Adriamycin Cellular Transport: Methodological
I.
HAVLIK, R. D. DANSEY, J. C. KEEPING,T. GOLOMBICK,
Aspects
AND
W. R. BEZWODA
A rapid and simple method for determination of cellular uptake of adriamycin is described. The method is based on the principle that active uptake is proportional to alterations of drug distribution, measured as a fraction of time, between suspending medium and cells, the volume of each having been accurately determined. Cellular drug uptake can be calculated by the use of a simple distribution formula. This method represents a compromise between indirect measurement of the loss of drug from suspending medium and direct measurement of drug uptake following cell separation, washing, and lysis. This method should be applicable to the measurement of cellular uptake of a wide range of drugs.
Key Words:
Adriamycin;
Cellular transport; Cancer cells
INTRODUCTION There
has been
adriamycin
considerable
interest
and in the correlation
in the determination
between
drug
uptake
of cellular
and cytotoxic
uptake effects
of (Se-
hested et al., 1987). While DNA binding appears to be the major mode of cytotoxicity of the anthracyclines, the amount of drug available for DNA interaction depends on whole
cell retention
the anthracycline
and distribution.
antibiotics
The uptake
thus appears
into and efflux
to be an important
from
cells of
determinant
antitumor action of these drugs (Gottesman and Pastan, 1988). The current methods employed for the determination of adriamycin pend on incubation of cells with different concentrations of adriamycin
of the
uptake defor defined
time intervals (Skovsgaard, 1978). After cells and supernatant have been separated (either by centrifugation or by filtration) (Dan@, 1973), intracellular drug accumulation is determined. ing to remove uptake
These separation
free
and efflux
drug.
Since
(processes
may introduce
a number
for determining
whole
procedures
intracellular
that both occur
of artifacts.
require
is a balance
rapidly),
the separation
We present
cell adriamycin
do, however,
retention
uptake
cell wash-
between
here a rapid and simple
that does not require
drug
and washing method
extensive
cell
washing.
From the
Department
of Pharmacology
G., W. R. B.), University Address rand,
reprint
Medical
Received
requests
School,
January
(I. H.), and the Department
of the Witwatersrand, to: Dr. I. Havlik,
Johannesburg
1989;
revised
2193,
Medical Department
South
and accepted
School,
of Medicine
Johannesburg,
of Pharmacology,
(R. D. D., J. C. K., T.
South
University
Africa. of the Witwaters-
Africa.
April 1989.
1 Journal of Pharmacological
Methods
0 1990 Elsevier Science Publishing
23, 1-6 (1990) Co., Inc., 655 Avenue of the Americas, New York, NY 10010
2
I. Havlik et al.
MATERIALS AND METHODS Chemicals and Drugs Adriamycin
was obtained
mCi/mmol)
was supplied
from
Farmitalia;
by Amersham
14C-adriamycin
International
(specific
Laboratories.
activity,
The buffer
56
used
for transport experiments was 150 mM KCl/50 mM Tris-HCI, pH 7.4. Radioactive counting procedures were performed in a Packard scintillation spectrometer with automatic
quench
settings.
correction.
The scintillant
Counting
was performed
used was Aquagel
at optimal
(Chemlab,
gain and window
Ltd.).
Cell lines Two cell lines were originally
obtained
studied.
from
has been propagated ered optimal developed
human
including with
in our laboratory ovarian
cisplatin,
fashion,
carcinoma
disease
adriamycin,
with
UWOVI
doubling
showed
Incubation Suitable volume and
mined Then,
cell line) was
(ATCC,
CCL 243) and
growth
cell line,
from
a patient
to and clogging
re-
in an ad-
Preliminary
of filters
and retention
with
chemotherapy
This line grows 72 hours.
consid-
was a recently
to combination
of approximately uptake
conditions
UWOVI,
It was obtained response
and cyclophosphamide. time
of unlabeled
of 0.1 $Yml)
kept
line.
that adherence
of 0.8 ml and
adjusted
leukemia
studies
was a significant
of adriamycin
using pre-
and Sampling Procedure
admixtures
preincubated
human Collection
since 1983 under malignant
after initial
problem in trying to assess cellular viously described methods.
centration
Type Culture
for this line. The second
lapsed and progressive herent
K562 (a nonadherent
American
were placed
at 37°C with
in suspension
by prior
+ 14C-adriamycin (at a constant conin Tris-KCI buffer to an exactly measured
in l-ml
capped
the lid closed. by continuous
so as to give a defined centrifugation
200~PI aliquots
adriamycin
diluted
packed
Eppendorf
Cells were stirring
prior
cell volume
in a microhematocrit
of the cell suspensions
were
tubes,
incubated to use.
of, e.g.,
which
had been
in the same buffer Cell
numbers
were
5% (by vol.) as deter-
centrifuge transferred
(Skovsgaard, to the tubes
1978). con-
taining drug solution, giving a final volume of 1 ml and a final packed cell volume of 1% when using a 5% cell suspension. Cells were then rapidly sedimented by centrifugation for 20 seconds at 3,500g using a Beckman microfuge. Exactly 0.9 ml of supernatant was removed using an adjustable pipette previously calibrated for precision. After removal of the 0.9 ml of supernatant, time is no longer critical. Adriamycin
was extracted
from the cell-free
supernatant
by addition
of 30 ~1 of 7.5
N HCI and 0.72 ml of 96% ethanol to a 100 ~1 aliquot as previously described (Bachur et al., 1970). The same extraction procedure was followed with the IOO-t.~l residue containing the pelleted cells. Extracts were quantitatively transferred to scintillation vials and counted after evaporation of the alcohol and addition of scintillant. The adriamycin content of the supernatant and that in the remaining 100 ~1 including the cell pellet was determined by isotope minute. Controls with no added cells were
dilution included
based on disintegrations in each experiment.
per
Adriamycin Cellular Transport
Calculations The calculation of adriamycin uptake is based on simple distribution of drug within the system. With a final cell volume of I%, the 100 ~1 remaining after removal of cell-free
supernatant
contains
90 PI of extracellular
fluid
plus IO ~1 of packed
cells. The drug concentration in the 90-t.~l extracellular volume is the same as in the 0.9 ml of cell free supernatant. The drug content in the 1 t.4 of packed cells can be calculated
as follows:
y = (A.X -
C-Z) (Pg4-4
100.B where
A = 100 t-d, B = IO ~1, C = 90 t.~l, X = concentration
t-d) determined supernatants,
in the remaining
100 ~1 of buffer
Z = concentration
of adriamycin
supernatant. Direct measurement
of cellular
method
(1978).
of Skovsgaard
jection
of the cell suspension
buffer.
Cells are then
twice
with
previously
drug uptake
In this method,
1 ml of ice-cold
(kg/l00
buffer.
~1) in 0.9 ml of separated
drug flux is terminated
for 20 seconds
after the
by rapid in-
tube containing
at 3,500g
14C-adriamycin
(kg/l00
of cell-free
in K562 cells was performed
(1 ml) into a centrifuge
centrifuged
of adriamycin
+ cells after removal
4 ml ice-cold
and the pellet
was extracted
washed
and counted
as
described.
and K, were generated by fitting the experCurves used for calculation of V,,, imental data obtained for the initial rate of uptake of adriamycin (measured over 1 minute) at different concentrations using unweighted nonlinear based on the Gauss-Newton computing algorithm (Terziivanov
RESULTS AND
DISCUSSION
Figure 1 shows the typical cell lines.
regression analysis et al., 1982).
time course
of adriamycin
Figure 2 shows the rate of cellular
drug concentrations.
uptake
In both cell lines, the initial
uptake
for each of the two
of 14C-adriamycin
rate of uptake
at different
of adriamycin
was
rapid, suggesting a facilitated or receptor-mediated uptake mechanism. Table I summarizes the V,,, and K, values obtained for each of the two cell lines. Comparison
of the two methods
the method
described
(extraction
in this paper)
of drug from the washed
showed
the adriamycin
cell pellet
content
versus
of the washed
cell pellet to be significantly lower than that expected from mass balance calculations based on the difference between the total quantity of adriamycin added, the amount present in the supernatant, and the amount recovered from the cell pellet after washing. An example of losses occurring during cell washing is provided in Table 2 for the K562 cell line. Under the experimental conditions described, the difference using the two methods amounted to 10.3 ? 1.41%. A difference of this magnitude must be due to drug explained
simply
losses resulting
from
on the basis of volume
the washing of fluid
steps,
trapped
since
it cannot
in intercellular
be
spaces
within the cell pellet. To illustrate this point, even if one were to assume a value of 20% for the volume of trapped fluid (an excessive assumption, in any case) the
3
4
I. Havlik et al.
20
IO
30
I
I
I
I
2
3
4
(minutes)
(hours)
Time FIGURE 1. lime course of uptake of adriamycin by K562 cell line ( W-0) and UWOVi cell line (A- - - - A) at 37°C. Suspensions of cells were incubated in 150 mM KccI50 mM Tris-HCI buffer, pH 7.4, at 2.4 &ml drug.
excess attributed
to cell uptake but in reality
due to adriamycin
content
of trapped
interstitial fluid could amount to only 0.16%. It appears, rather, that cell washing, which introduces a poorly controlled step, is responsible for a significant underestimate The method
of drug binding
and uptake.
aim of the current
investigation
for the measurement
technique the ability
of cellular
was to establish adriamycin
reproducible The
described has the advantages of speed, requiring no cell washing, and to work at room temperature. Centrifugation times, which were of short
duration, were included in the total incubation times, by time delay during centrifugation procedures. While
a simple,
uptake and accumulation.
the method
described
thus eliminating
here does not distinguish
errors
caused
between intracellular
and
membrane-bound adriamycin, it should be pointed out that any surface membranebound fraction, while it is probably quantitatively small, may yet be of considerable biologic significance in view of the active membrane binding and transport mechanisms involved. Furthermore, the method described here probably mimics the in vivo situation more closely, since under these circumstances there would be a gradient between extracellular and intracellular drug concentration. The method
described
represents
a compromise
between
indirect
measurement
of the loss of a drug from suspending medium and direct measurement of the cellular drug content (Skovsgaard and Nissen, 1982). The currently described method appears to give a truer reflection of cellular adriamycin uptake and retention
c I
10
I
I
I
I
20
30
40
Adriamycin
concentration
-I
50
(lg/ml)
FIGURE 2. The initial rate of uptake of adriamycin measured over 1 min at 37°C in 150 mM KCII’SOmM Tris-HCl buffer, pH 7.4, for different concentrations of adriamycin in K562 celf line @-----O) and UWOVl cell line cc>- - - - 0). TABLE 1 Estimated Kinetic Parameters for the Transport of Adriamycin CELL
LINE
V max (&C/MIN/&LL
CELLS)
UWOVl
0.57
t
W62
0.64
-c 0.029
Km
10.19
0.025
(ILCiML)
‘- 1.12
9.42 k 1.33
The data were derived from nonlinear regression analysis for a capacity-limited process of the Michaelis-Menten type: v = (c~V,,,,>/(K,
+ ci,
The experimental data were obtained for initial rate of uptake of adriamycin measured over 1 min at 37°C in 150 mM KC1150 mM Tris-HCI buffer, pH 7.4, for different concentrations of adriamycin. The values shown are the mean rt SD, (n = 8).
6
I. Havlik et al. TABLE 2
_.-_
-.
Comparison of Methods of Measurement
-
-
_.
.-_
I__
of Adriamycin Uptake
DRUG DISTRINJTION
DIRECT EXTKACTION
ASSAY
FROM WASHFI~ CFLL PELLET
_
Total kdpm added kdpm in cell-free superna~ant kdpm in cell pellet Average loss (% of total kdpm added)
179.9 84.1 + 0.27 95.4 i 0.41 0.22
--
^’
179.9 84.1 s 0.27 85.6 + 0.92 5.67
Experiment was done using K562 ceils, adriamycin concentration 2.4 pgiml, incubation time 60 minutes, and cell volume 0.32%. The values shown are the mean i SD (n = 5). kdpm = 1,000 disintegrations per minute.
than previously described methods. Expression of results in terms of adriamycin uptake per cell requires only simple counting procedures. This method should be applicable to the measurement of cellular uptake of other anthracycline derivatives. The method also lends itself to analysis of any changes that may occur in the rate of adriamycin uptake following manipulations that might affect adriamycin handling by cells.
REFERENCES Bachur NR, Moore AL. Bernstein JC, Liu A (1970) Tissue distribution and disposition of daunomycin (NSC-82151) in mice: Fiuorometric and isotpic methods. Cancer Cbemo~he~ Rep .54:8934. Dan@ I( (1973) Active outward transport of daunomycin in resistant Ehriich ascites tumor cells. Bioch;m 5iophys Acta 323:466-483. Gottesman MM, Pastan tiple chemotherapeutic cells. T/PS 9:54-58.
I (1988) Resistance of mulagents in human cancer
Sehested M, Skovsgaard T, van Deurs B, WintherNielsen N (1987) Increased plasma membrane traffic in daunorubicin resistant P388 leukaemic
cells. Effect of daunorubicin Cancer 56:747-75-l.
and verapamii. Br /
Skovsgaard T (1978) Carrier-mediated transport of daunorubicin, adriamycin and rubidazone in Ehrlich ascites tumour cells. Biochem fbarmaco~ 2?:1222-1227. Skovsgaard T, Nissen Nl (1982) Membrane transport of antrhacyclines, P~arm~co/ Ther 18:293311. Terziivanov D, Havlik I, fanku I (1982) Evidence for a saturable component in isoniazid transfer across rat small intestine in vitro. f Pharm Pharmad 34:817-819.
I.
HAVLIK, R. D. DANSEY, J. C. KEEPING,T. GOLOMBICK,
Aspects
AND
W. R. BEZWODA
A rapid and simple method for determination of cellular uptake of adriamycin is described. The method is based on the principle that active uptake is proportional to alterations of drug distribution, measured as a fraction of time, between suspending medium and cells, the volume of each having been accurately determined. Cellular drug uptake can be calculated by the use of a simple distribution formula. This method represents a compromise between indirect measurement of the loss of drug from suspending medium and direct measurement of drug uptake following cell separation, washing, and lysis. This method should be applicable to the measurement of cellular uptake of a wide range of drugs.
Key Words:
Adriamycin;
Cellular transport; Cancer cells
INTRODUCTION There
has been
adriamycin
considerable
interest
and in the correlation
in the determination
between
drug
uptake
of cellular
and cytotoxic
uptake effects
of (Se-
hested et al., 1987). While DNA binding appears to be the major mode of cytotoxicity of the anthracyclines, the amount of drug available for DNA interaction depends on whole
cell retention
the anthracycline
and distribution.
antibiotics
The uptake
thus appears
into and efflux
to be an important
from
cells of
determinant
antitumor action of these drugs (Gottesman and Pastan, 1988). The current methods employed for the determination of adriamycin pend on incubation of cells with different concentrations of adriamycin
of the
uptake defor defined
time intervals (Skovsgaard, 1978). After cells and supernatant have been separated (either by centrifugation or by filtration) (Dan@, 1973), intracellular drug accumulation is determined. ing to remove uptake
These separation
free
and efflux
drug.
Since
(processes
may introduce
a number
for determining
whole
procedures
intracellular
that both occur
of artifacts.
require
is a balance
rapidly),
the separation
We present
cell adriamycin
do, however,
retention
uptake
cell wash-
between
here a rapid and simple
that does not require
drug
and washing method
extensive
cell
washing.
From the
Department
of Pharmacology
G., W. R. B.), University Address rand,
reprint
Medical
Received
requests
School,
January
(I. H.), and the Department
of the Witwatersrand, to: Dr. I. Havlik,
Johannesburg
1989;
revised
2193,
Medical Department
South
and accepted
School,
of Medicine
Johannesburg,
of Pharmacology,
(R. D. D., J. C. K., T.
South
University
Africa. of the Witwaters-
Africa.
April 1989.
1 Journal of Pharmacological
Methods
0 1990 Elsevier Science Publishing
23, 1-6 (1990) Co., Inc., 655 Avenue of the Americas, New York, NY 10010
2
I. Havlik et al.
MATERIALS AND METHODS Chemicals and Drugs Adriamycin
was obtained
mCi/mmol)
was supplied
from
Farmitalia;
by Amersham
14C-adriamycin
International
(specific
Laboratories.
activity,
The buffer
56
used
for transport experiments was 150 mM KCl/50 mM Tris-HCI, pH 7.4. Radioactive counting procedures were performed in a Packard scintillation spectrometer with automatic
quench
settings.
correction.
The scintillant
Counting
was performed
used was Aquagel
at optimal
(Chemlab,
gain and window
Ltd.).
Cell lines Two cell lines were originally
obtained
studied.
from
has been propagated ered optimal developed
human
including with
in our laboratory ovarian
cisplatin,
fashion,
carcinoma
disease
adriamycin,
with
UWOVI
doubling
showed
Incubation Suitable volume and
mined Then,
cell line) was
(ATCC,
CCL 243) and
growth
cell line,
from
a patient
to and clogging
re-
in an ad-
Preliminary
of filters
and retention
with
chemotherapy
This line grows 72 hours.
consid-
was a recently
to combination
of approximately uptake
conditions
UWOVI,
It was obtained response
and cyclophosphamide. time
of unlabeled
of 0.1 $Yml)
kept
line.
that adherence
of 0.8 ml and
adjusted
leukemia
studies
was a significant
of adriamycin
using pre-
and Sampling Procedure
admixtures
preincubated
human Collection
since 1983 under malignant
after initial
problem in trying to assess cellular viously described methods.
centration
Type Culture
for this line. The second
lapsed and progressive herent
K562 (a nonadherent
American
were placed
at 37°C with
in suspension
by prior
+ 14C-adriamycin (at a constant conin Tris-KCI buffer to an exactly measured
in l-ml
capped
the lid closed. by continuous
so as to give a defined centrifugation
200~PI aliquots
adriamycin
diluted
packed
Eppendorf
Cells were stirring
prior
cell volume
in a microhematocrit
of the cell suspensions
were
tubes,
incubated to use.
of, e.g.,
which
had been
in the same buffer Cell
numbers
were
5% (by vol.) as deter-
centrifuge transferred
(Skovsgaard, to the tubes
1978). con-
taining drug solution, giving a final volume of 1 ml and a final packed cell volume of 1% when using a 5% cell suspension. Cells were then rapidly sedimented by centrifugation for 20 seconds at 3,500g using a Beckman microfuge. Exactly 0.9 ml of supernatant was removed using an adjustable pipette previously calibrated for precision. After removal of the 0.9 ml of supernatant, time is no longer critical. Adriamycin
was extracted
from the cell-free
supernatant
by addition
of 30 ~1 of 7.5
N HCI and 0.72 ml of 96% ethanol to a 100 ~1 aliquot as previously described (Bachur et al., 1970). The same extraction procedure was followed with the IOO-t.~l residue containing the pelleted cells. Extracts were quantitatively transferred to scintillation vials and counted after evaporation of the alcohol and addition of scintillant. The adriamycin content of the supernatant and that in the remaining 100 ~1 including the cell pellet was determined by isotope minute. Controls with no added cells were
dilution included
based on disintegrations in each experiment.
per
Adriamycin Cellular Transport
Calculations The calculation of adriamycin uptake is based on simple distribution of drug within the system. With a final cell volume of I%, the 100 ~1 remaining after removal of cell-free
supernatant
contains
90 PI of extracellular
fluid
plus IO ~1 of packed
cells. The drug concentration in the 90-t.~l extracellular volume is the same as in the 0.9 ml of cell free supernatant. The drug content in the 1 t.4 of packed cells can be calculated
as follows:
y = (A.X -
C-Z) (Pg4-4
100.B where
A = 100 t-d, B = IO ~1, C = 90 t.~l, X = concentration
t-d) determined supernatants,
in the remaining
100 ~1 of buffer
Z = concentration
of adriamycin
supernatant. Direct measurement
of cellular
method
(1978).
of Skovsgaard
jection
of the cell suspension
buffer.
Cells are then
twice
with
previously
drug uptake
In this method,
1 ml of ice-cold
(kg/l00
buffer.
~1) in 0.9 ml of separated
drug flux is terminated
for 20 seconds
after the
by rapid in-
tube containing
at 3,500g
14C-adriamycin
(kg/l00
of cell-free
in K562 cells was performed
(1 ml) into a centrifuge
centrifuged
of adriamycin
+ cells after removal
4 ml ice-cold
and the pellet
was extracted
washed
and counted
as
described.
and K, were generated by fitting the experCurves used for calculation of V,,, imental data obtained for the initial rate of uptake of adriamycin (measured over 1 minute) at different concentrations using unweighted nonlinear based on the Gauss-Newton computing algorithm (Terziivanov
RESULTS AND
DISCUSSION
Figure 1 shows the typical cell lines.
regression analysis et al., 1982).
time course
of adriamycin
Figure 2 shows the rate of cellular
drug concentrations.
uptake
In both cell lines, the initial
uptake
for each of the two
of 14C-adriamycin
rate of uptake
at different
of adriamycin
was
rapid, suggesting a facilitated or receptor-mediated uptake mechanism. Table I summarizes the V,,, and K, values obtained for each of the two cell lines. Comparison
of the two methods
the method
described
(extraction
in this paper)
of drug from the washed
showed
the adriamycin
cell pellet
content
versus
of the washed
cell pellet to be significantly lower than that expected from mass balance calculations based on the difference between the total quantity of adriamycin added, the amount present in the supernatant, and the amount recovered from the cell pellet after washing. An example of losses occurring during cell washing is provided in Table 2 for the K562 cell line. Under the experimental conditions described, the difference using the two methods amounted to 10.3 ? 1.41%. A difference of this magnitude must be due to drug explained
simply
losses resulting
from
on the basis of volume
the washing of fluid
steps,
trapped
since
it cannot
in intercellular
be
spaces
within the cell pellet. To illustrate this point, even if one were to assume a value of 20% for the volume of trapped fluid (an excessive assumption, in any case) the
3
4
I. Havlik et al.
20
IO
30
I
I
I
I
2
3
4
(minutes)
(hours)
Time FIGURE 1. lime course of uptake of adriamycin by K562 cell line ( W-0) and UWOVi cell line (A- - - - A) at 37°C. Suspensions of cells were incubated in 150 mM KccI50 mM Tris-HCI buffer, pH 7.4, at 2.4 &ml drug.
excess attributed
to cell uptake but in reality
due to adriamycin
content
of trapped
interstitial fluid could amount to only 0.16%. It appears, rather, that cell washing, which introduces a poorly controlled step, is responsible for a significant underestimate The method
of drug binding
and uptake.
aim of the current
investigation
for the measurement
technique the ability
of cellular
was to establish adriamycin
reproducible The
described has the advantages of speed, requiring no cell washing, and to work at room temperature. Centrifugation times, which were of short
duration, were included in the total incubation times, by time delay during centrifugation procedures. While
a simple,
uptake and accumulation.
the method
described
thus eliminating
here does not distinguish
errors
caused
between intracellular
and
membrane-bound adriamycin, it should be pointed out that any surface membranebound fraction, while it is probably quantitatively small, may yet be of considerable biologic significance in view of the active membrane binding and transport mechanisms involved. Furthermore, the method described here probably mimics the in vivo situation more closely, since under these circumstances there would be a gradient between extracellular and intracellular drug concentration. The method
described
represents
a compromise
between
indirect
measurement
of the loss of a drug from suspending medium and direct measurement of the cellular drug content (Skovsgaard and Nissen, 1982). The currently described method appears to give a truer reflection of cellular adriamycin uptake and retention
c I
10
I
I
I
I
20
30
40
Adriamycin
concentration
-I
50
(lg/ml)
FIGURE 2. The initial rate of uptake of adriamycin measured over 1 min at 37°C in 150 mM KCII’SOmM Tris-HCl buffer, pH 7.4, for different concentrations of adriamycin in K562 celf line @-----O) and UWOVl cell line cc>- - - - 0). TABLE 1 Estimated Kinetic Parameters for the Transport of Adriamycin CELL
LINE
V max (&C/MIN/&LL
CELLS)
UWOVl
0.57
t
W62
0.64
-c 0.029
Km
10.19
0.025
(ILCiML)
‘- 1.12
9.42 k 1.33
The data were derived from nonlinear regression analysis for a capacity-limited process of the Michaelis-Menten type: v = (c~V,,,,>/(K,
+ ci,
The experimental data were obtained for initial rate of uptake of adriamycin measured over 1 min at 37°C in 150 mM KC1150 mM Tris-HCI buffer, pH 7.4, for different concentrations of adriamycin. The values shown are the mean rt SD, (n = 8).
6
I. Havlik et al. TABLE 2
_.-_
-.
Comparison of Methods of Measurement
-
-
_.
.-_
I__
of Adriamycin Uptake
DRUG DISTRINJTION
DIRECT EXTKACTION
ASSAY
FROM WASHFI~ CFLL PELLET
_
Total kdpm added kdpm in cell-free superna~ant kdpm in cell pellet Average loss (% of total kdpm added)
179.9 84.1 + 0.27 95.4 i 0.41 0.22
--
^’
179.9 84.1 s 0.27 85.6 + 0.92 5.67
Experiment was done using K562 ceils, adriamycin concentration 2.4 pgiml, incubation time 60 minutes, and cell volume 0.32%. The values shown are the mean i SD (n = 5). kdpm = 1,000 disintegrations per minute.
than previously described methods. Expression of results in terms of adriamycin uptake per cell requires only simple counting procedures. This method should be applicable to the measurement of cellular uptake of other anthracycline derivatives. The method also lends itself to analysis of any changes that may occur in the rate of adriamycin uptake following manipulations that might affect adriamycin handling by cells.
REFERENCES Bachur NR, Moore AL. Bernstein JC, Liu A (1970) Tissue distribution and disposition of daunomycin (NSC-82151) in mice: Fiuorometric and isotpic methods. Cancer Cbemo~he~ Rep .54:8934. Dan@ I( (1973) Active outward transport of daunomycin in resistant Ehriich ascites tumor cells. Bioch;m 5iophys Acta 323:466-483. Gottesman MM, Pastan tiple chemotherapeutic cells. T/PS 9:54-58.
I (1988) Resistance of mulagents in human cancer
Sehested M, Skovsgaard T, van Deurs B, WintherNielsen N (1987) Increased plasma membrane traffic in daunorubicin resistant P388 leukaemic
cells. Effect of daunorubicin Cancer 56:747-75-l.
and verapamii. Br /
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