[CANCER RESEARCH 33, 2584-2587, November 1973]
Nitrogen Mustard Sensitivity and Choline Transport in Walker 256
Carcinosarcoma Cells in Vitro1
Gerald J. Goldenberg2
and B. K. Sinha
Departments of Medicine [G. J. G.] and Surgery [B. K. S.], University of Manitoba and the Manitoba Institute of Cell Biology, Winnipeg, Manitoba
R3E OV9, Canada
SUMMARY
MATERIALS
Single cell suspension cultures of Walker 256 carcinosarcoma were established in vitro in Fischer's medium
supplemented with 10% horse serum. The cells grew ex
ponentially with a doubling time of 16 to 17 hours, had a
cell volume (mean ±S.E.) of 2313 ±89 cu /tm, and were
cloned in soft agar with a cloning efficiency of 68.7 ±6.3%.
Sensitivity to the alkylating agent nitrogen mustard (HN2)
was comparable to that observed previously with LSI78Y
murine lymphoblasts; the D0 (the dose of drug reducing
survival to 37% of the initial cell population) was 5.35
ng/ml, and the extrapolation number, n, was 1.27. An
investigation of the interaction of hydrolyzed nitrogen
mustard and choline transport revealed that uptake of
choline-14C obeyed simple Michaelis-Menten kinetics,
proceeded "uphill" against a concentration gradient of over
30-fold, and showed a decrease in distribution ratio as
choline concentration increased and choline transport was
competitively inhibited by hydrolyzed HN2. The Michaelis
constant, Km, for choline transport was 2.90 x 10~5 M,the
transport capacity, Vmttx, was 4.83 x 10~17 moles/min/
cell, and the inhibition constant, K,, with hydrolyzed HN2
as inhibitor, was 7.83 x 10~5 M. These findings indicate
that choline transport by Walker carcinosarcoma cells is an
active carrier-mediated process and that choline and hy
drolyzed HN2 compete for the same transport mechanism.
Walker 256 carcinosarcoma (obtained from Dr. A. C.
Wallace, University of Western Ontario, London, Canada)
was maintained by weekly i.m. inoculation of 150- to 200-g
Wistar albino male rats (Canadian Breeding Laboratories,
St. Constant, La Prairie City, Quebec, Canada) with 1 ml
of tumor homogenate containing approximately 5 x IO6
cells in Hanks' balanced salt solution; 65 to 80% of the
cells were viable by trypan blue dye exclusion. Approxi
mately 7 days after transplantation the tumor was removed
aseptically and homogenized in Hanks' balanced salt solu
tion; cell cultures were incubated at 37°in Fischer's me
dium (Grand Island Biological Co., Grand Island, N. Y.)
supplemented with 10% horse serum at a final concentra
tion of 5 x IO5 cells/ml. Cell suspensions were diluted
with medium every 24 to 48 hr to maintain cell concen
tration within a range of 4 x IO4to 4 x IO5cells/ml. Cell
cultures were maintained for several months, and the cells
grew exponentially with a doubling time of 16 to 17 hr.
Attempts to culture cells in Dulbecco modified Eagle's
medium (Grand Island Biological Co.) supplemented with
10% fetal calf serum were unsuccessful. On occasion, cells
were frozen at the rate of l°/min to -70°in equal volumes
of tumor homogenate and preservative, consisting of 30%
dimethyl sulfoxide in Fischer's medium supplemented
with 10% horse serum. An i.m. inoculation of 1 ml of
rapidly thawed, frozen homogenate containing approxi
mately 5 x IO6cells consistently produced tumors.
The size of W256 cells was determined in a Coulter
Model B electronic particle counter, calibrated with giant
ragweed pollen (mean cell diameter, 19.5 ^m) and paper
mulberry spores (mean cell diameter, 12.5 /urn), both of
which were obtained from Coulter Diagnostics, Inc., Miami
Springs, Fla. The cell volume (mean ±S.E.) of W256 cells
on 7 determinations was 2313 ±89 cu /urn.
Sensitivity to HN2 was determined by treating W256
cells, at a concentration of 1 x 10s cells/ml, with HN2 for
5 hr at 37°;the treated cells were washed once with 5 ml of
medium, and dose-survival curves were obtained by the
cloning method of Chu and Fischer (1). The cloning effi
ciency (mean ±S.E.) on 12 determinations was 68.7 ±
6.3%. Linear regression analysis of the dose-survival curve
was performed, the regression equation being in the form
log ey = mx + b, where y = surviving cell fraction, x =
dose of HN2 in ¿¿g/ml,
m = slope of regression line, and
b = the y intercept or extrapolation number n, which de-
INTRODUCTION
Active, carrier-mediated transport of the alkylating agent
HN23 has been reported previously in L5178Y murine
lymphoblasts (4, 5) and in normal and leukemic human
lymphoid cells (6). The carrier involved was identified as
the transport carrier for choline, a close structural analog
of HN2 (4, 6). This report describes the establishment of a
cell line of rat Walker 256 carcinosarcoma in vitro and ex
amines sensitivity of those cells to HN2 and the interaction
of HN2-OH and choline transport.
'This
work was supported
by a grant from the National
tute of Canada.
2Clinical Research
Associate
Canada.
3The
used
abbreviations
are:
of the National
HN2,
nitrogen
Cancer
Cancer Insti
Institute
mustard;
hydrolyzed nitrogen mustard; W256, Walker 256 carcinosarcoma.
Received June 5, 1973; accepted July 9, 1973.
2584
HN2-OH,
of
AND METHODS
CANCER
RESEARCH
VOL. 33
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Properties
scribes the shoulder of the curve and is of little practical
value. Z)0 (the dose of drug reducing survival to \/e, i.e.,
37% of the initial cell population) was derived from the
negative reciprocal of the slope of the regression line.
Transport studies were performed on W256 cells in sus
pension cultures with the use of choline-1,2-14C chloride
(specific activity, 2.0 mCi/mmole) obtained from New
England Nuclear, Boston, Mass., with methods previously
described (5). Linear regression analysis of the LineweaverBurk plots was determined: the slope represents Km/Vmax,
the y intercept is l/Vmax, and the x intercept is -l/Km.
The inhibition constant K¡was determined from the Lineweaver-Burk plots with the formula: apparent Km (with in
hibitor) = Km[l + (/)/KJ, where Km was obtained without
inhibitor and (/) was the concentration of inhibitor, as pre
viously described (5). The hydrolyzed derivative HN2-OH
was prepared by alkaline hydrolysis in 0.1 N NaOH at 60°
for 2 hr.
of W256 Cells in Vitro
U
CI
o
5
30
60
90
120
TIME (MIN)
Chart 2. Time course of uptake of 0.1 mvi choline-"C chloride by
W256 cells in vitro. A 20-ml suspension of W256 cells at a concentration of
3.6 x 10scells/ml was incubated at 37°;2-ml aliquots containing 7.2 x 10s
RESULTS
A dose-survival curve of W256 cells treated with HN2 is
shown in Chart 1. The surviving cell fraction followed a
simple exponential function of HN2, and there was only a
slight shoulder on the dose-survival curve. The D0, the dose
of drug reducing survival to 37% of the initial cell popula
tion, was 5.35 ng/ml and the extrapolation number, n, was
1.27.
A time course of the uptake of choline-14C by W256
cells in vitro is shown in Chart 2. Uptake was linear for
60 min and thereafter decreased slightly; subsequent kinetic
0.01
0.02
0.03
0.04
HN2(/ig/ml)
Chart 1. Dose-survival of log-phase W256 cells treated in vitro with
HN2 for 5 hr and cloned in soft agar by the method of Chu and Fischer (1).
Composite plot of 2 experiments illustrated by separate symbols. - - -, an
extrapolation of the regression line to the y intercept to give the extrapola
tion number n. The regression equation was log,,^ = - 187.0360.x+ 0.2411
with a correlation coefficient of -0.9496. The confidence limits shown are
the standard deviation of the mean of 4 determinations at each point;
occasionally, these were too narrow to be illustrated.
NOVEMBER
cells were removed at the times indicated, and radioactivity was deter
mined by methods previously described (5).
analysis of choline transport was terminated at 60 min to
ensure that initial uptake velocity was being examined.
A kinetic analysis demonstrated that choline uptake
followed simple Michaelis-Menten kinetics. The linear re
gression equation of the Lineweaver-Burk plot for choline
transport in the absence of HN2-OH was y = 0.6002* +
2.0708 with a correlation coefficient of 0.9984, and that
with HN2-OH present was y = 1.3012* + 1.9051 with a
correlation coefficient of 0.9955. A t test comparing the
significance of the difference in slopes was highly significant
(p < 0.001). The Michaelis constant Km for choline trans
port was 2.90 x 10~5 M and the transport capacity Vmax
was 4.83 x 10 l7 moles/min/cell. In the presence of
0.1 mM HN2-OH, choline transport was characterized by a
2.35-fold increase of Km to 6.83 x 10~5 Mwithout any sig
nificant change in Vmax (5.25 x 10'17 moles/min/cell).
This indicated that HN2-OH acted as a competitive in
hibitor of choline transport, and the inhibition constant K¡
was 7.83 x 10~5 M.
Evidence that choline transport was an active process
was the fact that uptake proceeded "uphill" against a con
centration gradient of over 30-fold (Chart 3). Additional
evidence for a carrier mechanism was the finding that the
cell/medium distribution ratio decreased as choline con
centration increased. The competitive nature of HN2-OH
inhibition was also illustrated in this plot, in that inhibition
of choline transport was overcome at higher substrate con
centrations.
DISCUSSION
To our knowledge this is the first report of successful
single-cell suspension culture of Walker 256 carcinosarcoma in vitro. Several properties of W256 and L5178Y
murine lymphoma in vitro are compared in Table 1. Both
1973
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1973 American Association for Cancer Research.
2585
Gerald J. Goldenberg
Comparison
and B. K. Sinha
of properties
of log-phase L5I78Y
Table I
murine lymphoma
and rat Walker 256 carcinosarcoma
sensitivity00(ng/ml)
CellsWalker
ling
time
size(CU
(hr)16-17 fitn)2313
256
L5178Y"Doub
±
±6.3
41.5 ±2.6'HN2 7.72 ±0.34
10-11Cell 1273 ±89"28Cloning
cells in vitro
transport(xlO-â„¢
man(xlO-"
efficiency68.7
M)2.90
n5.35
mole/
min/celi)4.83
1.27
0.29 ±0.09Choline
2.55 ±0.23vY 2.90 ±0.21K,
(xlO
f7.38
5.36HN2-OHtransport,(xlO~sM
7.76
" HN2-OH used as inhibitor of choline transport.
"Mean ±S.E.
r N.D., not done.
" Data for LSI78Y cells were obtained
from Refs. 2 4.
' Recent experience with cloning of various LSI78Y cells lines in soft agar has shown wide variation in cloning efficiency ranging from 3 to 75%.
Q
5
5
30
2°
10
O
5
10xlO-5M
[CHOLINE -14C]
Chart 3. Uptake of choline-"C chloride by W256 cells incubated for
60 min at 37°in the absence (O) and presence (•)of 0.1 mM HN2-OH.
cell lines exhibit exponential growth curves; W256 cells
have a slower doubling time of 16 to 17 hr and are almost
twice as large, with a cell volume of 2313 ±89 cu ¿¿m.
The
cloning efficiency in soft agar, with the method of Chu and
Fischer (1), is at least as high for W256 cells; recent experi
ence with L5178Y lymphoblasts has demonstrated con
siderable variation in cloning efficiency (unpublished ob
servation).
Sensitivity to the alkylating agent HN2 was remarkably
similar for both W256 and LSI78Y cells. The surviving cell
fraction varied exponentially with dose of HN2, and the
dose-survival curves for both lines showed only a slight
shoulder with a low extrapolation number; a comparison
of DO showed that sensitivity of W256 cells in this study
was 1.44-fold greater than that previously reported for
LSI78Y cells (3). Sensitivity of W256 cells in vitro is con
sistent with early studies of HN2, which demonstrated
sensitivity of Walker carcinosarcoma in vivo (7).
The correspondence in HN2 sensitivity of W256 and
LSI78Y cells prompted an investigation of the interaction
of HN2-OH and choline transport in W256 cells. It was
previously shown that active transport of HN2 is mediated
by the choline carrier in LSI78Y lymphoblasts (4) and in
normal and leukemic human lymphoid cells (6). Evidence
that choline transport by W256 cells was an active, carriermediated process was that uptake followed simple
Michaelis-Menten kinetics, that it proceeded "uphill"
against a concentration gradient of over 30-fold, and that
2586
the cell/medium ratio decreased at higher substrate con
centrations. The transport Km for choline was similar in
W256 and LSI78Y cell lines, but the transport capacity
Vmax was 1.63-fold higher in W256 cells (Table 1). An
evaluation of Vmax must include a consideration of cell
size, and W256 cells were 1.82-fold larger in volume. How
ever, without the critical information on the density or dis
tribution of transport carriers on the cell membrane, at
tempts to relate Vmaxto cell volume or surface area are fu
tile (2). Thus the higher Vmaxin W256 cells may indicate
either a larger number of transport sites and/or faster car
rier mobility.
HN2-OH acted as a competitive inhibitor of choline
transport with a similar inhibition constant, K¡,for both
cell lines (Table 1). This finding suggests that, as with
L5178Y (3) and human lymphoid (6) cells, HN2 transport
by W256 cells is mediated at least in part by the choline
carrier. Of interest is the fact that the Km for HN2-OH
transport by L5178Y cells was 7.76 x 10~5 M, which is
similar to the K, of 7.38 x 10 5 M for HN2-OH acting as
inhibitor of choline transport by W256 cells. This similarity
of Km and K, for a compound acting as substrate or inhibi
tor confirms the competitive nature of choline and HN2OH transport and also suggests a similar binding affinity
between HN2-OH and the transport carrier in W256 and
LSI78Y cells.
W256 carcinosarcoma cultured in vitro has provided a
useful model for a study of HN2 sensitivity and the inter
action of HN2-OH and choline transport. Parallel attempts
to culture human carcinoma as single cell suspensions in
vitro may prove more rewarding.
ACKNOWLEDGMENTS
We thank Ruby Ng for technical assistance and Sally Barker for typ
ing the manuscript. HN2 hydrochloride (Mustargen) was kindly supplied
by Dr. W. Dorian (Merck, Sharp and Dohme, Dorval, Quebec, Canada).
REFERENCES
1. Chu, M. Y., and Fischer, G. A. The Incorporation of H3-Cytosine
Arabinoside and Its Effect on Murine Leukemic Cells (L5178Y). Biochem. Pharmacol., 17: 753-767, 1968.
2. Goldenberg, G. J., Lyons, R. M., Lepp, J. A., and Vanstone, C. L.
CANCER RESEARCH
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1973 American Association for Cancer Research.
VOL. 33
Properties of W256 Cells in Vitro
Sensitivity to Nitrogen Mustard as a Function of Transport Activity
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5. Goldenberg, G. J., Vanstone, C. L., Israels, L. G., Ilse, D., and Bihler,
I. Evidence for a Transport Carrier of Nitrogen Mustard in Nitrogen
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6. Lyons, R. M., and Goldenberg, G. J. Active Transport of Nitrogen
Mustard and Choline by Normal and Leukemic Human Lymphoid
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7. Stock, C. C., and Sugiura. K. Tumor-Inhibitory Effects of Alkylating
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NOVEMBER 1973
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2587
Nitrogen Mustard Sensitivity and Choline Transport in Walker
256 Carcinosarcoma Cells in Vitro
Gerald J. Goldenberg and B. K. Sinha
Cancer Res 1973;33:2584-2587.
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