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Effect of Tamoxifen on Cell Lines Displaying the Multidrug-Resistant Phenotype
By Ellin Berman, Matthew Adams, Rosemarie Duigou-Osterndorf, Loren Godfrey, Bayard Clarkson, and Michael Andreeff
We examined the effect of tamoxifen (Tmx), verapamil, and
daunorubicin (DNR) in two cell lines that displayed the
multidrug-resistant (MDR) phenotype and used laser flow
cytometry to quantitate intracellular DNR content. In the
vinblastine-resistant human lymphoblastic lymphoma cell
line CEM-VBL, simultaneous incubationof DNR with Tmx 10
pmol/L or Tmx 50 pmol/L increased intracellularDNR fluorescence in a dose-dependent manner and demonstrated an
uptake pattern similar to that seen with DNR and verapamil.
Similar results were obtained in the vincristine-resistant
human myeloid leukemia cell line HL-GO/RV+. Cellular retention of DNR was also measured in both cell lines and results
suggested that continuous exposure of the cells to Tmx
resulted in higher intracellular DNR content compared with
cells resuspended in fresh medium. No effect of Tmx or
verapamil was observed in the drug-sensitive parent cell
lines CEM or HL-60. Clonogenic experiments were then
performedto determine whether Tmx was itself inhibitory to
cell growth or whether Tmx potentiated DNR cytotoxicity.
Tmx 10 pmol/L did not significantly inhibit either CEM-VBL
or HL-GO/RV+ cells after a 3-hour exposure followed by
culture in methylcellulose. Tmx 50 pmol/L was significantly
more inhibitory in both cell lines. However, cells that had
been incubatedwith DNR and Tmx 10 pmol/L demonstrated
a marked incrementin growth inhibition compared with cells
that had been incubated with DNR alone or Tmx 10 pmol/L
alone. Based on the data presented here, we suggest that
clinical testing of Tmx and DNR be pursued in the setting
where MDR may play a role.
o 1991 by The American Society of Hematology.
D
line CEM" and its vinblastine-resistant subline CEMVBL," along with the myeloid leukemia cell line HL-6OZ3
and its vincristine-resistant subline HL-60/RV+'4 were
used in these studies. We first demonstrated that each
MDR subline expressed PGP by flow cytometric analysis
with the murine monoclonal antibody (MoAb) HYB-241,
which recognizes an external portion of the PGP molecule." We then performed (1) DNR uptake and retention
studies using laser flow cytometry to quantitate intracellular
DNR content26327
and (2) clonogenic assays to determine
whether Tmx was by itself toxic to these cells or whether it
potentiated the effect of DNR in vitro. Results of these
experiments demonstrate that Tmx, in doses that may be
achievable in vivo, can increase intracellular DNR concentration in MDR cells. Moreover, Tmx at low doses is not
cytotoxic but strongly potentiates DNR cytotoxicity.
RUG RESISTANCE TO chemotherapy represents a
major problem in oncology. One type of resistance,
multidrug resistance (MDR), can develop after exposure to
certain natural chemotherapeutic agents such as vinca
alkaloids, anthracyclines, dactinomycin, and epipodophyllotoxins.' Cells that display the MDR phenotype demonstrate
decreased intracellular drug acc~mulation'-~
because of an
170,000-d plasma membrane-associated glycoprotein (Pglycoprotein [PGP]) that functions as an active efflux
Membrane transport studies have demonstrated
that calcium channel-blocking drugs such as verapamil bind
directly to PGP, allowing for increased intracellular drug
Such studies have formed the rationale for
phase I trials combining verapamil with vinblastine,' adriamycin," or combinations" in patients with relapsed cancer. However, one major difficulty with the use of verapamil
in vivo is its extremely narrow therapeutic index; doses that
approach the in vitro levels required for reversal of the
MDR phenotype frequently cause significant degrees of
heart block and hypotension.'-''
dipyriOther types of drugs, such as cyclosporin
d a m ~ l e , ' ~reserpine,16
.'~
the calmodulin antagonist trifluoperazine,"~" and certain hormone^,''^'^ can also reverse the
MDR phenotype in vitro. In this study, we examined the
effect of the synthetic antiestrogen tamoxifen (Tmx) in
combination with daunorubicin (DNR) in two human
MDR leukemia cell lines. The lymphoblastic leukemia cell
-
From the Laboratory of Hematopoietic Cell Kinetics and The
Laboratory of Leukemia Cell Biology, Memorial Sloan-Kettering
Cancer Center, New York, Ny.
Submitted July 26, 1990; accepted October I I , 1990.
Supported in part by an American Cancer Society Research Career
Development Award No. 89-124 (E.B.).
Address reprint requests to Ellin Berman, MD, Leukemia Service,
Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York,
Ny 10021.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C.section I734 solely to
indicate this fact.
0 I991 by The American Society of Hematology.
0006-4971I9117704-0002$3.0OlO
818
MATERIALS AND METHODS
Cell lines. The parent cell lines HL-60 and CEM were obtained
from the American Type Culture Collection (Rockville, MD). The
HL-60 vincristine-resistant cell line (HL-60/RV+) was kindly
provided by Dr Melvin S. Center (Kansas State University, Manhattan, KS). The CEM vinblastine-resistant subline was originally
provided by Beck et al?* All cells were kept in RPMI 1640 (GIBCO
Laboratories, Grand Island, NY) supplemented with 10% (vol/vol)
heat-inactivated fetal calf serum (FCS; Hyclone, Salt Lake City,
Utah) and 1% L-glutamine (GIBCO) and incubated at 37°C with
5% CO, and 95% humidified air.
Drugs. Tmx citrate was kindly provided by IC1 Pharmaceuticals
(Wilmington, Delaware) and dissolved in dimethyl sulfoxide
(DMSO). The final concentration of DMSO in the Tmx suspension
was 0.1%. Daunorubicin was purchased from Adria Laboratories
(Dublin, OH), dissolved in sterile water, and diluted with RPMI
containing 10% FCS before each experiment. Verapamil was
obtained from Knoll Pharmaceuticals (Whippany, NJ) and dissolved in sterile NaCl with 1.57% DMSO before each experiment.
The final concentration of DMSO in the verapamil suspension was
0.008% in each experiment. Solutions containing verapamil were
protected from light at all times by tin foil wrapping.
Flow cytometry. For determination of flow cytometric intracellular DNR concentrations, a FACScan (Becton Dickinson, Mountain View, CA) equipped with an argon ion laser using a 488-nm
line operating at 15 mW was used. Light was collected through a
585142-nm filter for the DNR uptake and retention studies.
Cell uptake and retention studies. Cells incubated at a concentraBlood, Vol77, No 4 (February 15). 1991: pp 818-825
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819
TMX EFFECT ON MDR CELLS
tion of 2 x l@cells/mL were exposed to DNR 1 pg/mL alone or in
combination with the test drug. At time 0, 5, 30, 45, 60, and 90
minutes cells were washed twice in ice-cold phosphate-buffered
saline (PBS) without Caz+or Mg2+(GIBCO) and resuspended in
0.5 mL ice-cold PBS with 5% FCS. DNR fluorescence was
immediately measured using the FACScan.
Drug retention studies were performed in separate experiments.
Cells were first incubated with either DNR 1 pg/mL or the
combination of DNR and the test drug for 1hour. All samples were
then washed twice in ice-cold medium and resuspended at 37°C in
fresh medium containing the specific parameter, and DNR fluorescence was measured at time 0,5,15,30,60, and 120 minutes. DNR
fluorescence was measured using the same FACScan instrument as
in the uptake experiments.
All drug uptake and retention experiments were performed in
duplicate.
Clonogenic assays. Cells at a concentration of 2 x lo6 were
incubated with either medium, 0.1% DMSO, DNR 1 pg/mL, Tmx
10 pmol/L, Tmx 50 pmol/L, or the combination of DNR 1 pg/mL
plus Tmx 10 pmol/L or Tmx 50 pmoVL for 1hour. Aliquots of these
test samples were then washed twice and plated in triplicate at a
concentration of 250 cells per plate. To determine whether a more
prolonged exposure to Tmx had an enhanced effect on cytotoxicity,
separate aliquots were simultaneously resuspended in either medium, 0.1% DMSO, DNR 1 &mL, Tmx 10 pmol/L, or Tmx 50
pmol/L for an additional 2 hours, washed twice, and plated in
triplicate at the above concentration.
The effect of verapamil on clonogenic capacity was also examined. Cells were incubated with either medium, DNR 1 pg/mL,
verapamil 10 pmol/L, or the combination of DNR 1 &mL plus
verapamil 10 pmol/L for 1 hour. Aliquots were then washed twice
and plated in triplicate at the above concentration. Separate
aliquots were simultaneously resuspended in either medium, DNR
1 pg/mL, or verapamil 10 pmol/L for an additional 2 hours, washed
twice, and plated in triplicate at the above concentration.
Culture medium used for plating consisted of 1 mL of Iscove's
modified Dulbecco's medium (IMDM; GIBCO) containing 24%
FCS, 0.8% deionized bovine serum albumin (BSA; Sigma Chemicals, St Louis, MO),
mol/L 2-mercaptoethanol (Sigma), and
methylcellulose at a final concentration of 1.3% in 35"
Lux
tissue culture dishes (Miles Scientific, Naperville, IL). Culture
plates were incubated in a humidified atmosphere of 5% CO, in air
for 10 to 12 days and scored using an inverted microscope.
Aggregates 2 40 cells were considered colonies.
Controls for each experiment were as follows: medium alone
served as controls for experiments using DNR, medium with 0.1%
DMSO served as controls for experiments using Tmx, and medium
with 0.008% DMSO served as controls for experiments using
verapamil.
Quantitation of membrane binding with HYB-241. Samples of
cells to be studied for PGP expression were suspended at a
concentration of 1 X lo6 in 0.5 mL PBS. Two microliters of the
MoAb HYB-241 (kindly provided by Dr L. Rittmann-Grauer,
Hybritech Corp, San Diego, CA) was added to the test sample and
2 pg of the isotypic control antibody anti-gardenella GDJ352
(Hybritech) was added to the control sample. Both samples were
processed simultaneously. The cells were first incubated on ice for
1 hour and then washed in PBS. Fifty microliters of a 1:64 dilution
of ice-cold fluorescein isothiocyanate (FITC) conjugated goat
antimouse IgG (Sigma) was then added and incubated for 1 hour
on ice. Samples were then washed three times in PBS and
fluorescence was measured immediately on the FACScan. Consort
30 and Lysis software (Becton Dickinson, San Jose, CA) was used
for data analysis.
Estrogen and progesterone receptor analysis. Estrogen and pro-
gesterone receptor analysis was performed using the dextrancoated charcoal assay?8
RESULTS
HYB-241 binding to HL-6OIRV+ and CEM-VBL cells.
Figure 1A shows that the CEM-VBL cells incubated with
MoAb HYB-241 strongly fluoresce, indicating the presence
of PGP. Background fluorescence was determined by incubation with the control MoAb (GDJ352). No PGP-related
fluorescence was detected in the parent cell line CEM.
Figure 1B shows the same experiment performed with
the MDR subline HLdO/RV+ and its parent line HL-60.
The HLdO/RV+ cells strongly fluoresce after incubation
with HYB-241; no PGP-related fluorescence was detected
.+.HY6241
A
100
101
102
103
104
Fluorescence
HYB241
B
io0
101
io2
103
104
Fluorescence
Fig 1. (A) PGP expression in CEM and CEM-VEL cell cells using
flow cytometric analysis with the murine MoAb HYB-241 and a
secondary goat-antimouse FITC-conjugated antibody. The antigardenella GDJ352 was used as isotypic control. (E) PGP expression in
HL-60 and HL-6O/RV+ cells using the same technique as above.
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820
BERMAN ET AL
im
4
ONR+V
DNR+TMXSO
DNR+V
DNR+TMXlO
DNR+TMXlO
ONR+M
I
V
TMXW
TMxm
AD5
15
30
15
60
90
Tim in Minuka
:I
DNR+M
”
“1
I
dz
B10 0
5
30
15
6
nm in Mlnuka
in the HLdO/RV+ cells incubated with the control MoAb
or on the parent cell line HL-60 incubated with HYB-241.
Effect of different concentrations of verapamil on DNR
uptake. DNR plus verapamil 10 pmol/L, 25 pmol/L, and
50 pmol/L were incubated for 1 hour in both CEM-VBL
and HL-60/RV+ cells and analyzed for intracellular DNR
concentration. No significant differences were seen among
the different concentrations of verapamil used (data not
shown). The lowest dose of verapamil, 10 pmol/L, was used
as a control in the Tmx experiments.
Effect of Tmx on DNR uptake and retention in CEM-YBL
and HL-6O/RV+ cell lines. Figure 2A shows the results of
uptake studies in CEM-VBL cells after exposure to DNR 1
pg/mL alone or in Combination with Tmx 10 pmol/L, Tmx
50 pmol/L, or verapamil 10 pmol/L. Cells incubated with
Tmx alone or verapamil alone served as controls. The
simultaneous incubation of DNR and verapamil 10 kmol/L
demonstrated a fivefold increase in DNR uptake compared
w
Fig 2. (A and 8 ) DNR uptake
as measured by flow cytometric
analysis in CEM-VBL and HLbO/
RV+ cells after simultaneous incubation with Tmx (10 bmol/L
[TMXlO] or 50 bmol/L FMX5OI).
verapamil (V), or medium (M).
with control. Compared with the control, simultaneous
incubation of DNR and Tmx 10 pmol/L increased DNR
uptake nearly threefold. Uptake was increased nearly
fivefold with the combination of DNR and Tmx 50 pmol/L.
Intracellular retention of DNR was also studied in
CEM-VBL cells. Cells were incubated with DNR alone,
DNR with Tmx, or DNR with verapamil. After 1 hour of
incubation cells were resuspended in either medium or
medium containing the initial incubation concentration of
Tmx or verapamil, and DNR intracellular content was
subsequently measured at various time points.
The results are shown in Fig 3A. Cells incubated initially
with DNR and resuspended in medium for up to 2 hours
demonstrated a rapid decrease in intracellular DNR content. Similar results were observed when the combinations
of DNR plus Tmx 10 pmol/L and DNR plus verapamil were
incubated for 1 hour and resuspended in medium. The
combination of DNR plus Tmx 50 pmol/L incubated and
la
4
ONR+TMXSO
DNR+TMM
ONR+TMXlO
+M
+ TMXlO
+M
ONR+TMX10 + M
ONR+V
ONR
Fig 3. (A and B) DNR retention as measured by flow cytometric analysis in CEM-VBL and
HL-GO/RV+ cells after simultaneous incubation with Tmx (10
bmol/L IrMXlO] or 50 bmol/L
FMX5OD. verapamil (VI, or medium (M).
DNR+V
+ TMXSO
+V
DNR+TMXlO + WIO
DNR+TMXSO + M
+M
DNR+V + M
DNR+W10 + M
‘1
DNR + M
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821
TMX EFFECT ON MDR CELLS
medium alone or fresh medium containing the initial
incubation concentration of Tmx or verapamil. The pattern
of DNR retention in these cells was similar to that observed
with the CEM-VBL cell line and is shown in Fig 3B. Cells
incubated for 1 hour with DNR and either Tmx 10 kmol/L
or verapamil and then resuspended in fresh medium had
rapid efflux of intracellular DNR. Cells incubated with
DNR and Tmx 10 pmol/L and resuspended in Tmx 10
pmoVL had intracellular DNR concentrations similar to
cells incubated with DNR and Tmx 50 pmol/L and resuspended in medium. Intracellular DNR levels were lower in
both instances than cells incubated with DNR and verapamil and resuspended in verapamil.
As observed in the CEM-VBL line, HLdO/RV+ cells
incubated with DNR and Tmx 50 pmol/L and resuspended
in Tmx 50 pmol/L maintained high intracellular DNR
levels throughout the 2-hour period of reincubation.
Effect of T m on the drug-sensitive parent cell lines CEM
and HL-60. CEM and HL-60 cells were studied for DNR
uptake as described above. Tmx did not increase DNR
uptake in either of these cell lines (Figs 4A and B,
respectively). Additionally, incubation of these cells with
verapamil did not increase DNR uptake.
Effect of Tmx and verapamil on clonogenic capacity of
CEM-VBL and HL-6O/RV+ cells. Clonogenic assays were
then performed to determine the relationship between
DNR retention and cell kill. The first set of experiments
was performed after 60 minutes of incubation with DNR
and the drug under study. To determine whether prolonged
exposure to the drug resulted in increased cell kill over that
observed when cells were resuspended in fresh medium
alone, a second set of clonogenic experiments was performed after an additional 2 hours of resuspension in either
drug or fresh medium.
Figure 5A outlines the effect of Tmx on the clonogenic
resuspended in medium led to a threefold increase in DNR
retention.
To determine whether continuous exposure to verapamil
or Tmx was important to maintain increased intracellular
levels of DNR, a second set of samples was analyzed after
resuspension in fresh medium containing the same concentration of drug that was used for the initial 1 hour of
incubation; eg, cells that were incubated for 1 hour with
DNR plus Tmx 10 pmoVL were washed twice and resuspended in fresh medium containing Tmx 10 pmol/L. Retention was measured at specific time points and results from
these experiments are also shown in Fig 3A. Cells that were
exposed to DNR and Tmx 10 pmoVL and resuspended for 2
hours in Tmx 10 FmoVL had DNR retention similar to that
of cells exposed to Tmx 50 pmol/L for 1 hour and resuspended in medium. Cells exposed to DNR and verapamil
and resuspended in verapamil retained a high intracellular
DNR content as shown.
CEM-VBL cells that had been incubated with DNR plus
Tmx 50 pmol/L and then resuspended in medium containing Tmx 50 pmol/L demonstrated the highest intracellular
concentration of DNR. As shown in Fig 3A, intracellular
DNR concentration was nearly 10 times that of cells
resuspended in medium alone.
Effect of T m on DNR uptake and retention in HL-6O/RV+
cells. DNR uptake studies with Tmx and verapamil were
also performed using HLdO/RV+ cells. Figure 2B shows
that cells incubated for 1 hour with DNR and Tmx had
increased intracellular DNR levels compared with cells
incubated with DNR alone. Cellular uptake appeared
dose-dependent with Tmx 50 pmol/L demonstrating the
highest accumulation.
DNR retention experiments were also performed after 1
hour of exposure to DNR alone, DNR with Tmx, or DNR
with verapamil. Cells were then resuspended in either fresh
7
600
4wl
a-
200
2001w 80-
lr:::
Fig 4. (A and 8 ) DNR uptake
as measured by flow cytometric
analysis in CEM and HL-60 cells
after simultaneous incubation
with Tmx (10 kmol/L FMXlO] or
50 imol/L ~ M X 5 0 1 ) .verapamil
(VI, or medium (M).
I W0
a0
E
‘i
6
2
2
1
A
w:M
30
45
15Tima in Mlnutes
60
-
1
8
0
5
15
M
45
Time in Minutes
W
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-
a22
BERMAN ET AL
% Control Growth
A
0 10 20 30 40 50 60 70 80 90 tM)+lO
60’
I
I
I
I
I
I
(
I
l
l
(
1
1
,
(3)
TMX 10
-
(4) TMX 50
I
El--+
El+
-
(11) TMX 10
.1%
(12) TMX 10-TMX 10
(13) TMX 50
.1%
(14) TMX 50 -TMX 50
(15)
(16)
(17)
(18)
(19)
V-M
V-V
DNR+TMX 10- .l%
DNR+TMX 10- TMX 10
DNR+TMX 50-.1%
(20) DNA +TMX 50
(21) DNR+V-M
(22) DNA+V-V
-
-
TMX 50
v
(6)
DNA +TMX 10
(7)
(8)
DNR+TMX 50
DNR+V
I
E+-+
m
120’
(9) DNR-M
(10) DNR-DNR
(11) TMX 10
.1%
(12) TMX 10-TMX 10
I
I
I
L c - - + - - - C
-
(5)
I
-
(13) TMX 50
.l%
(14) T M X 5 0 d T M X 5 0
8-1
b
V-M
V-V
DNR+TMX 1 0 4 .l%
ONR+TMX IO-TMX 10
ONR+TMX 50- .l%
(20) DNR+TMX 50-TMX 50
I
B
Eb
(21) DNR+V-M
(22) DNR+V-V
El+
B+
m
i
I+
(15)
(16)
(17)
(18)
(19)
I
Fig 5. (A and B) Clonogenic growth of CEM-VBL and HL-6O/RV+ cells after incubation in DNR, Tmx, verapamil, or combinations. 0.1%
indicates control medium with 0.1% DMSO, which served as control mediumfor the Tmx experiments. Control growth is designated as 100%. See
text for details. Cells were incubated for 1 hour in medium or designated drug. Aliquots were then washed twice and plated in triplicate at a
concentration of 250 cells/plate. Colony growth was counted after 10 days of incubation. The percent inhibitioncompared with control is shown
in the bar graph under the column 60’. Each bar on the graph represents the mean of three experiments f standard deviation. Percent inhibition
was also evaluated after an additional 2 hours of incubation. Aliquots obtained after the initial 1 hour of incubation were resuspended for an
additional2 hours in either fresh medium or medium containingthe designated drug. Aliquots were then washed twice and plated in triplicate at a
concentration of 250 cellslplate. Colony growth was counted after 10 days of incubation. The percent inhibitioncompared with control growth is
shown in the bar graph under 120’. Each experimentrepresentsthe mean of three experiments standard deviation.
growth of CEM-VBL cells. Cells incubated in control
medium alone for 1 hour served as controls (100%) (panel
1). Tmx 10 pmol/L had no effect on the growth of these cells
after 1 hour of incubation (panel 3); an additional 2 hours
of resuspension in Tmx 10 p.mol/L did not result in any
further inhibition (panel 12). Clonogenic growth after 1
hour of incubation with DNR 1 kg/mL demonstrated 22%
inhibition (panel 2). However, after CEM-VBL cells were
exposed to the combination of DNR 1 pg/mL and Tmx 10
kmol/L for 1 hour, approximately 50% growth inhibition
was noted (panel 6). Resuspension for 2 additional hours in
medium (panel 17) or Tmx 10 pmol/L (panel 18) led to a
greater degree of inhibition, 62% and 72%, respectively.
Tmx 50 pmol/L was more inhibitory to CEM-VBL cells
than Tmx 10 pmol/L: 36% inhibition was noted after 1 hour
of incubation of CEM-VBL cells (panel 4), which increased
to 72% after an additional 2 hours of exposure in fresh
medium containing Tmx 50 kmol/L (panel 14). Cells
exposed to the combination of DNR 1 pg/mL and Tmx 50
pmol/L demonstrated a strong degree of inhibition, 86%,
after 1 hour of incubation (panel 7); an additional 2 hours
of resuspension in either fresh medium (panel 19) or fresh
medium containing Tmx 50 ymol/L (panel 20) also resulted
in a strong degree of growth inhibition, 82% and 99%,
respectively.
Figure 5B outlines clonogenic experiments performed
using HLdO/RV+ cells. As observed with the CEM-VBL
cells, Tmx 10 pmol/L had no inhibitory effect after 1hour of
exposure (panel 3). After an additional 2 hours of resuspension in either fresh medium (panel 11) or fresh medium
containing Tmx 10 p,mol/L (panel 12), a small amount of
inhibition was noted, 21% and 29%, respectively. Incubation of HL-60/RV+ cells with the combination of DNR 1
p,g/mL and Tmx 10 kmol/L resulted in a significant degree
of inhibition after 1 hour of exposure, 65% (panel 6);
resuspension for an additional 2 hours in either fresh
medium with 0.1% DMSO alone (panel 17) or fresh
medium with 0.1% DMSO containing Tmx 10 kmol/L
(panel 18), a similar degree of inhibition was noted, 76%
and 89%, respectively.
As noted with the CEM-VBL cells, incubation with Tmx
50 pmol/L resulted in -50% inhibition after 1 hour of
exposure (panel 4). Resuspension in fresh medium containing Tmx 50 kmol/L for 2 hours resulted in 95% inhibition
(panel 14). The combination of DNR plus Tmx 50 pmol/L
was also strongly inhibitory after 1 hour of exposure (panel
7) and after resuspension in either fresh medium (panel 19)
or fresh medium containing Tmx 50 pmol/L (panel 20); in
the latter instance, 100% inhibition was noted.
Growth inhibition after exposure to verapamil alone and
in combination with DNR was also examined in CEM-VBL
cells after 1 hour of exposure (Fig 5A, panels 5 and 8) and
-
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TMX EFFECT ON MDR CELLS
after 2 hours of resuspension in either medium alone (panel
15) or medium containingverapamil (panel 16). Results are
similar to those seen with Tmx 10 kmol/L the combination
of DNR and verapamil was much more inhibitory to cell
growth than either drug alone (panels 21 and 22). A similar
effect was noted in HL-60/RV+ cells (Fig 5B, panels 5 and
8 after 1 hour of incubation and panels 15, 16, 21, and 22
after 2 hours of resuspension).
Estrogen and progesterone receptor analysis of CEM-B L
and HL-6O/RV+ Cells. To determine whether the toxicity
of Tmx correlated with expression of either estrogen or
progesterone receptors on the cells, receptor analysis was
performed. CEM cells had no detectable estrogen or
progesterone receptors. CEM-VBL cells had no detectable
estrogen receptor but were positive for the progesterone
receptor (9.5 fentomole [fm]/mg protein). The HL-60
parent line and the HLdO/RV+ line were negative for both
estrogen and progesterone receptors.
DISCUSSION
In this report we demonstrate that Tmx can increase
DNR uptake and retention in two human leukemia cell
lines that display the MDR phenotype. The increase in
DNR uptake with Tmx was concentration dependent in
both the CEM-VBL cells (Fig 2A) and the HLdO/RV+
cells (Fig 2B) and is unrelated to Tmx's known antiestrogenic properties because estrogen receptors were absent
from both cell lines studied. Tmx had no effect on DNR
uptake in the drug sensitive parent cell lines (Figs 4A
and B).
Ramu et a1 first recognized that Tmx was able to reverse
drug resistance in a murine leukemia cell line in 198419and
postulated that, by virtue of its structure, the hormone
interacted with the membrane phospholipid domain in such
a manner as to alter the lipid packing density and thereby
the diffusion rate of certain drugs.29However, the exact
nature of its interaction with the cellular membrane has not
been clarified. Clarke et alM have suggested that the
plasticity of the membrane and, secondarily, its transport
systems may be affected by highly lipophilic hormones. This
group measured fluorescence of the fluorophore 1,6diphenylhexatriene in the human breast cancer cells lines
MCF-7 (estrogen receptor positive) and MDA-MB-436
(estrogen receptor negative) and demonstrated that both
estrogen and Tmx can significantly decrease the fluidity of
the cellular membranes.= Clarke et a13' have also suggested
that these membrane changes account for their own previous observation concerning the dose-dependent reduction
of the cytotoxic effects of methotrexate when 17b-estradiol
is added to MDA-MB-436 cells'* and for the decrease in
cytotoxicity induced by melphalan or fluorouracil when
Tmx is added to both the estrogen receptor positive and
negative breast cancer cell lines recently described by
Osborne et al.33
More recent evidence suggests that Tmx interacts with
specific enzyme and calcium transport systems. For example, it has been shown that Tmx antagonizes phospholipid
and calmodulin-activated protein kinase C.34,35
Significantly,
Greenberg et a1 have also shown that Tmx can inhibit
023
calcium influx and can compete for calcium binding sites in
the neurosecretory cell line PC12.36It is possible that Tmx
binds directly to PGP; that hormones can do so has been
~ , ~ ~et
recently demonstrated by two separate g r o ~ p s .Yang
alZ0have shown that progesterone significantly inhibited
[3H] vinblastine binding to membrane vesicles prepared
from MDR cells; hydrocortisone and testosterone did so to
a lesser degree. Moreover, progesterone enhanced vinblastine accumulation in these cells and increased their sensitivity to vinblastine.mNaito et aP7subsequently confirmed that
progesterone bound specifically to PGP.
That Tmx may also bind to PGP rather than exert a more
generalized effect on membrane function is suggested by
the results of the experiments presented here. If Tmx
altered membrane permeability such that DNR diffused in
more rapidly, then intracellular DNR retention would not
be expected to remain constant over time as, presumably,
efflux would be just as rapid. However, the retention
experiments in both the CEM-VBL (Fig 3A) and the
HLdO/RV+ (Fig 3B) cell show that intracellular DNR
concentrations remain relatively constant over time, suggesting that active efflux of the drug is blocked. Photoaffinity
labeling studies are planned that will hopefully determine
whether Tmx binds directly to PGP.
Data from the clonogenic experiments with both CEMVBL (Fig 5A) and HL-60/RV+ cells (Fig 5B) demonstrate
that Tmx 10 kmol/L had little inhibitory effect on cell
growth. As expected in these drug resistant cell lines, DNR
alone did not have a significant inhibitory effect on cell
growth. However, incubating cells for 1 hour with the
combination of Tmx 10 pmol/L and DNR resulted in a
marked enhancement in cytotoxicity compared with cells
exposed to DNR alone. When cells were subsequently
resuspended in fresh medium containing Tmx 10 pmol/L,
inhibition of cell growth increased further. This result
would suggest that continued exposure to Tmx results in a
higher percentage of cell death. The effect of verapamil on
growth inhibition is shown in Figs 5A and B. Similar to Tmx
10 kmol/L, the inhibitory effect of incubating cells with
verapamil and DNR is increased compared with incubating
cells in DNR alone.
Based on the in vitro data presented here we suggest a
potential new role for Tmx in patients with relapsed or
refractory leukemia (or any solid tumor that demonstrates
the MDR phenotype) whereby the hormone is given in high
doses for a short period of time, followed by administration
of a drug affected by the PGP pump such as DNR.
However, we recognize that significant difficulty may exist
in applying these results directly to the clinical setting. First,
not all patients with relapsed or refractory disease have
PGP expressed on their cells384o
and overexpression of PGP
may be heterogeneous within a given population of cells."
It is clear that further data regarding PGP expression is
needed before defining the extent of this form of drug
resistance and it will be extremely important to correlate
PGP expression with clinical resistance.
Second, results with the two cell lines tested here suggest
that a high concentration of Tmx, in the range of 10 p,mol/L,
is effective in increasing the intracellular concentration of
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
824
BERMAN ET AL
DNR in MDR cells. Both these cell lines are many-fold
resistant to DNR, and it may be that lesser amounts of Tmx
are needed in the clinical setting. However, it is important
to note that pharmacokinetic properties of Tmx allow for
high, prolonged serum levels: the half-life of the hormone is
biphasic, with an initial serum half-life of 7 t o 14 hours and
a secondary half-life of more than 7 days4' With a standard
dose of Tmx (20 to 40 mg/d), serum levels are in the range
of 300 ng/mL or 0.8 p,mol/L and may take up to 28 days t o
a~hieve.~'
Higher doses of Tmx, in the range of 200 mg/d,
have been used and serum levels in this instance are -5
~mol/L.~
By* administering an initial loading dose of Tmx
followed by high daily doses for a short period of time it may
be possible to achieve high serum levels within 1 week.43
Preliminary results of a phase I trial of high-dose Tmx
combined with vinblastine in patients with advanced renal
cancer suggest that such an approach is feasible.44
Third, Tmx does generate some degree of toxicity even a t
standard doses. The main side effects of Tmx are nausea
and hot f l a ~ h e s ; transient
~'
leukopenia and thrombocytopenia have also been
as have venous and arterial
thromboses, although the incidence of the latter two
complications is
High doses administered over a
prolonged period of time (240 t o 320 mg/d administered for
more than a year), can cause abnormalities of the cornea
and retina.'" Concern exists, however, that toxicity may be
altered when Tmx is administered to reverse the MDR
phenotype, given that PGP has been detected on a variety
of normal tissues such as normal human kidney, adrenal
~~~~'
Cordon-Cardo e t
gland, liver, and ~ o l o n . Additionally,
a149have shown that endothelial cells lining the trachea,
lung, endometrium, and prostate as well as acinar cells in
the pancreas all express the PGP. However, previous
studies combining verapamil with vinblastine,' adriamycin," or a vincristine-adriamycin-decadronregimen" have
not shown excessive toxicity apart from the anticipated
cardiac side effects of verapamil (hypotension and heart
block). Moreover, Fine e t a15' have shown that verapamil in
doses high enough t o reverse the MDR phenotype does not
inhibit normal marrow granulocyte-macrophage colony
forming unit. Whether Tmx in similar doses is toxic to
normal marrow is currently under study.
Lastly, it may be possible to combine a lower dose of both
Tmx and verapamil and achieve the same degree of PGP
reversal. Preliminary uptake experiments in HLdO/RV+
cells have shown that the combination of Tmx 5 pmol/L and
verapamil 2 p,mol/L are additive in their effect (Berman,
unpublished data). Further evaluation of other triphenylethylene derivatives such as toremifene is also warranted as
preliminary evidence also suggests that this compound is
also able t o reverse the MDR phenotype."
ACKNOWLEDGMENT
The authors thank Dr Melvin S. Center for his gift of HL-60I
RV+ cells and his many helpful comments, Azita Mairzadah for
the verapamil studies, and Dr Celia Menendez-Botet for performing estrogen and progesterone receptor analysis. The authors also
thank Duane Jones for his expert assistance in the preparation of
this manuscript.
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1991 77: 818-825
Effect of tamoxifen on cell lines displaying the multidrug-resistant
phenotype [see comments]
E Berman, M Adams, R Duigou-Osterndorf, L Godfrey, B Clarkson and M Andreeff
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