[CANCER RESEARCH 51. 1855-1859, April 1. 1991]
Decreasing Sensitivity to Cytotoxic Agents Parallels Increasing Tumorigenicity in
Human Fibroblasts1
Anne R. Kinsella2 and M. Sally Harán
Paterson Institute for Cancer Research, Christie Hospital and Holt Radium Institute, H'ilmsloK Road, Manchester, M20 9BX United Kingdom
ABSTRACT
Human embryo fibroblasts of common genetic origin but exhibiting a
range of phenotypes from normal to aggressively tumorigenic have been
used to study resistance to the cytotoxic drugs methotrexate and ¿V(phosphonacetyl)-i.-aspartate. Measurement of the intrinsic sensitivities
of these cells to the two drugs in standard survival assays, in normal fetal
bovine serum, showed increasing resistance to parallel increasing tumor¡genicity.Tumor cells were totally resistant to 10 HIM¿V-(phosphonacetyl)-L-aspartate whereas the 50% lethal dose for methotrexate for the
tumor cells was 500 IIM compared with 50 IIM for the normal diploid
parent cell line. The difference in resistance between the immortal and
tumorigenic cell lines was eliminated for both methotrexate and V(phosphonacetyl)-i.-aspartate, when the experiments were repeated in
the presence of dialyzed fetal bovine serum, but could be restored by the
addition of either hypoxanthine (100 MM) or uridine (10 MM)-This
suggested an important role for the salvage pathways of purine and
p\ rimirimi- biosynthesis in the increased resistance of the more tumori
genic cell lines. The implications of these data in relation to cancer
chemotherapy will be discussed.
INTRODUCTION
Resistance to cytotoxic drugs is generally accepted to be a
more common property of tumor cells than of normal cells.
This has been attributed in part to the ability of tumor cells to
more readily amplify their target genes than normal cells (Ref.
1 and references therein) and to progression-linked changes in
the pathways of purine and pyrimidine biosynthesis (2).
The availability of a series of human fibroblast cell lines of
common genetic origin that exhibit normal, immortalized, and
tumor phenotypes (3, 4), provides the ideal system in which to
address the following questions. Do tumor cells really have the
potential to be more resistant to cytotoxic drugs than normal
cells, and if so can this be correlated with the presence and
expression of a specific oncogene, general chromosomal insta
bility which might be a preliminary to gene amplificai ion (5), a
transformation-linked imbalance in the purine and pyrimidine
salvage pathways, difference in drug uptake, or some as yet
undefined property of the tumor phenotype?
To test these possibilities the human fibroblast cell lines were
exposed to MTX,' an inhibitor of purine biosynthesis used in
MATERIALS AND METHODS
Cell Lines. The cell lines chosen for study (Table 1) were the "normal"
KMS-6 human embryo fibroblast cell line, its 60Co-immortalized deriv
ative KMST-6 (3), the KN-NM cell lines established from the fibrosarcomas that resulted from activated N-ras infection of the immortalized
KMST-6 cell line (4), the SUSM-1 immortalized human fetal liver cell
line (3), and its N-ras-infected derivative (4). Normal diploid human
skin fibroblasts (FF,) and an activated N-ros-infected subclone were
used as controls. All cell lines were routinely maintained in Dulbecco's
modified Eagle's medium (GIBCO, Paisley, Scotland) supplemented
with nondialyzed fetal bovine serum (GIBCO; batch 980S). The same
serum batch was used for the entire series of experiments.
Measurement of Intrinsic Drug Sensitivities. To assay for drug resist
ance (sensitivity) 500 test cells were plated onto a feeder layer of 5 x
IO4irradiated (5000 rads) EJ human bladder carcinoma cells (5 plates/
point). The feeder cell line was really necessary only for the successful
cloning of the normal fibroblast cell lines but was included for all cell
lines to eliminate any complications that might arise from feeder cell
metabolites with regard to the supply of "salvage pathway" substrates.
MTX (Lederle, Pearl River, NY) and PALA (Drug Synthesis and
Chemistry Branch, NIH, Bethesda, MD) were added 4 h later and the
plates medium changed once a week, i.e.. twice during an incubation
period of 3 weeks. After 3 weeks resistant clones were isolated and
maintained under selection for slot blot analysis (11). The remaining
plates were fixed and stained. Identical experiments were performed
using dialyzed fetal bovine serum, and where appropriate hypoxanthine
(100 MM)and uridine (10 MM)and thymidine (50 MM)were added; all
were purchased from Sigma Chemical Co., St. Louis, Mo. All experi
ments were performed at least three times. All the illustrative data to
be presented under "Results" will be of representative experiments.
Experiments done within the same time frame were extremely repro
ducible, but generally the cells became more resistant with increasing
time in culture, although the differential resistance was always
maintained.
RESULTS
The chromosomally and phenotypically normal FF, cell line
and the "normal" KMS embryonic fibroblast cell line were
equally sensitive to MTX (Fig. 1). The chromosomally abnor
mal, immortalized derivative of KMS-6, KMST-6, showed in
creased resistance, which was increased still further in the
activated N-ras-infected KMST cell pool (Fig. 1), to a level
equal to that observed for the KN-NM tumor cell line derived
from a fibrosarcoma produced on injection of the KMSTthe clinic, resistance to which in vitro and in vivo can be due to
activated N-ras-infected cell pool into nude mice. The 50%
amplification of the gene for dihydrofolate reducÃ-ase(6, 7). The
lethal dose for the tumor KN-NM cells (Fig. 1) was 10 times
same cells were also exposed to PALA, a specific inhibitor of
that for the normal KMS cells.
de novo pyrimidine biosynthesis, in vitro resistance to which is
The differences in sensitivity were even more marked when
due almost exclusively to amplification of the CAD gene (8the same cell lines were tested for resistance to PALA, with the
10). For both drugs the intrinsic sensitivities of the different
KN-NM tumor cell line exhibiting absolute resistance to PALA
cell lines were compared with the phenotype and the resistant
concentrations of 10 mivi(Fig. 2). The resistance was unaffected
clones were tested for gene amplification.
by retroviral infection per se as seen for KMST PALA resistance
(Fig. 3), but in the case of the two normal fibroblast cell lines
Received 8/2/90; accepted 1/24/91.
' This work was funded by the Cancer Research Campaign.
KMS and FF,, resistance was increased by infection with the
2 Present address: Department of Surgery, University of Liverpool. Liverpool,
activated N-ras-cDNA (Fig. 2, inset). N-ras infection of the
L69 3BX United Kingdom.
SUSM-1 cell line, although it failed to induce tumorigenicity,
3The abbreviations used are: MTX, methotrexate; PALA, A'-(phosphonacetyl)L-aspartate.
resulted in increased resistance to MTX (Fig. 4). Similarly, all
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DRUG RESISTANCE IN HUMAN CELLS
Table 1 Growth characteristics
and phenotypic
properties
of the cell lines used
strainFF,FF,
Cell
growth0000±
in
nudemice00000+00
46.XYActivated
foreskin
FF,Human N-ras infection of
46,XXActivated
fetus
N-raiKMST-6KN-NMSUSM-1SUSM-N-rosOriginHuman
KMS-6*°Co-ray
N-rai-infection of
KMS-6Fibrosarcoma;
treatment
(0.2%)+±
KMSTHuman activated N-ras infection of
liverActivated
fetal
(0.06%)+Tumors
N-raj infection of SUSM-1MorphologyN"TNNrTTTLifespanFiniteFiniteFiniteFiniteImmortalImmortalImmortalImmortalAgar
N-raÃ-KMS-6KMS-6
" N, normal morphology;
T, transformed
morphology.
100j
100
10
1
0.1
Iff"
10"
1Ö6
IO'5
104M
IO M
[MTX]
Fig. 1. Intrinsic MTX sensitivities of the different cell strains in nondialyzed
fetal bovine serum. O, FF, normal fibroblasts; A. KMS normal fibroblasts; •
KMST immortalized fibroblasts; A, activated N-ros-infected KMST cell pool; •,
KN-NM tumor cells. Representative data from a single experiment (5 plates/
point).
anchorage-independent,
nontumorigenic clones derived from
activated N-ras infection of the KMST cell line exhibited the
same resistance to PALA as the tumorigenic KN-NM cell line
(data not shown).
Unfortunately, very few of the MTX- and PALA-resistant
clones isolated have been analyzed for amplification of their
target dihydrofolate reducÃ-ase(MTXR) or CAD (PALA") genes.
This was due to their lack of stable resistance (particularly in
the case of PALA" clones) to the drug concentration in which
they were originally isolated. Consistent with this unstable
resistance, none of the clones analyzed exhibited gene
amplification.
When the experiments to measure the intrinsic sensitivities
of the cell lines to MTX and PALA were repeated under
conditions in which dialyzed fetal bovine serum was substituted
for nondialyzed fetal bovine serum the sensitivities of the cell
lines to both drugs increased and the differential resistance of
the KMST and KN-NM cell lines was almost completely elim
inated (Figs. 5 and 6). However, the resistance of the KN-NM
cell line could be restored by supplementing the medium con
taining dialyzed serum with uridine (10 AIM)(Fig. 7). In the case
of MTX, the resistance of the KN-NM cell line could be
restored to approximately 80% of that seen in nondialyzed
serum by the addition of 100 /UM hypoxanthine (data not
shown). The presence of both thymidine (50 ^M) and hypoxan
thine ( 100 ßM)
made no difference to the survival seen following
Fig. 2. Intrinsic PALA sensitivities of the different cell strains in nondialyzed
fetal bovine serum. A, KMS normal fibroblasts; •KMST immortalized fibro
blasts; A. activated N-raj-infecled KMST cell pool; •,KN-NM tumor cells. Inset,
effect of activated N-ros infection of normal cells on intrinsic PALA sensitivity;
•.FF, normal fibroblasts; O, N-ras-infected FF, normal fibroblasts; A, KMS
normal fibroblasts; A, N-riu-infected KMS normal fibroblasts. Representative
data from a single experiment (5 plates/point).
100
10
10'
10"
10s
1Ö4
10 M
[PALAI
Fig. 3. Investigation of influence of retroviral infection per se on intrinsic
PALA sensitivity in nondialyzed fetal bovine serum. O, KMST immortalized
fibroblasts infected with pZIPneo vector alone; A, KMST immortalized fibro
blasts; X, activated N-ras-infected KMST cell pool. Representative data from a
single experiment (5 plates/point).
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DRUG RESISTANCE IN HUMAN CELLS
ity, as assessed by resistance to PALA and MTX, has recently
been studied in mouse fibrosarcoma (12) and rat liver cell lines
(5) exhibiting different degrees of tumorigenicity. In both stud
ies a striking parallel was observed between the acquisition of
drug resistance due to gene amplification and increasing tu
morigenicity. Although gene amplification need not necessarily
be distinct from "salvage pathway" effects which might facilitate
100 «ii-
the rescue of cells for long enough to facilitate gene amplifica
tion, it is not the mechanism operating in the present system.
100-«—'
0.1
10°
10°
10
[MTXl
Fig. 4. Investigation of the influence of activated N-ras infection on the intrinsic
MTX sensitivity of the immortalized SUSM-1 cell line. O, SUSM-1 immortalized
fibroblast cell line; A, activated N-ras-infected
immortalized
SUSM-1 cell pool.
Representative
data from a single experiment (5 plates/point).
10
100- (
1-
0.1
10 M
10 M
10"4M
10 M
10"2M
IPALAl
Fig. 6. Comparison
of the intrinsic PALA sensitivities of the immortalized
and tumor cell lines in dialyzed versus nondialyzed fetal bovine serum. A, KMST
immortalized
fibroblasts in dialyzed serum; • KMST immortalized
fibroblasts
in nondialyzed serum; D, KN-NM tumor cells in dialyzed serum; A, KN-NM
tumor cells in nondialyzed serum. Representative
data from a single experiment
(5 plates/point).
10
10°
10
10 M
[MTXl
Fig. 5. Comparison
of the intrinsic MTX sensitivities of the different cell
strains in dialyzed versus nondialyzed
fetal bovine serum: •, KMS normal
fibroblasts in dialyzed serum; •.KMS T immortalized
fibroblasts in dialyzed
serum; D, KMST immortalized
fibroblasts in nondialyzed serum; A, KN-NM
tumor cells in dialyzed serum; A, KN-NM tumor cells in nondialyzed serum.
Representative
data from a single experiment (5 plates/point).
10-
the addition of hypoxanthine alone. Thymidine alone (50 MM)
failed to restore the high level of PALA resistance of the KNNM cell line (Fig. 7) suggesting salvage pathway specificity.
Also, although the isolated MTXR and PALAR clones selected
in dialyzed serum were stably resistant to the drug concentration
in which they were originally isolated they again did not exhibit
amplification of their target genes. The increased resistance did
not correlate with an increase in growth rate (data not shown).
0.1
10 M
DISCUSSION
10 M
10 M
[PALAI
MTX and PALA are powerful inhibitors of de novo purine
and pyrimidine biosynthesis. Resistance to both drugs is com
monly due to amplification of the corresponding target gene.
The relationship between gene amplification and tumorigenic-
Fig. 7. Influence of uridine and thymidine on the intrinsic PALA sensitivity of
the KN-NM tumor cells in dialyzed serum. •,KN-NM tumor cells in nondialyzed
serum; O, KN-NM tumor cells in dialyzed serum; A, KN-NM tumor cells in
dialyzed serum plus 10 ^M uridine; A, KN-NM tumor cells in dialyzed serum
plus SO I¿M
thymidine. Representative data from a single experiment (5 plates/
point).
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DRUG RESISTANCE IN HUMAN CELLS
This is not surprising for the normal cells (1) but perhaps is
surprising for the highly chromosomally abnormal immortal
and tumorigenic cell lines (5). However, the increased sensitivity
of all the cell lines to both MTX and PALA when the experi
ments were performed in dialyzed fetal bovine serum suggests
an important role for the "salvage pathways" of purine and
pyrimidine biosynthesis in their original resistance. This is
confirmed by the restoration of the resistance of the K.N-NM
cell line to PALA to the levels observed in nondialyzed serum
by the addition of uridine (Fig. 7) and restoration of the MTX
resistance by hypoxanthine. Interestingly, thymidine alone had
no influence on MTX toxicity in dialyzed serum in contrast to
some clinical reports (12) and this observation coupled with
that for hypoxanthine and thymidine together might suggest
that excessive thymidine in the serum is masking the effect.
The elimination of the difference in resistance to MTX and
PALA between the immortal KMST-6 cell line and KN-NM
tumor cell line in dialyzed serum also suggests that the differ
ences in resistance observed (Figs. 1 and 2) are due to differences
in "salvage pathway" activity between the cell lines.
These data therefore, although preliminary, support the con
cept of a progression-linked change in the key metabolic path
ways of purine and pyrimidine synthesis (2). Not surprisingly,
inhibitors of de novo pyrimidine synthesis, such as PALA,
produce a reduction in the intracellular pyrimidine ribonucleoside (UTP and CTP) and deoxyribonucleoside (dCTP) triphosphate pools (14-17). Moreover, the antiproliferative, cytotoxic,
and antitumour effects of PALA can in large part be reversed
by the addition of exogenous uridine (18-22) and partially
reversed by exogenous deoxycytidine (23). What all these ex
ogenous agents have in common is that they are either sub
strates of "salvage pathway" enzymes or components of the de
novo pathway distal to the inhibitory block.
Interestingly PALA in preclinical trials showed a unique
spectrum of activity, being cytotoxic to several experimental
tumors (Lewis lung and B16 melanoma) but inactive against
several more rapidly spreading murine leukemias (24, 25).
However, in clinical trials PALA failed to show any significant
effect against a wide spectrum of tumor types in humans (2633). One explanation of this might be that human tumor cells
are able to use circulating uridine in the synthesis of pyrimidines, i.e., use their salvage pathways more efficiently than
mouse cells. The data from the present study strongly support
this interpretation. Certainly, Dentón et al. (34) demonstrated
a concurrent increase in the activities of the key enzymes of
both the de novo and salvage pathways of pyrimidine synthesis
in human colon tumors, while in studies of rat hepatomas (2),
the activities of the salvage enzymes were higher than the ratelimiting enzymes of de novo pyrimidine synthesis. Moreover, it
is well known that the relative rate of reduction of the purine
nucleotide and thymidylate pools in response to MTX varies
from cell type to cell type and is dependent on the partition of
the reduced folate into those forms required for inosinate versus
thymidylate synthesis (35) and the availability and ability of the
cells to utilize exogenous hypoxanthine and thymidine. Com
parison of the enzymic capacities of the de novo and salvage
pathways for purine biosynthesis in normal and neoplastic
animal tissues (36) showed the high activity and high affinity
for substrate of a key salvage pathway enzyme (purine phosphoribosyltransferase) when compared with a key enzyme of
the de novo synthesis pathway (amidophosphoribosyltransferase). All these data suggest an important role for salvage
pathway enzymes in the circumvention of the action of inhibi
tors of de novo purine and pyrimidine biosynthesis, supported
by studies using the transport inhibitor of nucleosides and bases,
dipyridamole (37).
Thus, we have demonstrated for the first time in isogenic
human cells, exhibiting a spectrum of phenotypes representative
of the stages in the progression to malignancy, the importance
of salvage pathways in the "rescue" of human cells from the
cytotoxicity of inhibitors of the de novo pathways of purine and
pyrimidine biosynthesis. The fact that the effect is in part Nras specific may reflect the influence mutant N-ras has on the
membrane and the transport of nucleosides and bases into the
cell.
The data therefore support strongly the hypothesis that the
concentrations of nucleosides in the plasma (38) and possibly
from adjacent necrotic cells might protect tumor cells from the
inhibitors of de novo synthesis. This confirms the need for
inhibitors of the salvage enzymes to be administered together
with inhibitors of the de novo pathway enzymes during cancer
chemotherapy (2, 34, 36-38). In a recent study in rat hepatoma
cells and human colon cells (39) azidothymidine inhibition of
thymidine kinase acted synergistically with MTX and 5-fluorouracil (inhibitors of de novo synthesis) to enhance
cytotoxicity.
ACKNOWLEDGMENTS
The authors would like to thank Michelle Boulter for typing the
manuscript.
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Decreasing Sensitivity to Cytotoxic Agents Parallels Increasing
Tumorigenicity in Human Fibroblasts
Anne R. Kinsella and M. Sally Haran
Cancer Res 1991;51:1855-1859.
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