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(CANCER RESEARCH 49, 5638-5643. October 15. I989|
Influence of Microenvironmental
pH on Adriamycin Resistance1
Oliver Alabaster,2 Tina Woods, Vilma Ortiz-Sanchez, and Saleem Jahangeer
Division of Hematology and Oncology, George Washington University, Washington, DC 20037
ABSTRACT
Resistance to Adriamycin (ADR) is frequently dependent upon en
hanced efflux associated with the expression of the IV!DR1-encoded P
membrane glycoprotein. Since enhanced expression of the MDR1 gene
in ADR-resistant cells may be the result of spontaneous genetic mutation
or amplification, it is presumed to be relatively stable and unalterable.
Yet, reducing ADR efflux could increase sensitivity, and has been at
tempted using calcium channel blockers and other drugs. However, since
the tumor cell microenvironment varies with respect to pH because of
differences in vascularization, oxygénation,and metabolite clearance, the
possibility exists that these factors could influence drug transport and
the critical biochemical pathways which determine cytotoxicity, even in
resistant cells.
Using flow cytometric analysis of ADR fluorescence, the influx and
efflux of IÃœMM ADR dissolved in MIS buffer (piI 6.5) and 4-(2hydroxyethylene)-l-pipera/ineethanesulfonicacid
buffer (pi I 7.5 and 8.5)
was measured in sensitive P388 and resistant P.388/R84 cells in vitro.
Substantially enhanced uptake of ADR was detected at alkaline pi I in
both cell populations, while the proportion of ADR-positive cells and the
level of ADR uptake was decreased at lower pH. Acidification reduced
ADR efflux, whereas alkalinization increased efflux when the uptake pi I
was 6.5 or 7.5. At uptake pH 8.5, the pH of the external buffer had little
effect, even in resistant cells. In resistant cells in an alkaline microenvi
ronment, ADR transport and retention were superior to that observed in
sensitive cells in an acidic microenvironment. No differences were ob
served in ADR transport when the transmembrane pH gradient was
equilibrated. These observations are especially relevant to the effect of
ADR on tumor cell subpopulations that are acidic, and in which drug
diffusion is inefficient. Efforts to alkalinize tumor cells prior to ADR
therapy might reduce ADR resistance, even of genetic origin.
INTRODUCTION
Chemotherapeutic cytotoxicity depends upon many factors
such as an adequate drug concentration in the tumor cell
microenvironment, the cell cycle distribution, the rate of bidi
rectional drug transport across the cell membrane, relative rates
of drug activation and deactivation, and the subsequent inhibi
tion of cell reproductive processes, with or without lethal met
abolic paralysis. Yet, even when these factors appear to favor
drug cytotoxicity a variable proportion of cells usually survive
drug treatment, particularly in solid tumors.
The most important reason for cell survival following cytotoxic drug exposure is the presence or development of genetic
variants that are relatively drug resistant. Rapid mutation of
malignant cells has been hypothesized to account for the devel
opment of resistant clones that finally emerge as predominant
when sensitive cell subpopulations are eradicated during treat
ment (1). Apart from such spontaneous mutations, evidence
exists for the transfer of drug-resistant genes between cells
within a cell population (2); a mechanism that would also
enhance survival of cell subpopulations within a tumor sub
jected to chemotherapy. Another important genetic mechanism
Received 12/14/88; revised 6/29/89; accepted 7/13/89.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported in part by NIH Grant RO1-CA32756.
2To whom requests for reprints should be addressed, at Division of Hematology and Oncology, Suite 421, Ross Hall, George Washington University, Wash
ington, DC 20037.
of drug resistance is the development of pleiotropic drug resist
ance following drug exposure (3, 4). At the cellular level, this
is characterized by the development of a wide range of cross
resistance to anticancer drugs, usually of natural plant origin,
that is induced by previous drug exposure. Activation of the
multidrug-resistant gene (MDR1) is associated with the expres
sion of a specific membrane glycoprotein of M, 170,000 (5, 6).
The effect of this energy-dependent membrane pump protein is
to enhance the efflux of intracellular drugs such as vincristine
and ADR.' Further studies have demonstrated that as the gene
which encodes this protein is amplified, drug efflux is increased
(7). The underlying mechanism by which this protein increases
drug efflux has been elucidated in a number of studies that have
demonstrated binding of vinblastine and anthracyclines to this
P170 membrane glycoprotein (8, 9), and precipitation of the
drug-protein complex by a monoclonal antibody to the PI70
glycoprotein (10). Although expression of the P170 membrane
glycoprotein has been demonstrated in selected patients with
ovarian cancer (11), it is not yet known how frequently such
expression occurs or is amplified in tumor cell populations
during clinical chemotherapy.
Nevertheless, there is increasing interest in finding ways to
reverse pleiotropic drug resistance by increasing drug uptake
and reducing drug efflux. Specifically, ADR resistance has been
diminished by the administration of verapamil, a calcium an
tagonist (12), and by certain drugs such as phenothiazines which
bind to calmodulin, the calcium regulatory protein (13-15).
In evaluating the effect of drugs on ADR transport, the flow
cytometric technique (16-20) has the advantage of predomi
nantly measuring the free ADR in individual cells. This tech
nique not only permits the detection of cells which contain
measurable levels of ADR, but also the detection of cell subpopulations with different ADR levels (18, 19), and the detec
tion of cells that do not contain free ADR.
In this study, we used flow cytometric analysis to measure
the influence of extracellular pH (and intracellular pH) on the
uptake and efflux of ADR in sensitive P388 and resistant P388/
R86 cells (18, 19). These studies indicate that the microenvironmental pH influences the uptake of ADR, the proportion of
cells that contain ADR, the relative intracellular ADR concen
tration, and the rate of efflux of ADR: all factors that can
influence the cytotoxic process. Furthermore, to contrast the
effects of intracellular and extracellular pH, the transmembrane
pH gradient was equilibrated to determine the effect of intra
cellular pH on ADR influx and efflux.
MATERIALS AND METHODS
The P388/R84 and P388 murine leukemia cell lines were maintained
as suspension cultures in RPMI 1640 medium supplemented with 10%
fetal bovine serum, antibiotics (penicillin and streptomycin), and 5 n\\
2-mercaptoethanoI. The cells were passaged every 2 to 3 days by diluting
one third of the suspension culture with fresh medium. For determi
nation of ADR uptake and efflux, log phase cells were removed from
culture flasks, washed twice with ice-cold PBS, using centrifugation at
1,000 rpm for 5 min at 4°C.Equal numbers of cells were resuspended
1The abbreviations used are: ADR. Adriamycin; PBS. phosphate buffered
saline; MES. (2[/V-Morpholmo]Ethane Sulfonic Acid): HEPES, 4-(2-hydroxyethylene)-1 -piperazineethanesulfbnic acid.
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INFLUENCE OF MICROENVIRONMENTAL
pH ON ADR RESISTANCE
in three flasks containing prepared buffers of pH 6.5, 7.5, and 8.5. The
pH 6.5 buffer was prepared by dissolving MES in RPMI so that the
final buffer concentration was 25 mivi MES. The pH was adjusted by
adding HC1. The pH 7.5 buffer was prepared by dissolving HEPES in
RPMI so that the final concentration was 25 mM HEPES. The pH was
adjusted by the addition of NaOH. Finally, the pH 8.5 buffer was also
prepared with HEPES in the same way as the pH 7.5 buffer, except
that NaOH was added until the pH was 8.5. ADR stock solution (1
IHM;Adria Laboratories, Inc., Columbus, OH) was added to each buffer
to obtain a final ADR concentration of 10 nM. The final osmolarities
of the three buffers were 307, 310, and 327 mOsm/liter; respectively.
Triplicate flasks containing P388 and P388/R84 cells in each of the
three ADR-containing buffers were then transferred to a water bath at
37°Cand the cells were allowed to incubate for varying time periods.
Trypan blue viability exceeded 90% in all experiments during the
selected time periods, which also correlated with viability by light scatter
analysis (21). At predetermined time points, a small aliquot of cells was
removed from each flask, washed twice in ice cold PBS, and resuspended in a small volume of ice-cold PBS prior to immediate flow
cytometric analysis, using a FACS IV (Becton Dickinson) equipped
with a 4-W argon laser at 488 nm. Using the well-established technique
for measuring intracellular ADR by flow cytometry (16-20), cells were
counted and analyzed by amplified measurement of forward-angle light
scatter and simultaneous fluorescence, which distinguished cells that
were fluorescence negative from those that were positive. Fluorescence
emission was only collected beyond 520 nm using an appropriate band
pass filter. At least 50,000 cells were measured during each sample
analysis; and each sample was analyzed several times to ensure stability
and reproducibility of the histograms. Data were collected on a PDP11/34 computer (Digital Equipment Corporation) using NIH analytical
software. Data analysis included measuring median ADR fluorescent
intensity and counting the number of ADR-positive and -negative cells.
Each complete experiment was repeated at least three times, but some
variations in the time intervals made exact data comparisons impossi
ble. Note that before these experiments were performed, drug-free cells
were analyzed to determine the possible influence of autofluorescence.
Any background fluorescence was then eliminated from the data analy
sis by establishing a fluorescence threshold for the presence of ADR,
which in turn was correlated with an appropriate cell-light-scatter
signal. In addition, FACS amplifier gain settings were standardized to
ensure fluorescence comparability between all samples.
Initial uptake experiments monitored ADR concentration in cells
after incubation for 15, 45, 75, 105, and 135 min, respectively. The
effect of drug concentration was evaluated at ADR concentrations of
5, 10, and 50 ^M respectively (data not shown). Comparable pH effects
on ADR uptake were evident at all ADR concentrations, but 10 juM
was arbitrarily selected for the complete range of experiments on influx
and efflux described here.
The effect of pH 6.5, 7.5, and 8.5 (for 90 min at 37°C)on ADR
uptake was then measured, and the cells were washed once in cold (4°C)
matching drug-free buffers before being resuspended in warm (37°C)
drug-free buffers of pH 6.5, 7.5, and 8.5, respectively. ADR efflux was
then measured in each cell population after 30 min and 90 min,
respectively, using flow cytometric analysis of residual cell fluorescence.
RESULTS
Experiments with sensitive and resistant cells were always
done concurrently so that differences between cell lines could
be evaluated under standardized conditions. However, when
experiments were repeated weeks or months apart, variations
in ADR uptake were sometimes detected that may have resulted
from differences in cell growth phase at the time cells were
harvested for experiments. Typical examples of data are there
fore presented.
Flow cytometric analysis of ADR uptake in P388/S (Fig. 1,
top) and P388/R84 (Fig. 1, bottom) cells after 105 min, is
shown in Fig. 1. In these examples of fluorescence histograms,
there is clear evidence of a considerable difference in the inten-
PH6.5
PH7.5
pHS.5
RELATIVE
FLUORESCENT
INTENSITY
[ADR]
pH6.5
pH7.5
PH8.5
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RELATIVE
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150
200
INTENSITY
[ADR]
250
Fig. 1. Flow cytometric histograms from sensitive P388 (top) and resistant
P388/R84 (bottom) cell lines following exposure of these cells to ADR (10 MM)
for 105 min in buffers of pH 6.5, 7.5, and 8.5, respectively. Note the shift in
fluorescence intensity derived from different levels of intracellular ADR. Subpopulations of greater ADR concentration are seen in P388 at pH 6.5, and P388/
R84 at all pH values. Although all experiments were repeated several times, small
variations in time intervals precluded comparative statistical analysis.
sity of ADR fluorescence depending upon the pH of the drugcontaining buffers in both sensitive and resistant cell popula
tions. Furthermore, it is possible to discern two subpopulations
that have differing fluorescent intensity among sensitive cells
in a pH 6.5 buffer, and among resistant cells that were exposed
to ADR in all three buffers.
Fig. 2 indicates the proportion of P388 cells (Fig. 2, top) and
P388/R84 cells (Fig. 2, bottom) that have taken up ADR. The
median level of ADR per cell is indicated by the median channel
number (fluorescence intensity) of ADR uptake for the cell
population. In these experiments, the proportion of P388 cells
that take up ADR at pH 6.5 is lower than that at pH 7.5 and
8.5 and the median intensity increases significantly with in
creasing pH. However, in this particular experiment, the uptake
of ADR by P388/R84 cells at pH 6.5 revealed a smaller
proportion of ADR-positive cells, but with a comparable me
dian fluorescent intensity. At pH 7.5, the median fluorescence
remained the same, while the proportion of ADR-positive cells
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INFLUENCE
OF MICROENVIRONMENTAL
pH ON ADR
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Fig. 2. Top and bottom, progressive uptake of 10 /IM ADR in buffers of pH
6.5, 7.5, and 8.5 in P388 and P388/R84 cell lines, respectively. D, percentage of
cells with detectable ADR levels; M, median concentration of ADR in the cell
population; areas above open bars, proportion of ADR-negative cells. In P388/
R84 cells at pH 8.5, ADR uptake at 105 min and 135 min revealed two
subpopulations. The majority of the cells are in the right column and have lower
median fluorescence intensity; the minority of cells are in the left column, with
higher median fluorescence intensity.
90
30
90
Time, Minutes
increased. After 105 and 135 min at pH 8.5, there were two
distinctive subpopulations of differing fluorescence intensity.
These subpopulations were analyzed separately and consisted
of a minority population of relatively high median fluorescence,
and a majority population of lower median fluorescence. How
ever, when combined, these two subpopulations consisted of
100% of the cell population.
Fig. 3 reveals the influence of pH on the uptake and subse
quent efflux of ADR in sensitive P388 cells (Fig. 3, top) and
resistant P388/R84 cells (Fig. 3, bottom). After 90 min of
incubation in a buffer of pH 6.5, ADR was taken up by nearly
the whole sensitive cell population, but only by about 80% of
the resistant cells. Cells were then transferred to buffers of pH
6.5, 7.5, and 8.5, and the efflux was measured at 30 and 90
min, respectively. After 30 min of efflux in a buffer of pH 6.5,
the proportion of both P388 and P388/R84 cells that contained
ADR was nearly halved; by 90 min the fraction of ADR-positive
cells was halved again. The median intensity of ADR fluores
cence was also approximately halved during this period, but
remained constant as the fraction of ADR-positive cells de
clined.
Efflux of ADR was greater at pH 7.5 and 8.5 At efflux pH
7.5, in both sensitive and resistant cell populations, about half
as many cells were positive for ADR as were seen at efflux pH
6.5, while the median intensity of fluorescence remained essen
tially unchanged. Within 30 min at efflux pH 8.5, there were
again about half as many cells positive for ADR than were seen
at efflux pH 7.5, and ADR efflux was greater still, being
virtually complete by 90 min in both sensitive and resistant
populations. Interestingly, at 30 min efflux was not diminished
in P388/R84 cells at pH 8.5, since the median ADR concentra
tion was similar to that found in the P388 cell population.
Fig. 3. Top and bottom, uptake of 10 (¿M
ADR after 90 min by P388 and
P388/R84 cells in a buffer of pH 6.5, followed by efflux in separate buffers of pH
6.5, 7.5, and 8.5, for 30 and 90 min, respectively. In P388 cells at efflux pH 8.5,
the solid bar within the shaded area represents the proportion of ADR positive
cells. A similar situation exists at pH 7.5 and 8.5 in the P388/R84 cells. In both
cell populations an acidic microenvironment delayed efflux, and an alkaline
microenvironment accelerated efflux. Among resistant cells fewer contained
ADR, although the median ADR concentration was similar to that found in
sensitive cells.
In Fig. 4, ADR uptake occurred at pH 7.5 and involved
nearly 100% of both cell populations. However, the median
ADR concentration was higher in P388 cells (Fig. 4, top).
Differences in efflux were seen between sensitive and resistant
cells. At efflux pH 6.5, it was evident that most P388 cells
remained ADR positive, although there was a decline in the
median drug concentration. In contrast, while many P388/R84
cells (Fig. 4, bottom) rapidly became ADR negative, the median
ADR concentration declined by about a third and then stabi
lized.
At efflux pH 7.5, a similar response was seen. Only at efflux
pH 8.5 did substantial differences occur between the sensitive
and resistant cell populations. Although there was a decline in
the number of ADR-positive P388 cells, there was a much
greater reduction in the proportion of ADR-positive P388/R84
cells. Despite this, the median drug concentration in P388/R84
cells remained comparable to that in sensitive P388 cells. By
90 min, all ADR had been lost from resistant cells.
Finally, in Fig. 5, it is evident that all cells took up ADR at
pH 8.5, although the median ADR concentration was lower in
P388/R84 cells (Fig. 5, bottom). Relatively little efflux was seen
at any pH among P388 cells (Fig. 5, top) compared to the efflux
seen in P388/R84 cells. Furthermore, there was little effect of
time on efflux in the sensitive population. Again, among resist-
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INFLUENCE OF MICROENVIRONMENTAL
pH ON ADR RESISTANCE
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Time, Minutes
Time, Minutes
PH8.5
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Fig. 4. Top and bottom similarly represent the uptake of 10 >JMADR by P388
and P388/R84 cells after 90 min in a buffer of pH 6.5, 7.5, and 8.5, respectively.
At this uptake pH there was greater contrast between sensitive and resistant
populations, with many more sensitive cells retaining ADR than resistant cells.
The median ADR concentration remained relatively high among resistant cells,
although the relative number of ADR-positive cells declined substantially.
ant cells, the greatest effect on efflux was seen in the declining
fraction of ADR-positive cells, with comparatively less effect
on the median ADR concentration.
DISCUSSION
There is an assumption which is implicit in many pharma
cological studies of chemotherapeutic drug action and resist
ance, that the degree of cytotoxicity is dependent upon stable
and predictable biochemical pathways within tumor cells. Fur
thermore, it is presumed that intrinsic sensitivity and resistance
is predominantly determined by variations in gene expression
that occur as a result of a high spontaneous mutation rate in
tumor cell populations (1). After all, variations in drug sensitiv
ity occur much less frequently in more stable normal cells,
which happen to be the specific site of toxicity. The greater
predictability of the response of specific normal cells to cytotoxic drug action may not only result from more genomic
stability, but also from a much better regulated cell microenvironment. Although the tumor cell microenvironment is known
to be much more variable than that of normal cells, it is less
clear to what extent this microenvironment can influence the
degree of drug resistance, particularly when it is of genetic
origin.
Despite known differences in the MDR1 expressed P-glycoprotein level in the P388 and P388/R84 cell lines (19), the data
reveal differences in drug transport and efflux which are not
Fig. 5. The top and bottom panels also represent the uptake of 10 JJMADR by
P388 and P388/R84 cells, respectively, after 90 min in buffer of pH 8.5, followed
by efflux in separate buffers of pH 6.5, 7.5, and 8.5, respectively. The median
ADR uptake concentration was highest in this alkaline microenvironment in both
sensitive and resistant cell populations, with 100% of cells being ADR positive.
Uptake of ADR in resistant cells at pH 8.5 is greater than that at pH 6.5 in
sensitive cells. Resistant cells which took up ADR at pH 8.5 displayed efflux
kinetics that revealed more ADR retention than that seen in sensitive cells which
took up ADR at pH 6.5.
easily explained simply on the basis of a modified efflux pump.
In particular, it is now evident that the microenvironmental pH
during the uptake of ADR influences subsequent efflux. Yet, it
remains to be determined whether pH variation can also influ
ence the effect of calcium channel blockers on ADR transport.
Other factors apart from the intracellular drug concentration
influence the relative sensitivity of cells to anthracyclines: the
capacity for DNA repair, the activity of DNA topoisomerase II
and the activity of the redox cycle may have varying importance
in different cell populations or subpopulations (22-25). How
ever, the influence of pH on these factors was not measured. It
remains possible that any change in the degree of ADR cyto
toxicity induced by variations in the pH of the microenviron
ment may be modulated not only by changes in ADR transport,
but also by the influence such variations in the extracellular pH
may have on the intracellular pH, which could modulate such
critical cell functions.
The flow cytometric method of analyzing ADR uptake and
efflux, although predominantly measuring free intracellular
ADR, is also an effective means to measure the heterogeneity
of ADR uptake and efflux within a cell population. The meas
ured heterogeneity of ADR transport suggests that relatively
resistant cells exist within the sensitive P388 cell population,
and that relatively sensitive cells exist within the resistant P388/
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INFLUENCE OF MICROENVIRONMENTAL
R84 cell population. Although enhanced efflux is not the only
basis for ADR resistance, this interpretation is supported by
evidence that intracellular ADR levels as measured by the flow
cytometric method have been shown to correlate closely with
subsequent cytotoxicity (26).
When evaluating these data, it is important also to exclude
any potential artefacts or other factors that might have caused
changes in cellular fluorescence. Since the fluorescence emis
sion intensity of ADR is known to be influenced by DNA
binding, it is important to assess an effect of pH itself on the
proportion of ADR bound to DNA. In fact, no significant effect
of pH within the range 6.4-7.8 has been observed on ADR
binding to DNA (27). Neither was any effect of pH observed
on the quantum efficiency of ADR during these experiments.
Loss of cell viability and cell breakdown could also potentially
distort fluorescence measurement, but the fluorescence signals
were only measured from cells that generated an adequate lightscatter signal, which is an effective means of distinguishing
viable from nonviable cells and of estimating cell volume (21).
This procedure ensured that fluorescence was derived only from
viable intact cells.
The data presented here provide information unavailable
from standard pharmacological drug transport studies. In par
ticular, the histograms reveal the proportion of ADR-negative
cells (within the limits of detection); the proportion of ADRpositive cells; and the median fluorescent intensity, which is
directly proportional to the intracellular drug level. The various
pH levels were designed to model variations in the in vivo tumor
cell microenvironment that might influence both ADR uptake
and efflux, and hence cytotoxicity.
The data indicate that ADR efflux is most sensitive to an
alkaline microenvironment when the uptake pH is acidic in
both sensitive and resistant cells. In contrast, when ADR uptake
occurs at pH 7.5, the proportion of ADR-positive cells falls
more rapidly and the efflux is increased by an alkaline microen
vironment (pH 8.5), especially in resistant cells. However, when
the uptake of ADR occurs in an alkaline microenvironment
(pH 8.5), efflux is delayed in both cell lines, even when the pH
of the efflux microenvironment is also alkaline (pH 8.5). In
fact, in an alkaline microenvironment, ADR transport and
retention in resistant cells was superior to that observed in
sensitive cells in an acidic microenvironment. Furthermore, no
additional differences in influx or efflux were observed when
the pH gradient across the cell membrane was equilibrated by
the addition of ammonium acetate (data not shown).
These observations suggest the possibility that alkalinization
of ADR prior to cell entry altered the drug in a way that was
not achieved by the alkalinization of the intracellular compart
ment after drug entry. They also suggest that ADR efflux,
which is only significantly enhanced by an alkaline microenvi
ronment when the uptake pH is acidic or neutral, may be the
result of an effect at the level of the cell membrane rather than
on ADR itself. Interestingly, there is evidence that ADR uptake
into unilamellar vesicle systems may be increased in response
to transmembrane pH gradients (interior acidic) (28). Other
studies have suggested that ADR uptake is dependent upon
passive diffusion, which is influenced by the permeability of the
cell membrane to unionized molecules (27). This might partly
account for the influence of pH on ADR uptake, but probably
does not explain the pH effects seen on energy-dependent ADR
efflux.
Although it is not possible to explain all these pH effects on
ADR transport, it is possible to consider the implications of
such effects within tumors where the microenvironment varies
pH ON ADR RESISTANCE
with respect to pH. Inevitably, drug concentrations are higher
near blood vessels within solid tumors, but because of variations
in oxygénationand metabolite clearance, it is likely that this
microenvironment is closer to neutral pH than more distant
sites where drug concentrations are lower and the pH more
acidic; conditions that favor cell resistance to ADR.
Clearly, the effects of pH on ADR transport support the
conclusion that intrinsic drug resistance could be modified by
changes in the tumor cell microenvironment. The alkalinization
of tumors prior to ADR administration, if technically feasible,
might lead to enhanced destruction of otherwise resistant cell
subpopulations.
ACKNOWLEDGMENTS
The authors would like to express their sincere appreciation to Dr.
Awtar Krishna who generously provided the P388 cell lines.
REFERENCES
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
Goldie, J. H.. and Goldman. A. J. The genetic origin of drug resistance in
neoplasms: implications for systemic therapy. Cancer Res., 44: 3643-3653,
1984.
Cadman, E., Wong. D., and Liu, E. Drug-resistant genes can be sponta
neously transferred among malignant cells. In: T. C. Hall (ed.), Cancer Drug
Resistance, pp. 11-20. New York: Alan R. Liss, 1986.
Daño,K. Active outward transport of daunomycin in resistant Erlich ascites
tumor cells. Biochim. Biophys. Acta, 323: 466-473, 1973.
Myers, C., Cowan, K., Sinha, B., and Chabner. B. The phenomenon of
pleiotropic drug resistance. In: V. T. DeVita. Jr.. S. Hellman, and S. A.
Rosenberg (eds.). Import Advances in Oncology, pp. 27-38. Philadelphia: J.
P. Lippincott Company. 1987.
Juliano. R. L., and Ling, V. A surface glycoprotein modulating drug perme
ability in Chinese hamster ovary cell mutants. Biochim. Biophys. Acta, 455:
152-162, 1976.
Ling. V. Genetic basis of drug resistance in mammalian cells. In: N. Bruchowsky and J. H. Goldie (eds.). Drug and Hormone Resistance in Neoplasia,
pp. 1-19. Boca Raton, FL: CRC Press, 1982.
Fojo, A. T., Akiyama, S., Gotesman, M. M., and Pastan, I. Reduced drug
accumulation in multiple drug-resistant human KB carcinoma cell lines.
Cancer Res., 45: 3002-3007, 1985.
Cornwell. M. M., Safa, A. R., Felsted, R. L., et al. Membrane vesicles from
multidrug-resistant human cells contain a specific 150-170 kDa protein
detected by photo-affinity labeling. Proc. Nati. Acad. Sci. USA, 83: 38473850, 1986.
Safa, A. R., Glover, C. J., Meyers, M.B., et al. Vinblastine photoaffinity
labeling of a high molecular weight surface membrane glycoprotein specific
for multidrug-resistant cells. J. Biol. Chem., 261: 6137-6140. 1986.
Kartner, N., Evernden-Porelle, D., Bradley. G., et al. Detection of P-glycoprotein in multidrug-resistant cell lines by monoclonal antibodies. Nature
(Lond.), 316: 820-822, 1985.
Bell. D. R., Gerlach, J. H., Kartner, N., et al. Detection of p-glycoprotein in
ovarian cancer: a molecular marker associated with multidrug resistance. J.
Clin. Oncol., 3: 313-315, 1985.
Tsuruo. T.. Lida. H., Nojioi, M., Tsukagoshi, S., and Sakurai, Y. Circum
vention of vincristine and Adriamycin resistance in vitro and in vivo by
calcium influx blockers. Cancer Res., 43: 2905-2910, 1983.
Prozialeck, W., and Weiss, B. Inhibition of calmodulin by phenothioazines
and related drugs: structure-activity relationships. J. Pharm. Exp. Ther., 222:
509-516. 1982.
Tsuruo, T.. Lida, H., Tsukagoshi. S.. and Sakurai, Y. Increased accumulation
of vincristine and adriamycin in drug-resistant p388 tumor cells following
incubation with calcium antagonist and calmodulin inhibitors. Cancer Res.,
42:4730-4733, 1982.
Ganapathi, R., and Grabowski, D. Enhancement of sensitivity to adriamycin
in resistant p388 leukemia by calmodulin inhibitor trifluoperazine. Cancer
Res., 43: 3696-3699, 1983.
Krishan, A., and Ganapathi, R. Laser flow cytometry and cancer chemother
apy: detection of intracellular anthracyclines by flow cytometry. J. Histochem. Cytochem., 27: 1655-1656, 1979.
Krishan, A., and Ganapathi, R. Laser flow cytometric studies on intracellular
fluorescence of anthracyclines. Cancer Res.. 40: 3895-3900, 1980.
Krishan, A., Sauerteig, A., and Wellham. L. Flow cytometric studies on
modulation of cellular adriamycin retention by phenothiazines. Cancer Res.,
45: 1046-1051, 1985.
Krishan, A.. Sauerteig. A., Gordon, K., and Swinkin, C. Flow cytometric
monitoring of cellular anthracycline accumulation in murine leukemia cells.
Cancer Res.. 46: 1768-1773. 1986.
Luk, C. K., and Tannock, I. F. Flow cytometric analysis of doxorubicin
accumulation in cells from human and rodent cell lines. J. Nati. Cancer Inst.,
81: 55-59, 1989.
5642
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1989 American Association for Cancer Research.
INFLUENCE OF M1CROENVIRONMENTAL
21. Alabaster, O., Woo, W. B.. and Magrath. I. T. Separation of viable and
nonviable human lymphoma cells influences flow cytometric analysis of DNA
content. In: Flow Cytometry IV, pp. 91-95. Universitetsforlaget, Norway,
1980.
22. Batist. G.. Tulpule. A., and Sinha. B. K., el al. Overexpression of a novel
MIIn MIu glutathione transferase in multidrug resistant human breast cancer
cells. J. Biol. Chem.. 261: 15544-15549. 1986.
23. Deffie, A. M., Alam, T., and Seneviratine, C., et al. Multifactorial resistance
to Adriamycin: relationship of DNA repair, glutathione transferase activity.
drug efflux, and P-glycoprotein in cloned cell lines of adriamycin-sensitive
and resistant P388 leukemia. Cancer Res.. 48: 3595-3602. 1988.
24. Kramer. R. A.. Zakher.J.. and Kim, G. Role of the glutathionine redox cycle
25.
26.
27.
28.
pH ON ADR RESISTANCE
in acquired and de novo multidrug resistance. Science (Wash. DC), 241: 694697, 1988.
Moscow, J. A., and Cowan, K. H. Multidrug resistance. J. Nati. Cancer Inst.,
SO: 14-20, 1988.
Durand. R. E., and Olive, P. L. Flow cytometry studies of intracellular
Adriamycin in single cells in vitro. Cancer Res., 41: 3489-3494. 1981.
Skovsgaard, T. Transport and binding of daunorubicin, Adriamycin. and
rubidazone in Erlich Ascites Tumor Cells. Biochem. Pharmacol., 26: 215222, 1977.
Mayer, L. D., Bally, M. B.. and Cullis. P. R. Uptake of Adriamycin into
large unilamellas vesicles in response to a pH gradient. Biochim. Biophys.
Acta. 857: 123-126, 1986.
5643
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1989 American Association for Cancer Research.
Influence of Microenvironmental pH on Adriamycin Resistance
Oliver Alabaster, Tina Woods, Vilma Ortiz-Sanchez, et al.
Cancer Res 1989;49:5638-5643.
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