Human melanomas are naturally resistant to methotrexate (MTX). The mechanism of intrinsic drug resistance has been explored in 3 melanoma cell lines not previously exposed to tins agent. All 3 lines exhibited relative MTX resistance with ID50 values of greater than 1 μm. Drug uptake studies were performed over an extracellular concentration range of 0.1 to 10 μm MTX. The uptake was linear over the initial 10min at all concentrations and subsequently reached plateau level only at the 10 μm Concentration. Lineweaver-Burke transformations yielded apparent Km (uptake) values of 1.4 to 5 μm, similar to data obtained from other human cell lines. The level of dihydrofolate reductase (DHFR) in the human melanoma cells ranged between 8.42 to 11.98 pmoles/mg protein. The melanoma DHFR levels are several fold higher than in MTX-sensitive human tumor lines and up to a hundred-fold higher than that measured in human brain tumor cells by our assay. The intrinsic resistance of these melanoma lines has therefore been attributed to elevated intracellular levels of DHFR

Abelson, Herbert T.

Kufe, Donald W.

Wick, Michael M.

Elsevier - Publisher Connector 

Natural Resistance to Methotrexate in Human Melanomas 

0022-202X/80/7504-0357$02.00/0 
THE JOURN AL OF INVESTIGATIVE DERMATOLOGY, 75:357-359, 1980 
Copyright © 1980 by The Williams & Wilkins Co. 
Vol. 75, No. 4 
Printed in U.S.A. 
Natural Resistance to Methotrexate in Human Melanomas 
DONALD W. KUFE, M .D ., MICHAEL M. WICK, M .D., PH.D. , AND HERBERT T. ABELSON, M.D. 
Divisions of Pharmacology and Medical Oncology (DWK and MM-w,J Sidney Farber Cancer Institute; Division of Pediatric Oncology (HTA) 
Sidney Farber Cancer Institute; and Departments of M edicine (DWK), Dermatology (MM"I¥,) and Pediatrics (HTA) Harvard Medical 
School, Boston, Massachusetts, U.S.A. 
Human melanomas are naturally resistant to metho-
trexate (MTX). The mechanism of intrinsic drug resist-
ance has been explored in 3 melanoma cell lines not 
previously exposed to this agent. All 3 lines exhibited 
relative MTX resistance with ID5o values of greater than 
1 fLM. Drug uptake studies were performed over an extra-
cellular concentration range of 0.1 to 10 ILM MTX. The 
uptake was linear over the initial 10 min at all concen-
trations and subsequently reached plateau levels only at 
the 10 ILM concentration. Lineweaver-Burke transfor-
mations yielded apparent Km (uptake) values of 1.4 to 5 
p.M. similar to data obtained from other human cell lines. 
The level of dihydrofolate reductase (DHFR) in the hu-
man melanoma cells ranged between 8.42 to 11.98 
pmoles/mg protein. The melanoma DHFR levels are sev-
eral fold higher than in MTX-sensitive human tumor 
lines and up to a hundred-fold higher than that measured 
in human brain tumor cells by our assay. The intrinsic 
resistance of these melanoma lines has therefore been 
attributed to elevated intracellular levels of DHFR. 
Human melanomas are frequently disseminated at the time 
of diagnosis. This poses a significant clinical problem since 
melanomas are resistant to treatment with most chemothera-
peutic agents [1]. We have, therefore, chosen human melano-
mas as a model for the study of natural drug resistance. This 
report deals with mechanisms of methotrexate (MTX) resist-
ance in these cells. 
MTX inhibits the enzyme dihydrofolate reductase and de-
pletes cells of tetrahydrofolate, a cofactor required for the de 
novo synthesis of thymidylic acid and purines [2-4]. The re-
sultant cytotoxicity of MTX, however, can be limited by the 
a bility of malignant cells to develop resistance to the drug. 
Mechanisms of MTX resistance have been characterized by 
increased intracellular levels of dihydrofolate reductase or by 
decreased transport of the drug [5-8]. The resistance of human 
neoplasms and a variety of cultured cells has been attributed to 
impaired drug transport and/or increased levels of intracellular 
dihydrofolate reductase (DHFR) [9-13]. The increase in en-
zyme level in certain murine cells has been shown to be due to 
selective multiplication of DHFR genes [14]. 
Although MTX resistance has been primarily studied in cells 
selected by prior exposure to the drug, it is also relevant to 
determine natural modes of resistance in unselected cell popu-
lations. We show that three human melanomas ru·e relatively 
Manuscript received January 15, 1980; accepted for publication April 
2, 1980. 
This work was supported by Grant CA-06516 from the National 
Cancer Institute. 
Dr. Kufe is a recipient of an American Cancer Society Junior Faculty 
Research A ward. 
Dr. Abelson is a recipient of Research Career Development Award 
CA 00075 from the National Cancer Institute. 
Reprint requests to: Dr. Michael M. Wick, Sidney Farber Cancer 
Institute, 44 Binney Street, Boston, MA 02115. 
Abbreviations: 
· DHFR: dihyd.rofolate reductase 
MTX: methotrexate 
resistant to the effects of MTX in vitro. The intracellular 
uptake of MTX in these lines is comparable to other human 
tumor cell lines. The mechanism of MTX resistance is attrib-
uted to elevated endogenous levels of DHFR. 
MATERIALS AND METHODS 
Melanoma Growth Studies 
The growth characteristics of melanoma cell lines have been previ-
ously described [15]. Each line was established from metastatic mela-
noma and continues to have morphologic characteristics of melanin-
producing cells. Fu.rthermore, these lines are tumorigenic in nude mice. 
AU cell lines were maintained for at least 6 mo in McCoy's 5A medium 
supplemented with 15% fetal calf serum, 100 units of streptomycin per 
milliliter, and 100 !Lg penicillin per milliliter. Single cell suspensions 
were inoculated into Linbro multiwell tissue cultu.re plates (106 cells/ 
well) and were allowed to attach for 24 hr prior to exposu.re. Cells were 
in log phase growth at the t ime of drug exposu.re. Methotrexate (Lederle 
Laboratories Division, Pearl River, NY) was freshly prepared in me-
dium without serum at a concentration of 0.1 mM. The drug was 
sterilized by Millipore fl.l.tration and serial dilu tions were made to give 
final MTX concentrations of w-• to 10- 7 M. Cells were then exposed to 
various concentrations of the drug for 48 h.r. Cells were harvested and 
counted in a Model Z coulter coun ter. Results are expressed as the 
percentage of growth inhibition according to the formula: [(number of 
cells-number of treated cells) / number of control cells] x 100 after 
correction for initial densities. Values represent mean ± standard 
deviation of the mean for 5 to 6 determinations. 
MTX Uptahe Studies 
MTX uptake studies were performed in exponentially growing cells 
at 72 hr after seeding. The cells were first washed 3 tin1es with cultw-e 
media without serum at 4°C. The [3H)MTX (Amersham Searle, Pu:-
lington Heights, Ill.), specific activity 20 Ci/mmole, was initially diluted 
20 fold with unla beled MTX and subsequently added to the cells at 
various concentrations in culture media without serum. Incubations 
were performed at either 37°C or 4°C dw-ing a tim e cou.rse up to 40 
min. The cells were then washed 10 times with 3 ml of cold phosphate 
buffered saline. This procedu.re has been shown to remove extracellular 
and adsorbed MTX without effecting intracellular (free or enzyme-
bound) MTX [16]. The intracellular MTX was measu.red by dissolving 
the monolayer in 2 ml ofO.l M NaCl, 0.01 M Tris-HCl (pH= 7.4), 0.001 
M EDT A and 0.5% sodium dodecyl sulfate and counting the recovered 
radioactivity in a Beckman Model LS-335 liquid scin tillation counter 
after the addition of 13 ml of Aquasol (New England Nuclear, Boston, 
MA) . Aliquots for each determination were assayed for protein using 
the Bio-Rad Protein Assay (Bio-Rad Laboratories, Richmond, CA) 
( 17). All assays were performed in duplicate and repeated twice. 
DHFR Assays 
DHFR levels were determined on cells in logarithmic growth by 
measu.ring the extent of complex formation with [3H]MTX in a ligand-
binding assay [18]. 
RESULTS 
Figure 1 shows the growth inhibition of 3 different human 
m elanoma cell lines 48 hr after continuous exposure to various 
concentrations .of MTX. Melanomas NH (Fig !A) and G361 
(Fig l C) have similar growth inhibition curves with IDso values 
of 4 X w-G M and 2 X w-G M, respectively. HM-1 cells (Fig lB) 
were relatively more resistant, however , and failed to reach 50% 
growth inhibition even at w-• M MTX. 
MTX uptake studies for the 3 melanoma lines are shown in 
357 
358 KUFE ET AL 
A NH 8 HM-1 C G361 
r 
BO BO 
60 60 § 60 
"' ~ 40 
.!; 
* 20 
40 40 
20 20 
[MTX) 
FIG 1. Growth inhibition of human melanoma cell lines at 48 hr 
after continuous exposure to the various concentrations of MTX. 
A NH Melanoma B HM -1 Melanoma C G 361 Melonomo 
40 2!1 
32 20 
10 
. --·-·--~ 
---·- ·- ....-
0o 5 K> 20 lO ao- 0o 5~o zo 30 40 
Mmules 
FIG 2. MTX uptake studies for the 3 melanoma cell lines in the 
presence of extracellular MTX concentrations of 10 p.M (0---0), 1p.M 
(x--x), and 0.1 J.LM (e----e). 
A NH 8 HM -1 C G361 
1 '0 
"0 
" / I ~~ ,. 40 290 
~ 12 / 30 210 
i ' 20 140 ~ Km • l4p.M Km • 5 ,..M l( m &S,... M 
_.:;; ' Vmo o : Q83 10 Vmol s l l 70 Vmo• • O.II 
" 
1/ rMTXJ( fLM ) 
FIG 3. Lineweaver-Burke transformations for the 3 melanoma lines 
calculated from the initial 10 min of linear MTX uptake for concentra-
tions of 0.1, 0.5, 1.0, 3.0, 5.0 and 10.0 J.LM. 
Fig 2. Constant extracellular MTX concentrations were main-
tained over a range . of 0.1 to 10.0 J.LM. All determinations were 
performed at both 37°C and 4 oc to differentiate the carrier-
mediated/ facilitated process from passive diffusion or nonspe-
cific adsorption. Values obtained for uptake at 4 °C varied 
directly with the extracellular MTX concentration. At 1.0 J.LM 
extracellular MTX, this constituted approximately 20% of the 
total uptake [19]. The data in Fig 2 is, therefore, corrected for 
uptake obtained at 4°C for each concentration shown. MTX 
uptake is linear through 10 min, following which the rate of 
accumulation slows at the higher extracellular MTX concentra-
tions and reaches equilibrium. All 3 lines continue to accumu-
late drug at extracellular concentrations of 0.1 and 1.0 J.LM over 
the 40-min time period. 
The intracellular MTX appears to achieve a plateau level 
with an extracellular MTX concentration of 10.0 J.LM. This 
suggests that MTX influx and efflux rates are equivalent. 
However, at the lower extracellular MTX concentrations, the 
continued accumulation of drug suggests a higher influx than 
efflux velocity. MTX efflux studies for the 3 melanoma lines 
were performed following exposure to an extracellular concen-
tration of 10 J.LM MTX. The efflux was rapid during the initial 
5 min and reached plateau levels at 40 min of 14.6, 10.6 and 7.2 
pmoles/ mg protein for the HM-1, G361 and NH lines, respec-
tively. 
The Lineweaver-Burke transformations of MTX uptake for 
each of the 3 melanoma lines are shown in Fig 3. The apparent 
Km and Vmax values were calculated during the initial10 min 
Vol. 75, No. 4 
Dihydrofolate reductase levels in human melanoma cells 
Line DHFR level" 
NH 
HM-1 
G361 
" p moles DHFR/ mg protein. 
11.98 
8.90 
8.42 
of linear MTX uptake for 6 different MTX concentrations of 
0.1, 0.5, 1.0, 3.0, 5.0 and 10.0 J.LM . The line plotted in each case 
was determined using a least squares linear regression analysis. 
The correlation coefficient for the individual plots is 0.99 for 
NH, 0.99 for HM-1 and 0.95 for G361. 
Dihydrofolate reductase levels were also measured in these 
cells harvested during a period of logarithmic growth. The 
amount of enzyme activity was normalized to the protein con-
tent of the cell extract to give the levels in terms of pmoles 
DHFR/mg protein. The results listed in the Table for each line 
represent those obtained for duplicate samples. Assays per-
formed on these cells following several months of continuous 
passage in culture yielded comparable results. 
DISCUSSION 
Human melanomas are clinically resistant to MTX even in 
the high dose protocols [19]. The 3 established melanoma cell 
lines used in this study were obtained from patients who had 
not received MTX therapy. These cell lines demonstrate sub-
stantial resistance to MTX in vitro based upon IDso values 
similarly obtained for a variety of human and mammalian cell 
lines [20]. The IDso values of 2 X 10- 6 M for the NH and G361 
cell lines are 10 to 100-fold higher than values previously 
reported [20] while the HM-1line is even more resistant, failing 
to reach an ID:;o value at 10- • M MTX. 
To delineate the mechanisms of intrinsic resistance to MTX, 
we have explored both MTX uptake as well as the intracellular 
levels of DHFR, the putative cytotoxic target of MTX. The 
MTX resistance in these melanoma lines is not due to a 
transport deficiency. All 3 lines transport and accumulate at 
least 20 picomoles of MTX per mg of protein when exposed to 
extracellular MTX concentrations of 10 J.LM for a period up to 
40 min. This degree of intracellular MTX accumulation is 
consistent with that previously reported for studies using MTX-
sensitive 3T6 cells [16]. Further, more recent studies using the 
MTX-sensitive human breast carcinoma line, MCF -7, have 
yielded MTX transport and accumulation data comparable to 
that obtained with the melanoma cells (data not shown). These 
intracellular MTX levels reflect only the carrier-mediated or 
carrier-facilitated uptake in that they have been corrected for 
any nonspecific uptake by passive diffusion or adsorption as 
measured at 4 °C. The degree of "uptake" at 4 oc with extracel-
lular MTX concentrations of 10- 6 M is consistent with that 
previously published for human tumor cells [9]. 
The apparent Km (uptake) values obtained for all 3 mela-
noma lines (1.4 to 5 J.LM) are consistent with values previously 
published for other human tumor cells [9,10]. MTX enters 
these cells by an active transport process and reaches an equi-
librium or steady-state after exposure to extracellular MTX 
concentrations of 10 mM for 30 min. The steady-state levels 
indicate that the rates of MTX influx and efflux are equivalent. 
The efflux process may be limited by binding MTX to intra-
cellular DHFR and by polyglutamation, thus preventing the 
transport of the drug back to the extracellular medium. The 
efflux studies indicate rapid MTX loss during the initial 5 min. 
All 3 lines then reach plateau MTX levels by 40 min and retain 
between 9 and 15 picomoles MTX/ mg of protein intracellularly. 
These nonexchangeable MTX levels are presumably due to 
that bound to DHFR. 
The resistance of these cells, therefore, appears to be unre-
lated to the intracellular uptake or accumulation of MTX and 
suggests a mechanism more directly related to DHFR. Mea-
surements of intracellular DHFR in these human melanomas 
Oct. 1980 RESISTANCE TO METHOTREXATE IN HUMAN MELANOMAS 359 
have shown that the levels are higher than in a variety of other 
human cells [18,22]. These levels are also 2 to 3-fold higher 
than that obtained with human brain tumor cells [18]. Further, 
there may be a relationship between the DHFR level and 
degree of MTX resistance. The NH line which had the highest 
DHFR level was also most resistant to MTX growth inhibition. 
However, the less than 30% elevation in DHFR level would not 
fully explain the marked increased resistance of the NH cell 
line over the other 2 melanoma cell lines. It is known that the 
Ki values for MTX inhibition of DHFR vary over a wide range 
from the most sensitive to the more resistant lines [22]. An 
intrinsic alteration in the Ki for MTX, in addition to the higher 
levels of intracellular enzyme, may therefore help account for 
the resistance of these cell lines and the NH cell line in partic-
ular. 
The high enzyme levels in these lines provide a source for 
the purification of human DHFR. It should also be feasible to 
enhance these already high endogenous DHFR levels by con-
tinuous exposure to appropriate concentrations of MTX. The 
melanoma cell lines could therefore serve as a source to purify 
the mRNA for human DHFR which could lead to the devel-
opment of probes to examine gene copy number in human 
tumors sensitive and resistant to MTX. DHFR gene duplication 
has been identified in sarcoma 180 cells resistant to MTX 
(14]. Since human melanomas are resistant to most chemother-
apeutic agents, these cell lines may also be useful for studying 
intrinsic resistance to a variety of other drugs. 
The authors appreciate the excellent technical assistance of L. Sar-
gent, J. Ratliff, E. Egan, and C. Gorka. 
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Natural Resistance to Methotrexate in Human Melanomas

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