gained con
cerning cross-resistance and collateral sensitivities
in the neoplasm L1@10, for example, even to other
lymphocytic neoplasms of the mouse.
Knowledge that resistance may arise by muta
tion, that there appears no known method for de
creasing mutation rates (although, as mentioned
previously, some progress in this direction has been
achieved), and that it is unlikely that the host is
able to alter the process of spontaneous mutation
suggests a familiar and feasible approach, the use
of combinations of agents in attempts to eliminate
mutants to one drug by means of a second drug
simultaneously present. The principle of combined
therapy is unusually successful against the L1@10
lymphocytic
neoplasm with two compounds
known to have independence of action, A-rnethop
term and 8-azaguanine,
and in L5178 with
A-rnethopterin plus 6-mercaptopurine. However,
it would appear most difficult to predict effective
combinations
without an understanding
of drug
interactions
involving cross-resistance,
collateral
sensitivity, synergism, antagonism, etc. Moreover,
as shown in bacterial studies, the bacterial species
affect, to some extent, the cross-resistance and
sensitivity patterns (86). This points up again the
danger in generalizing from results gained with a
single neoplasrn.
Hitchings
and colleagues
(88) have shown
com
binations of drugs, with suitably related mecha
nisms of action, to be synergistic, regularly and
predictably, in inhibiting the growth particularly
of L. casei and S. faecalis. The compounds used
were analogs of purine and pynimidine bases and
folic acid analogs, all involved in a major biochem
ical pathway leading to nucleic acid synthesis.
This pathway for mammalian cells may be repre
sented as follows:
PGA CF Precursors
Polynucleotide
+
Thyrnine
I
I
+
I
purines
+
X—+Y±Coenzyrnes
F
+
ic
—t-Serine
t
Methionine
Preformed Histidine
purines
and Etc.
pyrimidines
x, v, andF represent
intracellularforms.PGA
is supplied exogenously and the transformation
from X to V (leucovorin, citrovoruin factor) is
followed by modifications leading probably to sev
eral coenzymes F concerned with the incorporation
of one carbon (1C) fragments into the products
shown. The incorporation of preformed purine and
pynirnidine bases is shown as an alternative
pathway.
Our studies with lymphocytic neoplasms, and
other lymphornas, have been limited arbitrarily to
antimetabolites
related to this biochemical path
way.
The probability of success appears equally good
with other limited systems, for example, the B-6dependent system. Recently certain pyrimidine
analogs, especially 6-azauracil and 6-uracil methyl
sulfone, have been shown to be effective inhibitors
of several lymphocytic neoplasms (@4). The mech
anism of inhibition of growth in bacterial systems
of this group of compounds has been detailed by
Welch and colleagues (see Welch [93]), and cogent
reasons exist for the development of effective ura
cil and orotic acid antagonists for chernotherapeu
tic application.
It should be pointed out here that the emphasis
placed on this whole group of compounds as nude
ic acid inhibitors is not because they are unique in
this respect or that other tissue components are
not equally affected. There are, however, many
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713
L@@w—Evolutionof Drug Resistance
known reactions concerned with the biosynthesis
of the basic components of nucleic acids, and the
possibilities of elucidating the mechanisms of ac
tion of these compounds in mammalian tissues
seem especially encouraging, particularly for the
antifolic compounds and for 6-mercaptopurmne and
azaserine.
One answer to the danger of resistance in micro
organisms is the employment
of associations of
drugs. This practice has been unusually successful
in certain situations. Clearly, without knowledge of
the pattern of resistance or the mechanism of drug
action, predictability of success of combinations of
drugs is uncertain. In practice this is borne out by
observations
of a range from true synergism to
simple additive effects, indifference, or even an
tagonism of drugs. Where resistance in neoplasms
is shown to be analogous to the penicillin pat
tern of resistance in microorganisms,
that is, de
veloping in discrete, stepwise fashion, and pre
sumably the result of an influence of a polygenic
system, it may be predicted that simultaneous ad
ministration
of such drugs as antifolics and anti
purmnes (or antipynimidines)
will show potentia
tion of effects. This possibility can be determined
in experimental neoplasms with the advent of new
drugs where treatment may be initiated early and
continued at effective concentrations. The host
limitations may be so serious, however, as to pre
dude adequate tests.
In consideration of the effective use of drugs in
the treatment, for example, of acute lymphocytic
leukemia, a drug known to select for resistance in
a discrete, stepwise fashion should be used at an
effective concentration and continually. Decrease
of the concentration below the effective level will
permit the accumulation
of resistant first-step
mutants and allow the occurrence of higher levels
of resistance, and so on. Control of these neoplastic
cells is made more difficult. In view of the toxic
effects of commonly used antileukemic compounds
on rapidly regenerating epithelium of oral surfaces
and intestine and on rapidly dividing cells of the
bone marrow, it is questionable that effective
levels of such drugs are ever attained. Further, the
difficulties in maintaining adequate levels of the
drugs commonly used are apparent. It is unlikely
at present that a regimen of drug therapy, de
signed for effective elimination of resistant neo
plastic cells, can be attained in chronic neoplastic
conditions.
The use of combinations of drugs, one of which
might be expected to select neoplastic cells exhib
iting a streptomycin pattern of resistance, occur
ring in some cells to a high level in a single step,
clearly would not be too efficacious.
Resistance in S. faecalis to A-methoptenin,
on
the basis of present information, appears to be the
result of a mechanism different from that deter
mining A-methopterin resistance in leukemic cells.
The definitive experiment of a comparison of
“sonicates―of resistant and sensitive leukemic
cells with intact cells in their abilities to convert
PGA to CF, however, has not been feasible. Re
sistance to sulfonamides within a single species of
bacteria, Staphylococcus aureus, is known to be
associated with more than one type of change re
lated, apparently, to changes in enzyme patterns
(1). Also, a number
of growth
and enzymatic
dif
ferences have been noted in S. faecalis among
variant lines resistant to the same antimetabolite
(36).
The
relationship
of such
difference
to actual
mechanisms of resistance is not yet clear. It re
mains to be determined whether resistance, for ex
ample, in acute lymphocytic neoplasms of the
mouse, results in each case from the same rnecha
nism. This information is necessary if one is pro
pared to transfer knowledge gained in studies of
experimental neoplasms to analogous situations in
man.
In summary, our current knowledge of leuke
mia (neoplasms) at the cellular level leads to the
consideration
of leukemic (neoplastic) cells as a
population
free to vary genetically within the
limits set by point mutation,
recombination,
changes in ploidy, and possibly by transformation
and transduction. A comprehensive understanding
of leukemia such as to enable its control therapeu
tically requires that all these parameters be de
fined and their influences assessed quantitatively
in terms of therapeutic response. In contrast to in
vitro bacterial systems, complex regulatory mecha
nisms of the host are imposed upon this population
of leukemic cells, probably leading to variations in
the population which are little understood at pros
ent. It would appear that primary attention, how
ever, should be directed toward the genetic status
of the leukemic cell both as regards mechanism of
drug resistance and response to therapy. A con
comitant problem is the exploitation of the host's
limitations in any attempt to control leukemic
cells by therapeutic measures.
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Differences between Cancers in Terms of Evolution of Drug
Resistance
L. W. Law
Cancer Res 1956;16:698-716.
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