The Role of Cellular Accumulation in Determining Sensitivity to Platinum-Based Chemotherapy*

Abstract

The platinum (Pt) drugs cisplatin and carboplatin are heavily employed in chemotherapy regimens; however, similar to other classes of drugs, a number of intrinsic and acquired resistance mechanisms hamper their effectiveness. The method by which Pt drugs enter cells has traditionally been attributed to simple passive diffusion. However, recent evidence suggests a number of active uptake and efflux mechanisms are at play, and altered regulation of these transporters is responsible for the reduced accumulation of drug in resistant cells. This review suggests a model that helps reconcile the disparate literature by describing multiple pathways for Pt-containing drugs into and out of the cell.

Full text

ANRV333-PA48-18 ARI 4 December 2007 16:1
The Role of Cellular
Accumulation in
Determining Sensitivity
to Platinum-Based
Chemotherapy∗
Matthew D. Hall, Mitsunori Okabe,
Ding-Wu Shen, Xing-Jie Liang,
and Michael M. Gottesman
Laboratory of Cell Biology, National Cancer Institute, National Institutes
of Health, Bethesda, Maryland 20892-4255; email: mgottesman@nih.gov
Annu. Rev. Pharmacol. Toxicol. 2008. 48:495–535
First published online as a Review in Advance on
October 15, 2007
The Annual Review of Pharmacology and Toxicology is
online at http://pharmtox.annualreviews.org
This article’s doi:
10.1146/annurev.pharmtox.48.080907.180426
Copyright c
2008 by Annual Reviews.
All rights reserved
0362-1642/08/0210-0495$20.00
∗The U.S. Government has the right to retain a
nonexclusive, royalty-free license in and to any
copyright covering this paper.
Key Words
cisplatin, carboplatin, resistance, cellular accumulation, drug
uptake
Abstract
The platinum (Pt) drugs cisplatin and carboplatin are heavily em-
ployed in chemotherapy regimens; however, similar to other classes
of drugs, a number of intrinsic and acquired resistance mechanisms
hamper their effectiveness. The method by which Pt drugs enter cells
has traditionally been attributed to simple passive diffusion. How-
ever, recent evidence suggests a number of active uptake and efflux
mechanisms are at play, and altered regulation of these transporters
is responsible for the reduced accumulation of drug in resistant cells.
This review suggests a model that helps reconcile the disparate liter-
ature by describing multiple pathways for Pt-containing drugs into
and out of the cell.
495
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ANRV333-PA48-18 ARI 4 December 2007 16:1
INTRODUCTION
The development of resistance to platinum (Pt)-based chemotherapy in the clinic is
a major challenge for cancer chemotherapy. Although the phenomenon of multidrug
resistance against natural product drugs exemplified by the ATP-dependent efflux
pump P-glycoprotein and other transporters is well characterized (1), the cellular
responses that confer resistance to cisplatin (DDP, cis-[PtCl2(NH3)2], Figure 1) are
multifactorial and less well understood (2, 3). This is particularly so for the reduced
drug accumulation commonly reported in Pt drug-resistant cell lines (4). The gener-
ally accepted intracellular mechanisms by which cells acquire resistance to cisplatin
and its congeners are (a) increased detoxification of drugs by the thiols glutathione
and metallothionein; (b) improved repair of, and tolerance to, nuclear lesions, leading
to a concomitant reduction in apoptosis; and (c) diminished accumulation of cisplatin
(3, 5).
Pt
NH
2
(H
2
C)
6
H
2
N
H
3
N
NH
3
Cl
Pt
NH
2
(H
2
C)
6
H
2
NNH
3
H
3
N
Pt
H
3
NNH
3
Cl
3+
Pt
H
3
N
H
3
N
Cl
Cl
Pt
H3N
H3N
O
O
O
O
Pt
H
2
N
N
H
2
O
O
O
O
Cisplatin Carboplatin Oxaliplatin
Pt
H
2
N
N
H
2
Cl
Cl
Cl
Cl
Pt
H
3
N
N
Cl
Cl
CH
3
Tetraplatin
Pt
H
2
N
H
3
N
Cl
Cl
O
O
CH
3
O
CH
3
O
Satraplatin
Pt
H
3
N
H
2
N
Cl
Cl
JM118
ZD0473
BBR3464
PtH
3
NCl
Cl
H
2
N
H
N
O
O
Pt-1C3
Figure 1
Platinum complexes
described in the text.
496 Hall et al.
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ANRV333-PA48-18 ARI 4 December 2007 16:1
0
20
40
60
80
100
120
0 5 10 15 20 25 30 35 40
Fold resistance
Cisplatin accumulation (%)
Figure 2
Plot of cisplatin-resistant
human-derived cell lines
showing the correlation
between the fold-resistance
of cell lines and the percent
decrease in cisplatin
accumulation compared
with the parental cell line.
Adapted from data in
References 8 and 9,
representing nine unique
parental cell lines and
fourteen selected cell lines.
Studies reported over the past 30 years have assessed the ability of Pt-containing
drugs to accumulate in cancer cell lines and measured the ability of new compounds to
accumulate in resistant cells (thereby circumventing resistance), and they have consis-
tently demonstrated that accumulation of drug is a determinant of cellular sensitivity
(6, 7). In a survey of human- and rodent-derived cisplatin-resistant cell lines reported
by Andrews & Howell, most cell lines demonstrated reduced cisplatin accumulation
relative to their parental strains (adapted with new data for Figure 2), and accumula-
tion defects were more common than increased glutathione expression (8). Johnson
et al. reported a strong correlation (r =0.98) between cisplatin accumulation and
relative cisplatin resistance for a series of increasingly resistant lines derived from the
BEL7404 human hepatoma cell line (9). Koga et al. examined seven primary bladder
cancer cell lines derived from untreated transitional cell cancer of the urinary bladder
(10) and found a positive correlation between cisplatin accumulation and sensitivity
(r =−0.778) among the intrinsically resistant cell lines, but no correlation with GSH
levels or expression of a range of proteins (10). Figure 2 demonstrates this general
trend for a range of resistant cell lines.
It is important to note that the diminished accumulation1of drug with increasing
resistance is not unique to cisplatin, and it has been observed for the clinical analogs
carboplatin ([Pt(CBDCA)(NH3)2]) (11) and oxaliplatin ([Pt(oxalato)(R,R-chxn)],
L-OHP) (12) and compounds in clinical trials such as tetraplatin ([PtCl4(chxn)]) (13)
and ZD0473 (cis-[PtCl2(NH3)(2-methylpyridine)]) (14). In addition, cross-resistance
among Pt complexes was observed at an early stage in Pt drug research (15), and in
1There is some variability in the literature in the terms used to describe drug flux into and out of cells, and
total intracellular drug levels. Accumulation is used here to avoid the possible ambiguity of the term uptake.
The term uptake can be interpreted to reflect only the passage of drug into cells irrespective of the mechanism
of cell entry, whereas the net total drug levels within a cell at the end of a given period of exposure is the
accumulation of drug. The exit of drug from cells is termed efflux. Uptake and efflux of a molecule can be
by passive diffusion down the concentration gradient, by facilitated transport down a concentration gradient,
or by active (energy-dependent) transport insensitive to the concentration gradient. Reduced uptake versus
increased efflux is not as easy to demonstrate as might be supposed because total accumulation is most often
used as a parameter in the literature and efflux can be rapid enough to mimic reduced uptake.
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ANRV333-PA48-18 ARI 4 December 2007 16:1
some cases reflects reduced accumulation of these compounds (although there are
exceptions to this, see below) (11).
There are a number of complicating factors associated with examining cisplatin-
accumulation defects; a wide range of cell lines from different tissue types have been
utilized in studies, with resistant cell lines usually selected by chronic exposure to
physiologically unrealistic levels of Pt drug to elicit a measurable alteration in cisplatin
accumulation. Further, the relative importance of each of the resistance mechanisms
described in vitro has not yet been demonstrated to correlate with intrinsic or acquired
resistance mechanisms in clinical samples. Stewart et al. examined Pt concentrations
in human autopsy samples and demonstrated that patients whose tumors responded
to Pt-containing therapies had higher tumor Pt concentrations than those that failed
to respond, seeming to indicate that Pt accumulation is an important factor for clinical
efficacy (16).
The major questions relating to Pt drug accumulation are:
1. How does cisplatin enter cells?
2. Is cisplatin actively extruded from cells, and if so how?
3. What are the cellular changes in acquired resistance associated with reduced
cisplatin accumulation?
The diverse and often disparate literature on the mechanisms by which cisplatin
and its congeners enter and leave cells has not been assessed in detail since an ex-
cellent review by Gately & Howell published in 1993 (6) [more recent results have
been summarized in part by Andrews (17)]. The conclusions of this survey were that
cisplatin enters the cell by passive diffusion and through gated channels (6). Early
studies noted that cellular uptake of cisplatin was linear, concentration dependent,
and nonsaturable—all hallmarks of passive (rather than actively mediated) drug entry
into the cell. Barnett Rosenberg, the discoverer of cisplatin, stated that, “the drug
is passively transported across the cellular membrane—no active transport is nec-
essary,” and this paradigm seems to have ruled drug design approaches (18). This
review explores more current data in the literature and from our own laboratory,
and we conclude that accumulation of cisplatin occurs by a variety of mechanisms,
including passive diffusion and facilitated transport by multiple transport systems.
To reduce drug accumulation to a significant extent, or to confer cross-resistance
to multiple cytotoxic Pt-containing drugs, cells must simultaneously inactivate more
than one of these transport systems, and this pleiotropic response occurs in cells se-
lected for cisplatin resistance in vitro. These uptake systems vary depending on the
species of Pt drugs under consideration, so we begin with a detailed consideration
of the chemistry of Pt species [see below and Supplemental Section on Speciation
of Platinum Drugs (follow the Supplemental Material link from the Annual Reviews
home page at http://www.annualreviews.org)].
SPECIATION OF PLATINUM DRUGS
The question of how Pt drugs enter cells is inextricably tied to an understanding of
what specie(s) enter cells. One of the main assumptions of studies into cellular uptake,
498 Hall et al.
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ANRV333-PA48-18 ARI 4 December 2007 16:1
and associated cisplatin resistance mediated through diminished drug accumulation,
is that the neutral, intact drug is the species that traverses the lipid bilayer by passive
diffusion. Conventional wisdom has been that neutral cisplatin is prevented from
aquation/hydrolysis outside the cell owing to the high chloride levels present in
plasma and media (19). Recent results question the need for a neutral Pt complex for
efficacy (20). Farrell and colleagues have reported a series of “rule-breaking” multi-
nuclear complexes that show increasing accumulation with increasing positive charge
(21), and a number of transporters have been implicated in Pt drug accumulation
defects that do not necessarily transport intact drug. It may be that one or more
biotransformation products of Pt drugs contribute to the pool of drug that enters
cells by a number of different pathways (see below).
The key mechanistic step in the activation of cisplatin is the first aquation step;
the replacement of a chloride leaving group by water (H2O) gives a singly positively
charged aquachloro species (2in Figure 3) that can then be hydrolyzed to a hydroxo
ligand (OH) yielding the neutral chlorohydroxo complex (4in Figure 3). Note that 2
is neutral, and could also potentially diffuse across the lipid bilayer. The proportion of
aquachloro (2) to chlorohydroxo (4) species is dependent on the pKaof the hydroxo
ligand, and hence the pH of the solution (22). It is the aquated species (2) that is
more reactive (labile) and can subsequently bind to the N7 of guanine and adenine
on DNA in the cell, the generally accepted cytotoxic target of cisplatin. Speciation
studies using a range of techniques, particularly those pioneered by Appleton and
Berners-Price/Sadler using isotopically labeled 15N cisplatin (see Reference 23 for an
excellent review of these techniques and experimental examples), have demonstrated
that the aquation of cisplatin is suppressed (but not prevented) in a high-chloride
environment (∼104 mM) of blood plasma and equivalent media, compared with the
low (4 mM) chloride concentration in the cytoplasm. Jennerwein & Andrews have
examined the intracellular concentration of chloride in a range of human carcinoma
cell lines, normally stated to be 4 mM, and shown it to range from 20–55 mM (24).
A number of studies have examined cisplatin aquation at varying pHs, concentra-
tions of chloride, and temperatures (reviewed in References 25, 26), incorporating
equilibria and acidity constants determined primarily by House and coworkers (22).
These have been used to determine the relative proportions of the aquation species
present at high and low chloride concentrations (though the dynamic nature of plasma
means a true equilibrium never exists). Figure 3 includes two sets of data from the
literature showing the percent of each of the aquation products of cisplatin present
at pH 7.4.
The two sets of values cited in Figure 3 for high chloride concentration are in
good accord with one another and reveal that at a high chloride concentration, the
two dominant species are cisplatin (1) and the chlorohydroxo species (4), both of
which are neutral and relatively unreactive. The only other significant species is the
reactive aquachloro (2) complex that would be expected to undergo rapid reaction and
deactivation in biological media. Given the pKaequilibrium that exists between 2and
4in solution, experimental determination of the lipophilicity of 4is difficult. However,
using experimentally verified theoretical prediction (27), it has been calculated that
the lipophilicity of cisplatin (1in Figure 3, log Poct =−2.4) is actually similar to
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ANRV333-PA48-18 ARI 4 December 2007 16:1
Pt
H
3
N
H
3
N
Cl
Cl
Pt
H
3
N
H
3
N
OH
2
Cl
Cl
–
H
2
O
Pt
H
3
N
H
3
N
OH
2
OH
2
Cl
–
H
2
O
+2+
123
Pt
H
3
N
H
3
N
OH
Cl
4
Pt
H
3
N
H
3
N
OH
OH
2
+
5
Pt
H
3
N
H
3
N
OH
OH
6
67
4
<1
26
<1
1
104 mM (26)
68
7
<1
24
<1
<1
100 mM (22)
3
5
<1
30
28
35
4 mM* (26)
1
2
3
4
5
6
Compound
*Note that reported fractions total to >100%.
Figure 3
Schematic showing the
stepwise aquation and
hydrolysis of cisplatin in
aqueous solution. The
percentage of each species
calculated to exist at
equilibrium (pH 7.4) in
high- (100 mM and
104 mM) and low-chloride
(4 mM) concentrations is
also shown in the table
below.
that of the hydroxo species cis-[PtCl(OH)(NH3)2](4, log Poct =−2.7), and as such
both species have a similar potential for diffusing across a lipid bilayer (28).2
The assumption that cisplatin diffuses into the cell carries the implicit expecta-
tion that charged species could not enter the cell by such means. While the doubly
aquated species 3,5, and 6are observed in simple aquation studies, their forma-
tion in biological media is doubtful given the rapid reaction of the singly aquated
2Although it may seem initially surprising that displacement of chloro with hydroxo gives a similarly lipophilic
complex, Lewis acidity barely affects lipophilicity. For example, ethanol and dimethylether have similar log
Poct values.
500 Hall et al.
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ANRV333-PA48-18 ARI 4 December 2007 16:1
species with biomolecules in the extracellular medium and cell membrane. Ham-
bley and coworkers observed only the monoaquated species when using HSQC
NMR to monitor the reaction of cisplatin with DNA, as this reacted with nucle-
obases at a rate greater than that which would lead to formation of the diaqua species
(29).
Given the near ubiquitous use of cell lines for studies of Pt uptake, it is important
to consider that the concentration of chloride in blood plasma is usually reported
as around 104 mM, and the chloride concentration in RPMI growth medium is
108 mM and phosphate buffered saline (PBS) is 137 mM. PBS is usually employed
for short-term exposure of drug by researchers who want to minimize aquation and
side reactions with components of media. The levels of phosphate in PBS (∼9 mM)
do not appear to interfere with the primary aquation products of cisplatin, although
Davies et al. report that the formation of phosphate products with the diaqua species
of cisplatin occurs after 3 h in NMR spectra (30). The commercial formulation of
cisplatin (Platinol in the United States) is supplied in 154 mM NaCl solution (31).
Significantly, Dabrowiak and coworkers examined the aquation of cisplatin in
RPMI growth medium and observed that the major long-lived metabolite of cis-
platin is, in fact, a carbonato complex, cis-[PtCl(OCO2)(NH3)3](7in Figure 4) (31).
In carbonate buffer (but not growth medium), the bicarbonato complex (8) is also de-
tected. Given that the concentration of carbonate in growth media and blood plasma is
∼24 mM, and the propensity of carbonate as a ligand for metal ions (32), it is surprising
that these species were not considered sooner. When Jurkat T-lymphocyte cells were
added to the growth media, the signal corresponding to 7disappeared rapidly, sug-
gesting that the negatively charged complex may be rapidly accumulated by the cells.
Pt
H3N
H3N
Cl
Cl
Pt
H3N
H3N
OH2
Cl
Pt
H3N
H3N
OCO2
Cl
+
–
12
7
Pt
H3N
H3N
OH
Cl
4
8
Pt
H3N
H3N
OCO2
OCO2
2–
Figure 4
The formation of carbonato
species. (7)
cis-[PtCl(OCO2)(NH3)3]
can form in both growth
medium and carbonate
buffer; however, the
dicarbonato (8) is only
observed in carbonate
buffer. Adapted from
Reference 31.
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Similar considerations come into play with carboplatin and other Pt compounds (see
Supplemental Section on Speciation of Platinum Drugs and References 33–38).
As well as exchange reactions with electrolytes and small molecules, cisplatin can
bind covalently and noncovalently (reversibly) with proteins present in cell growth
media. It is generally reported that protein binding renders Pt drugs inactive (39), and
cisplatin preincubated with serum protein is not cytotoxic in vitro (40) or active in vivo
(41). Preincubation in cell culture medium also diminishes cisplatin cytotoxicity to a
lesser extent (42). Hambley and coworkers have shown that after 6 h in RPMI medium
supplemented with FCS at 37◦C (standard cell line growth media conditions), 63%
of the cisplatin analogue cis-[PtCl2(en)] was free (passed through a 30 kDa filter),
and 25% was free after 24 h (43). The ultrafilterable portion has been shown to
be cytotoxic (44). Although reactions with proteins occur in media, a considerable
portion of the drug is free to enter cells during short (0–4 h) exposures. Pt binding to
human plasma proteins is also to some extent reversible (45), although Melvik et al.
have shown that protein-bound Pt cannot enter cells (40).
That metabolites of cisplatin can enter the cell is supported by a number of studies
(46–49). Cisplatin accumulates in cells more rapidly at pH 6 than at pH 7.4 (extracel-
lular tumor pH is often lower than 7.4), suggesting that a species other than cisplatin
(which does not have a pKain the pH range 4–8) can be taken into cells (50), and
resistant cells show less pH dependence on uptake than sensitive cells (51). Species
that cannot hydrolyze, such as [Pt(en)2]2+, are not detectable in cells in short-term
experiments (47). Jennerwein & Andrews found that aquated cisplatin accumulated
in the OV 2008 human ovarian carcinoma cell line at the same rate as intact cis-
platin, and that 1.9 times more DNA binding was found in cells treated with aquated
cisplatin. Using the same cell line, Shirazi et al. prepared a range of cisplatin metabo-
lites, aquated cisplatin, mono- and bis-methionine products, and ultrafiltered human
plasma incubated with cisplatin, and examined their activity alongside cisplatin (48).
The aquated cisplatin (created by dissolving cisplatin in water) was three times more
active than cisplatin after 1 h exposure, and the mono-methionine and ultrafilterable
cisplatin preparations showed similar potency to cisplatin (the bis-methionine prepa-
ration was inactive). Again, the accumulation of the metabolites after 1 h correlated
with their activity (r =−0.997), and the aquated cisplatin showed a threefold in-
crease in accumulation compared with cisplatin, suggesting that an aquated species
can be preferentially accumulated (48). That the aquated, mono-methionine and
plasma ultrafiltered preparation all showed greater or equipotent activity to cisplatin
demonstrates the complicated nature of the mechanisms of cisplatin accumulation
and activation. These observations are supported by Ehrsson and coworkers, who
isolated pure mono-aquated cisplatin (2) and showed it to be more active against the
U-1285 small-cell lung cancer cell line than cisplatin (49).
An understanding of the specific cisplatin metabolites that form and the nature of
those that can enter cells is essential. Indeed, given the dynamic nature of aquation
and the compartmentalization of drug into proteins, it is possible that a number of
species enter the cell by different routes with varying kinetics, and determining the
routes of entry of active drug into the cell may enable improved drug design.
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ARGUMENTS FOR PASSIVE DIFFUSION
As noted above, early experiments incubating cells with increasing concentrations of
cisplatin and analogues revealed linear uptake that was not saturable against either
time or drug concentration (up to 3 mM), consistent with passive diffusion down a
concentration gradient (52–56). In one of the first papers on uptake, using tritiated
cis-[PtCl2(py)2] (py =pyridine), Gale et al. showed linear uptake in Ehrlich ascites
tumor cells was limited only by solubility, which was confirmed by a double-reciprocal
plot of concentration against uptake that intersected with the origin (54). Gale (54)
and Ogawa (57) found little difference in accumulation and potency at 4◦C and 37◦C
and increased uptake at 60◦C, also suggestive of passive entry into cells. Similar studies
with everted rat intestine, L1210 mouse leukemia cells, and OV2008 human ovarian
carcinoma cells supported these conclusions (52, 56, 58, 59).
Gale and Andrews also showed that other compounds, including Pt compounds,
could not lower accumulation by competitive inhibition as would be expected if a
unique active transporter were at play, whereas compounds that compromised mem-
brane integrity increased accumulation (51, 54). Other compounds believed to enter
the cell by passive diffusion alone, such as mannitol, show similar uptake profiles
in cisplatin-sensitive and -resistant cells (60), and long-term exposure to Pt drugs
correlates well with drug log Poct values, probably because it allows for equilibra-
tion via passive diffusion (27, 61); however, in vivo peak plasma drug concentrations
persist for a short period of time, and uptake kinetics during short-term exposure
are more likely to be relevant to drug efficacy. Results showing nonsaturability of
uptake of cisplatin are consistent either with a major component of passive diffusion
at high cisplatin concentrations or uptake via other nonsaturable systems, such as
fluid-phase endocytosis. The latter process, which allows compounds to enter cells
via membrane-lined invaginations, would also be expected to be energy dependent,
as has been demonstrated (see below).
CHANGES IN PLASMA MEMBRANE COMPOSITION
AND BIOPHYSICS
If plasma diffusion is the major mechanism of cisplatin entry into cells, then cell lines
that show reduced uptake (or accumulation without increased efflux) should have
alterations in the biophysical properties of the plasma membrane. There have been a
number of studies into the interactions of cisplatin, primarily with erythrocyte mem-
branes, showing that cisplatin perturbs membranes (reviewed partially in Reference 62
and summarized in the Supplemental Section on Changes in Plasma Membrane
Composition and Biophysics), although few focus on the nature of cisplatin’s passage
across the cell membrane. Few or no changes have been seen in biophysical stud-
ies of plasma membrane function in cisplatin or carboplatin-resistant cell lines (see
Supplemental Section on Changes in Plasma Membrane Composition and Bio-
physics and References 63–65). Collectively, there is evidence that a drug can en-
ter a cell by passive diffusion; however, given that resistant cells demonstrate only
small changes (if any) in their membrane composition and biophysics, the lowered
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ANRV333-PA48-18 ARI 4 December 2007 16:1
accumulation must be due to other alterations. The relative amount of drug entering
a cell by passive diffusion is dependent on the concentration of drug: at low drug
concentrations, active transporter(s) may mediate the uptake of the majority of drug,
but at high concentrations most uptake would be via passive diffusion.
The ability of drugs to enter cells by passive diffusion led to the design of lipophilic
Pt complexes, such as satraplatin, that are capable of circumventing cisplatin resistance
through increased accumulation in resistant cells, reducing the need for any active
component of uptake (66). It may be that Pt(IV) complexes can only enter cells
by passive diffusion. Hall et al. showed that a series of relatively hydrophilic Pt(IV)
complexes demonstrated lower rates (versus concentration and time) of accumulation
than cisplatin in A2780 cells; however, although cisplatin accumulation is halved in
the A2780 cisR line, the Pt(IV) accumulation is marginally reduced, suggesting that
removing the active transport does not affect Pt(IV) uptake (67). This effect is also
observed in the CH1/CH1cisR pair, where the accumulation of cis- and trans-Pt(IV)
complexes and satraplatin [a Pt(IV) drug] was unaffected. Resistant lines generated
from the 41M and CH1 human ovarian carcinoma cell lines showed a range of cellular
alterations (increased MT and DNA repair), but accumulation was unaffected in both
resistant lines, suggesting that no alterations in membrane composition occur in these
cell lines to reduce Pt(IV) drug uptake (66).
A number of membrane-disrupting agents, such as the detergent gemcitidine and
the antifungal drug amphotericin B, have been shown to enhance Pt drug accumu-
lation in vitro (68, 69) and in vivo (70) by facilitating passage of a drug across the
cell membrane. Other compounds, such as spermidine, have been shown to increase
cellular accumulation by an as-yet-unidentified mechanism (the agents are detailed
in the Supplemental Section on Membrane Disrupting Agents).
EARLY EXPERIMENTS ON CARRIER-MEDIATED UPTAKE
The first postulation that carrier-dependent processes were involved in cisplatin up-
take was made by Byfield & Calabro-Jones in 1981, who observed that proliferating
T-lymphocytes were more sensitive to cisplatin than resting cells, as were other drugs
known to be carrier-mediated, such as melphalan (amino acid carriers) and nitrogen
mustard (choline carrier) (71). This was in contrast to carrier-independent drugs, such
as mitomycin C, which showed identical survival curves for resting and proliferating
cells. The authors argued this effect was due to enhanced import of drugs in cycling
cells. However, the authors did not consider cell phase-specific events, the actual
accumulation of drug was not measured, and as cisplatin was prepared in double-
distilled water, a significant amount of drug would have been aquated (Figure 3),
which may have exaggerated any transport effect.
Early experiments into the accumulation of cisplatin employed compounds that
nonspecifically disrupted membrane protein function. Dornish et al. conducted a
series of early experiments with cisplatin and found that the protein reactive agent
benzaldehyde inhibited cisplatin’s activity in the NHIK 3025 human cervical carci-
noma line (72), and cellular accumulation was shown to be halved in the presence of
the aldehydes benzaldehyde, pyridoxal, and pyridoxal 5-phosphate (73, 74). When
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ANRV333-PA48-18 ARI 4 December 2007 16:1
cells were electropermeabilized, benzaldehyde no longer affected accumulation, and
structural analogs not containing a reactive aldehyde functional group did not af-
fect accumulation (73). Aldehydes form Schiff base imine bonds with protein amine
groups (75), and given that the cell-impermeable aldehyde pyridoxal 5-phosphate
also lowered drug accumulation, it is likely that it deactivates transporters and pre-
vents active accumulation, although no controls (other drug classes or endogenous
substrates) were employed to confirm this general effect (74).
The implication that transporters can mediate a portion of cisplatin accumulation
in cells was reinforced by observations that the metabolic inhibitors dinitrophenol or
sodium fluoride alone did not affect accumulation, but a combination with iodoacetate
did reduce accumulation by almost half in 2008 cells, but to a lesser degree in resistant
2008/DDP cells, suggesting the active component of uptake is already lost in the
resistant cells (52). Shen et al. also showed that carboplatin uptake in 7404 cells was
temperature dependent, and ATP depletion by antimycin A or oligomycin reduced
uptake in 7404 cells to levels observed in 7404-CP20-resistant cells (11).
Andrews et al. noted that preincubation with the Na+,K+-ATPase-specific in-
hibitor ouabain reduced cisplatin accumulation in both the sensitive and resistant
2008 cells by up to 50% (52, 76). Na+,K+-ATPase maintains the sodium gradi-
ent across the cell membrane (pumps sodium out and potassium into the cell), and
a number of facilitated transporters are dependent on this gradient for function.
Na+,K+-ATPase also regulates cell volume, although cell volume was not affected
at the concentrations used (that would artificially alter Pt accumulation values) (76).
Short-term accumulation experiments showed that drug accumulation was imme-
diately reduced on exposure, suggesting Na+,K+-ATPase inhibition affects influx;
replacement of sodium with choline in cell growth media reduced cisplatin uptake by
a similar amount as ouabain; and cisplatin uptake increases with extracellular sodium
concentration, suggesting it is the sodium gradient dissipation rather than Na+,K+-
ATPase inhibition that lowers cisplatin uptake (76). Andrews did not find a difference
in Na+,K+-ATPase expression or function between 2008 and 2008/DDP cell lines,
but Fujimara did observe lowered function in PC-9/CDDP cells (77).
Interestingly, tissues subject to cisplatin toxicity, such as kidney and the inner
ear, do express high levels of Na+,K+-ATPase (76, 78, 79). How the observation that
decreased osmolarity enhances cisplatin accumulation relates to the requirement for a
high sodium gradient remains to be determined (80). D-methionine reverses cisplatin
toxicities associated with Na+,K+-ATPase owing to its antioxidant properties (e.g.,
81), although in actuality, this is probably due to methionine chelation of cisplatin
and subsequent inactivation, as a number of sulfur-containing amino acid analogs
have been shown to be capable of inhibiting nephrotoxicity (82). This may be related
to the observation that cisplatin inhibits tetraethylammonium uptake by organic base
transporters [see Organic Cation Transporters (The SLC22 Family) below] that are
highly expressed in kidney tissues, but cisplatin-methione complexes do not (83).
Perhaps SLC transporters present in tissues require Na+cotransport, or a high Na+
gradient for cisplatin transport.
Sharp et al. showed in two human ovarian carcinoma cell lines (41M and CH1)
that low temperature or ouabain reduced accumulation. However, accumulation was
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ANRV333-PA48-18 ARI 4 December 2007 16:1
not affected in the resistant lines (41McisR6 and CH1cisR6, respectively), suggesting
active/facilitated transport was lost in these selected lines that probably required a
Na+gradient for optimal function (84). Others have subsequently shown a greater
decrease in accumulation with ouabain or ATP-depleting conditions in a range of
parental cells compared with resistant lines (85–87). Kishimoto found that expres-
sion of the Na+,K+-ATPase A1 subunit was reduced in H4-II-E/CDDP-resistant
cells. Bando et al. demonstrated that the expression of Na+,K+-ATPase alone is not
sufficient for increased cisplatin accumulation because, even though Na+,K+-ATPase
was expressed in SCLC and NSCLC cell lines, ouabain inhibition lowered accumu-
lation in three NSCLC lines, but not in three SCLC lines (88).
Experimental evidence indicates that, in part, Pt drug uptake can be mediated
by active or facilitated influx. Nonspecific aldehydes prevent membrane transporters
from functioning and energy-depleting agents lower accumulation either directly or
indirectly. Cisplatin is not a substrate for Na+,K+-ATPase (89), and the inhibition of
Na+,K+-ATPase by ouabain reduces the Na+gradient across the cell membrane that
drives active/facilitated transport of cisplatin, although the specific transporters that
rely on this sodium gradient have not been identified. The observations of Bando et al.
suggest that the mechanisms of drug uptake are tissue-specific (88) and are dependent
on the endogenous expression of transporters. An understanding of tissue-specific
transporter expression may allow for tailored therapies that enhance cisplatin uptake
and efficacy.
THE SEARCH FOR TRANSPORTERS
If a single transporter is at play in the accumulation of cisplatin, then either satu-
ration of accumulation kinetics and/or substrate specificity would be expected. Al-
though saturable kinetics has not been generally observed in native or transfected cells
(discussed above), this could be attributed to a number of transporters, with various
affinities for cisplatin, operating collectively to accumulate drug. Adequately designed
experiments provide evidence of influx transport kinetics. When synthetic diaquated
cisplatin (6in Figure 3) was incubated with cells, the initial rate of uptake was 40-
fold higher than that for cisplatin (but equal in both cell lines), greater at pH 6 than
pH 7.4, but most importantly, tended to be saturable, suggesting the active transport
of aquated cisplatin species can occur (47). Analysis of uptake kinetics yielded a kM=
1.9 mM indicative of a low-affinity transporter, which certainly accounts for the lack
of saturable uptake in studies that limit concentrations to no higher than 500 μM.
Gately and Howell proposed that cisplatin accumulation could be facilitated via
a gated channel, but cisplatin’s minimum cross-section (3.97 ˚
A by 6.92 ˚
A) is greater
than that of most channel pores (17), with the possible exception of aquaporin 9
(AQP9), recently reported (by two-dimensional crystallography) to have a pore size
of approximately 7 ˚
Aby12 ˚
A (90). AQP9 has been shown to permit the passage
of neutral molecules, such as glycerol, urea, purines, and pyrimidines (91), and its
expression correlates with As2O3(arsenite) accumulation in myeloid and lymphoid
leukemia lines (92). Although the ability for the aquaporins to transport Pt drugs
has not been directly demonstrated, we note here (Supplemental Figure 3) that
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ANRV333-PA48-18 ARI 4 December 2007 16:1
Pt-resistant lines have reduced expression of AQP2 and AQP9, presenting a potential
new Pt drug transport family.
Influx selectivity of Pt drugs has been demonstrated: Helleman et al. showed
that the A2780-Pt ovarian carcinoma cell line displayed reduced accumulation of
the cis Pt drugs cisplatin, carboplatin, tetraplatin, and oxaliplatin, but transplatin
accumulation was unaffected (93). This loss of cis-specific drug uptake in resistant
cells is probably not uncommon, but trans complexes are examined less often owing
to the (erroneous) assumption that trans complexes are inactive (94). The unchanged
transplatin accumulation is similar to the effect described above for Pt(IV) complexes,
and suggests that the A2780-Pt cells are lacking a transporter or transporters on the
cell surface that the parental line possesses.
More recently, proteomic and mRNA microarray studies comparing cisplatin-
sensitive and -resistant cell lines have rarely identified altered gene or protein ex-
pression associated with cellular accumulation (either influx- or efflux-related), and
there is a lack of significant cross-over in hits between studies (93, 95–103). It should
be noted that this feature is not unique to transport proteins, and no dominant bio-
chemical pathway emerges from analyses, although mRNA and proteomic screens
do not take account of alterations in protein localization (discussed below) (95). The
only statistically significant alterations in transporter expression reported in these
publications are an increase in SLC27A2 and SLC2A3 and the general decrease in
the mRNA levels of five transport proteins (none of which are implicated in drug re-
sistance) between the Tca8113- and Tca/cisplatin-resistant human oral squamous cell
carcinoma cell lines (102),and a small change in ABCA13 and SLC22A2 expression
in the IGROV-1/CP ovarian cancer cell line (101). SLC22A2 is discussed below.
CTR1 COPPER INFLUX TRANSPORTER
CTR1 (copper transporter 1, SLC31A1) is an evolutionarily conserved copper influx
transporter present in plants, yeast, and mammals, and is the main copper importer in
mammalian cells. The human version, hCTR1, is expressed in all tissues and is a key
player in the exquisite homeostatic regulation of intracellular copper levels to ensure
that nutritional delivery of copper to enzymes such as cytosolic Cu,Zn-superoxide
dismutase and mitochondrial cytochrome oxidase is maintained while surplus copper,
which can cause toxicity, is avoided (104). Human hCTR1 is a 197–amino acid protein
containing three transmembrane domains residing mainly in the plasma membrane,
transporting Cu in a temperature-, pH-, and K+-dependent (but Na+-independent)
manner with saturable kinetics that are not energy-dependent (105). The transporter
can trimerize, forming a channel-like pore that facilitates substrate transport (106).
An extracellular amino terminus of CTR1 containing a methionine- and histidine-
rich domain has been implicated in initial Cu binding (note that both Met and His
side groups are good Pt ligands), followed by Cu internalization by a little-understood
mechanism that is believed to result in lysosomal Cu sequestration prior to cytosolic
trafficking.
The role of CTR1 in cisplatin accumulation has been reviewed, and only the key
observations pertaining to Pt drug influx are described here (107, 108). Deletion of the
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Saccharomyces cerevisiae CTR1 gene lowers accumulation of, and increases resistance
to, cisplatin, and coadministration with copper inhibits cisplatin uptake in wild-type
(Ctr1p expressing) strains (109, 110). Yeast Ctr1p was also demonstrated to transport
oxaliplatin, carboplatin, and ZD0473 (110), and mouse mCtr1 knockout strains also
show reduced cisplatin accumulation (109). Although transfection of hCtr1 cDNA
into SCLC cell lines conferred sensitivity, Song et al. showed that of five resistant
small-cell lung cancer (SCLC) cell lines, only one (SR2) showed a slight reduction
in hCtr1 protein expression, along with a reduction in the accumulation of cisplatin,
carboplatin, and oxaliplatin (111). Alternatively, Zisowsky et al. reported a reduction
in hCtr1 mRNA levels in A2780cis-resistant ovarian and HeLaCK cisplatin-resistant
cell lines (112). Collectively, this suggests that the contributions of CTR1 to Pt drug
accumulation and the development of resistance are tissue-specific phenomena.
Although CTR1 has been shown to be capable of transporting a number of Pt
analogs, there are clear exceptions that suggest some structural discrimination is ex-
ercised by the transporter. Howell and coworkers have shown that while cisplatin and
carboplatin accumulate with high affinity in mouse embryonic CTR+/+cells (com-
pared with CTR−/−cells), oxaliplatin accumulation appears not to be as dependent
on CTR1 for uptake into cells, although CTR−/−cells still accumulate drug, albeit
at a lower rate (113). It has also been shown that the Pt(II) analog of satraplain, JM118
[cis-ammine(aminocyclohexyl)dichloroplatinum(II)] is not a CTR1 substrate (114).
CTR1 can transport metal ions other than Cu but with much lower affinity,and the
nature of the metal ion speciation is unclear. It may be that a hydrated metal ion passes
through the trimeric pore, although given the requirement for the Cu(I)-selective ex-
tracellular methionine residues (copper-sensing domain) for transport (115, 116), it
seems likely that the Cu is chelated by methionine residues before internalization of
copper. Is copper transported by CTR1 in the same fashion as Pt drugs? Their coordi-
nation chemistry is remarkably different; Cu has two readily accessible oxidation states
under physiological conditions (+I and +II), whereas Pt(II) drugs remain in their +II
oxidation states; their geometries are different; and Pt complexes are more inert to
ligand exchange. Cisplatin treatment promotes a stable homotrimeric form of CTR1,
probably owing to intrameric cross-linking by Pt, especially given that Pt(II) also has
a high affinity for the thioether sulfur donor contained in methionine residues (117).
Given that Pt drugs may be binding to methionine residues as part of CTR1-
mediated transport, one must question whether the Pt pool imported is cytotoxic,
or if it loses the ligands required for activity. Cells with increased CTR1 expression
demonstrate increased Pt accumulation (109, 113), and in most instances increased
sensitivity to cisplatin (109), yet DNA platination did not increase in A2780 trans-
fected to express 20-fold more hCTR1 than parental cells (118). It seems that Pt is
transported into cells, but perhaps the am(m)ine ligands are displaced in binding to
the extracellular methionine or histidine residues prior to internalization, rendering
the Pt center inactive and unable to effectively bind to DNA. Howell argues that this
observation may be due to internalization by macropinocytosis of hCTR1 on exposure
to cisplatin, the Pt later being released from hCTR1 within the cell (118). However,
the nature of the Pt species is not probed. While CTR1 is capable of internalizing
cisplatin in cells, it seems that this Pt is not necessarily contributing to the cytotoxic
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pool of Pt in cells, or that yeast and mouse CTR1, but not human CTR1, are capable
of transporting Pt drugs.
What of the lowered CTR1 expression of the influx transporter in resistant cells
then? How can it be that lowered CTR1 expression correlates with reduced cel-
lular accumulation and DNA binding in resistant cell lines (112), yet transfecting
A431/Pt cervical squamous cell carcinoma-resistant lines with CTR1 did not recover
the accumulation defect (but did recover copper accumulation)? It may be that the
accumulation defect in resistant lines is not due to downregulation of CTR1, but that
it is downregulated or mislocalized in resistant lines as part of a pleiotropic resistance
mechanism present in selected resistant cells (119).
ORGANIC CATION TRANSPORTERS (THE SLC22 FAMILY)
The solute carrier (SLC) gene series encodes a large family of passive trans-
porters, ion-coupled transporters, and exchangers. To date, 45 gene families
(SLC1–SLC45) with approximately 350 transporter genes have been identified
(http://www.gene.ucl.ac.uk/nomenclature, 120). As discussed above, SLC31A1,
which encodes the copper transporter CTR1, is an SLC family member. The SLC22
transmembrane transporters have also been shown to play a role in the uptake of Pt
compounds. The human SLC22 family consists of 18 genes (SLC22A1–18), which
include the organic cation transporters (OCTs), organic cation/carnitine transporters
(OCTNs), and organic anion transporters (OATs). The transporters are predicted to
contain 12 transmembrane helices with a large extracellular loop between helices 1
and 2 (121). These transporters are generally involved in the absorption and/or ex-
cretion of various endogenous and exogenous compounds in organs, including the
kidney, liver, intestine, brain, lung, heart, and bone marrow (121).
The SLC22 family members OCT1, 2, and 3, encoded by SLC22A1,2, and 3,
respectively, were first investigated as candidate uptake transporters of Pt compounds
owing to their expression in tissues associated with cisplatin toxicities, and early
observations that cisplatin inhibited the active uptake of tetraethylammonium (TEA),
a prototypical substrate for OCTs, in rat kidney slices (122). OCT1 is expressed
primarily in the liver (and also the intestine), OCT2 is found in the kidney, and
OCT3 is detected in a range of tissues, such as the placenta and heart (121). Because
OCT2 is expressed mainly in the proximal tubules of the kidney, which is the major
site of cisplatin-induced renal injury, it has been assumed that OCT2 is one of the
main transporters of cisplatin.
Given that there is some divergence in the observations on Pt drugs as OCT
substrates, the results reported to date are detailed in the Supplemental Section
on Organic Cation Transporters (the SLC22 Family) (123–131) and summarized
in Table 1. Differences in the expression levels of OCTs in transfected cells (often
not demonstrated in the reports), the specificity of inhibitors, media used for assays,
and the types of drug sensitivity assays employed may be reasons for the discordant
data. A further complication may arise from the cell lines employed for the above
studies; kidney cell lines, by nature of their function, have a high background level
of influx and efflux transporter expression (such as the ABC and MATE transporters
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Table 1 Observations on cisplatin, carboplatin, and oxaliplatin
substrate specificity for OCT1, OCT2, and OCT3 influx
transporters (rat and human)a
Pt
H3N
H3N
Cl
Cl
Cisplatin OCT1 OCT2 OCT3
Pan et al. (128) — Yes —
Yonezawa et al. (129) No Yes —
Briz et al. (123) No No No
Ciarimboli et al. (124) No Yes —
Yonezawa et al. (130) Yes (weak) Yes No
Zhang et al. (131) Yes (weak) No No
Pt
H3N
H3N
O
O
O
O
Carboplatin OCT1 OCT2 OCT3
Ciarimboli et al. (124) — No —
Yonezawa et al. (130) No No No
Zhang et al. (131) Yes (weak) No No
Pt
H2
N
N
H2
O
O
O
O
Oxaliplatin OCT1 OCT2 OCT3
Ciarimboli et al. (124) — No —
Yonezawa et al. (130) No Yes Yes (weak)
Zhang et al. (131) Ye s Yes No
aPositive or negative observations based on increases in drug accumulation
(rather than sensitization) observed in transfected cell lines.
mentioned elsewhere), which may interfere with drug accumulation and account for
discrepancies in the observations summarized in Table 1.
Cisplatin’s clinical dose-limiting toxicity is predominantly renal toxicity, which
correlates with the high renal expression of OCT2, whereas carboplatin and oxali-
platin (and other less reactive Pt drugs) are not generally OCT2 substrates, and their
toxicities do not correlate with the OCT transporter tissue expression (carboplatin
dose-limiting toxicity: thrombocytopeniea/myelosuppression; oxaliplatin: sensory
neuropathy) (132). One of the common characteristics of SLC22 family members
(OCTs, OCTNs, and OATs) is that certain members transport some of the same
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compounds. Presumably, several SLC22 family members participate in the uptake of
Pt compounds, some being more effective than others. To shed more light on the
uptake of Pt compounds, a clear demonstration of drug uptake in a dose-dependent
and saturable manner needs to be provided for each candidate uptake transporter of
Pt compounds.
ARGUMENTS FOR ACTIVE EFFLUX
The reduced accumulation of cisplatin in resistant cell lines and the early evidence
pointing toward passive diffusion into cells meant the search for a molecular cause
of accumulation defects was not undertaken. The identification of P-glycoprotein
(P-gp), the multidrug resistance transporter encoded by MDR1, probably slanted
initial investigations toward the search for increased expression of an efflux transporter
in resistant cells [it has been demonstrated that cisplatin is not a substrate of P-gp
(133)].
A number of reports of increased efflux in cisplatin-resistant lines exist in the
literature (summarized in the Supplemental Section on Arguments for Active Ef-
flux), although the nature of the effluxed species (intact drug versus metabolites) has
rarely been examined. The main complicating factor in the interpretation of these
efflux studies (134–137) is the possibility that observed drug efflux is unreal passive
efflux that only is observed in cells exposed to high doses (all three reports of efflux
described in the Supplemental Section on Arguments for Active Efflux used ∼500
mM cisplatin). To examine efflux, cells are generally rapidly washed to remove ex-
tracellularly associated drug, and then incubated in drug-free media for proscribed
periods of time until the cells are harvested and their Pt content examined. In this
situation, the drug concentration gradient favors movement out of the cell into the
media, and this may account for an initial rapid efflux that would not occur when the
cell is bathed in drug media. To examine this, cells initially treated with radiolabeled
“hot” drug could then be treated with media containing cold drug, and efflux of hot
drug in the presence of unchanged extracellular drug concentration could provide
insight into whether efflux actually occurs against a concentration gradient.
There are also several reports of no difference in the exodus of cisplatin from
sensitive and resistant cell pairs: L1210/0 and L1210/DDP (SRI) mouse lymphocytic
leukemia (59), 2008 and 2008/DPP human ovarian carcinoma (treated at lower doses,
10 mM) (52), BEL7404 and 74049CP20 human hepatoma (9), H4-II-E and H4-II-
E/CDDP rat hepatoma (87), A431 and A430/Pt human squamous carcinoma (138),
A2780 and A2780cis ovarian cancer (112), and HeLa and HeLaCK cervical carci-
noma (112) cell lines. This has also been shown for carboplatin in the BEL7404 and
7404 CP20 pair and is not temperature-dependent, further suggesting passive efflux
(11).
ATP7A/7B COPPER EFFLUX TRANSPORTERS
In tandem with the copper influx transporter CTR1 described above, the copper ef-
flux transporters ATP7A and ATP7B have also been examined—again reviews exist
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in the literature, and only the key observations are discussed here (107–139). ATP7A
and 7B are functionally conserved P-type ATPases involved in the sequestering and
extrusion of excess Cu ions. They are homologous in structure, sharing eight trans-
membrane domains and ∼65% amino acid sequence. However, their tissue expression
differentiates them (ATP7A, intestinal epithelium of copper absorption; ATP7B, liver
and kidney), and mutations in 7A and 7B are responsible for the Cu-related diseases
Menkes and Wilson’s disease, respectively (104). Under normal (Cu replete) condi-
tions, ATP7A/B resides in the trans-Golgi network, where it receives Cu from the
chaperone Atox1 and translocates it to the luminal side for incorporation into en-
zymes. When excess Cu exists in the cell, ATP7A/B is trafficked to the cell surface to
directly efflux Cu from the cell (104). In both instances, the Cu is transferred directly
to ATP7A/B by a metallochaperone; O’Halloran and coworkers have proposed that
there are no free Cu atoms in the cell (140). ATP7A/B possesses six metal-binding
sequences of ∼100 amino acids each that include a GMTCXXCIE motif on the cyto-
plasmic side required for metal ion translocation, with a methionine and two cysteines
capable of coordinating Pt (139).
Akiyama and coworkers transfected KB-3-1 cells with ATP7B cDNA and demon-
strated resistance to both cisplatin and Cu, which coincided with diminished accu-
mulation of cisplatin that could be recovered by ATP depletion (141). PC-5 prostate
carcinoma cells were also shown to express increased levels of ATP7B relative to
the sensitive PC-3 line and the revertant PC-5R. Nakayama subsequently examined
ATP7B mRNA expression and cisplatin cytotoxicity in nine parental ovarian cancer
cell lines and showed that cells with increased ATP7B expression were less sensitive
to cisplatin (142).
Katano et al. showed that three selected resistant ovarian lines (2008/C13∗5.25,
IGROV-1/CP, and A2780/CP) expressed greater levels of either APT7A or 7B protein
relative to their respective parental lines (143). Whereas increased Pt and Cu efflux
was demonstrated in the 2008/C13∗5.25, expression of other Pt efflux pumps, such as
the GS-X family (described below), was not examined to deconvolute other potential
contributors to efflux. There is not complete agreement on ATP7A/B expression
changes in resistant lines; Zisowsky et al. observed that cisplatin-selected A2780cis
cells show modest increases in expression of both ATP7A and 7B (by RT-PCR),
whereas resistant HeLa cervical carcinoma cells expressed less of each gene in selected
cells, rather than more (112), the authors concluding that ATP7B did not contribute
to resistance in the lines examined.
Cells transfected with ATP7B accumulate less cisplatin and carboplatin than cells
transfected with empty vector (144), whereas cells with ATP7A transfected into
them are resistant to cisplatin, carboplatin, and oxaliplatin. However, the ATP7A
transfectants had increased Pt accumulation, which coincided with the cell vesicular
fraction, suggesting Pt is extruded into vesicles and effectively deactivated by ATP7A.
This seems to suggest that simple overexpression of ATP7A alone may be sufficient
to compartmentalize Pt, but not be enough to lower accumulation. ATP7A is not
relocalized to the plasma membrane for Pt efflux as it is for Cu efflux, suggesting the
Cu sensing domain associated with trafficking cannot sense Pt. This was confirmed
by confocal microscopy using fluorescent Pt compounds (see below) that colocalized
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with ATP7A signal in vesicles (145). The role of ATP7A/B is also Pt drug-type de-
pendent; cells selected with the satraplatin analog JM118 did not show expression
of either ATP7 gene (93). It was also recently shown that ATP7A transfection into
Chinese hamster ovary cells conferred cross-resistance to a large range of organic
drugs (etoposide, vincrisine, paclitaxel, doxorubixin, SN-38, CPT-11), reduced drug
accumulation, and compartmentalized doxorubicin into Golgi vesicles only in ATP7A
expressing cells, suggesting that ATP7A confers multidrug resistance (146). It may
be that ATP7 gene expression alters underlying cellular processes (or vice versa), par-
ticularly given that cellular copper status regulates proteins, such as XIAP (X-linked
inhibitor of apoptosis), that inhibit proapoptotic caspase-3 and -7 in the presence of
Cu (147, 148).
The vesicular accumulation of Pt drugs by ATP7A/B presents a challenge to the
traditional decreased influx and increased efflux model of drug transporter-dependent
resistance. Increased vesicular sequestration of drug does not necessarily affect global
cell Pt levels, although this should be measurable as decreased DNA platination as
the cytosolic drug pool is depleted. The mechanism of Pt drug trapping with these
vesicles is also a matter for debate and may rely on acidic pH trapping of charged drug
as described elsewhere. Elimination of this vesicular Pt pool is achieved (although
perhaps not necessary to confer resistance) by trafficking along the secretory pathway.
The secretory pathway inhibitors wortmannin (inhibitor of endosomal maturation)
and H89 (blocks Golgi vesicular secretion) both increase cisplatin accumulation dra-
matically in 2008 cells (145). In keeping with a lysosomal trapping and efflux of Pt,
2008/C13∗5.25-resistant cells have significantly fewer lysosomes than parental cells
and greater exosomal Pt levels than in wild-type cells (149). Significantly, steady-state
drug levels in sensitive and resistant cells demonstrated little difference. However,
once placed in drug-free media, resistant cells eliminated drug at a more rapid rate
(149), suggesting that vesicular sequestration is responsible for lowered drug activity.
Unlike other putative transporters described already, there is a significant amount
of clinical evidence for ATP7B expression relating to outcome prognosis in a range of
human solid carcinomas (150), and some data is available for ATP7A (146), although
prognostic significance has not been demonstrated (clinical observations are summa-
rized in the Supplemental Section on ATP7A/B as Markers for Chemoresistance).
For example, cisplatin is regularly used in ovarian cancer chemotherapy, and patients
with APT7B-positive tumors demonstrated an inferior response to chemotherapy
compared with ATP7B-negative patients (median survival of 33 months for ATP7B-
positive versus 66 months for ATP7B-negative) (151).
Given the in vitro observation that ATP7A and ATP7B expression can result
in lowered accumulation and/or efficacy of cisplatin, and the evidence that ATP7B
expression in solid carcinomas is generally an indicator of poor response to
chemotherapy, it is possible that disruption of ATP7A/B function may sensitize cells
to Pt drugs. Although no known inhibitors exist (other than the obvious indirect
ATP-depletion or vanadate inhibition of ATPase activity), Cu could act as a compet-
itive substrate that would increase Pt drug accumulation. Farrell and coworkers have
reported that coincubating subtoxic Cu concentrations with cisplatin increase cis-
platin accumulation in A2780 and HCT116 cells, suggesting that Cu may be effluxed
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ANRV333-PA48-18 ARI 4 December 2007 16:1
preferentially over cisplatin. However, A2780 cells expressed elevated ATP7B with
little effect on cisplatin uptake and cytotoxicity (152). Also interesting is that cells
transfected with ATP7A/B do not result in the same Pt accumulation and efflux
properties as selected cell lines, leading one to wonder whether ATP7A/B expression
and Cu resistance to parallel cisplatin resistance in Pt-resistant cells is symptomatic
of a broader upregulated detoxification response (153), and that ATP7A/B is more of
a marker of resistance than the cause of it.
GLUTATHIONE-CONJUGATED (GS-X) EFFLUX
TRANSPORTERS
In considering Pt drug efflux, one must consider the active efflux of glutathione-
conjugated Pt, part of a larger generic detoxification pathway for d-block and the
so-called heavy metals, along with metallothionein. The chelation of the Pt drugs by
glutathione is generally accepted to be a deactivation pathway, and one which drugs
can be designed to circumvent, as is the case with the sterically bulky ZD0473 that
has reduced reactivity with glutathione.
Both Pt(II) and the glutathione thiol sulfur are relatively soft [according to Pear-
son’s Hard-Soft-Acid-Base theorem (154–156)], and form highly stable 2:1 GS-Pt
complexes (157). Many Pt-resistant cell lines express increased levels of glutathione
(owing to increased expression of γ-glutamylcysteine synthetase and γ-glutamyl
transpeptidase synthetic enzymes) (158), and once the Pt drug is chelated (nonenzy-
matic conjugation) by glutathione, the glutathione-Pt complex is effluxed from the
cell in an ATP-dependent fashion by a transporter family termed the GS-X pumps
(157). The GS-X efflux pumps are responsible for the elimination of a range of
glutathione-conjugated drugs, including metal-glutathione chelates.
Ling and coworkers first reported the overexpression of a 200-kDa plasma mem-
brane glycoprotein in a murine thymic lymphoma cell line selected for resistance
to cisplatin (termed CPR-200), and showed that its expression correlated with the
degree of resistance (159). Although CPR-200 has not been further characterized, it
is probably the same 200-kDa membrane protein that is overexpressed in cisplatin-
resistant HL-60 human leukemia cells and is associated with the GS-X pump (160).
GS-X pumps are ATP-dependent organic anion transporters, belonging mainly to
the ABCC (or MRP) family [see Supplemental Table 1 for list of the ABCC (MRP)
transporters].
The first member of the family identified was the multidrug resistance-associated
protein (MRP, MRP1, ABCC1), a 200-kDa transmembrane glycoprotein capable of
effluxing a range of glutathione-conjugated molecules (hence the term GS-X), and a
member of the ABC family of drug efflux transporters (161, 162). It is highly expressed
in lung, adrenal, heart, and skeletal muscle tissue, and weakly expressed in liver and
brain tissue (162), and its overexpression in multidrug-resistant tumors and cell lines
was shown to confer resistance to natural product drugs (163).
Following the observation that Pt-conjugated glutathione is effluxed from
cisplatin-treated cells in an ATP-dependent manner (157), Ishikawa et al. examined
cisplatin-resistant human promyelitic leukemia HL-60/R-CP cells that expressed
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high levels of glutathione, showing overexpression of a 200-kDa protein now known
as MRP1 (160). The cells did not show altered P-glycoprotein expression, and doxoru-
bicin was equipotent against both the parental and resistant cells. Membrane vesicles
generated from the R-CP cells showed a marked increase in tritiated glutathione-
conjugated Pt, and confocal microscopy revealed vesicular localization of the MRP1
substrate monochlorobimane [syn-(ClCH2,CH3)-1,5-diazabicyclo-[3.3.0]-octa-3,6-
dione-2,8-dione], suggesting that MRP1 may sequester substrates into vesicles that
then fuse with the plasma membrane, ejecting their contents (160).
Cole et al. showed that HeLa cells transfected with MRP1 were not cross-resistant
to cisplatin (164), suggesting that the efflux transporter alone is not sufficient to con-
fer resistance to Pt drugs. Hamaguchi et al. examined a series of seven cell lines
increasingly resistant to cisplatin established from A2780 cells by step-wise exposure
to cisplatin (9–400-fold cytotoxicity resistance) and found no relationship between
MRP1 gene expression and resistance to cisplatin, but they did observe that intracel-
lular levels of glutathione correlated with resistance (accumulation was not assessed)
(165) and depletion of cellular glutathione with buthionine sulfoximine (BSO) in-
creases sensitivity to cisplatin (166). Ikuta et al. also found no relationship in NSCLC
cell lines between MRP1 expression and cisplatin cytotoxicity or accumulation (167),
and a number of investigations found no clinical correlation between MRP1 expres-
sion and Pt therapy outcome (7, 168, 169).
Seven more MRP1 homologs have subsequently been identified (MRP2–MRP8).
MRP2, initially termed canalicular multispecific organic anion transporter (cMOAT),
shares 49% sequence homology with MRP1, resides in the hepatocyte canalicular
membrane, and exports glucuronide-conjugated bile acid and glutathione conjugates
such as leukotriene C4into the bile (170, 171). Taniguchi initially reported cMOAT
as a putative cisplatin efflux pump after isolating MRP2 cDNA from three cisplatin-
resistant cell lines (172). A vector containing MRP2 antisense cDNA transfected
into KB epidermoid carcinoma cells increased both sensitivity to, and accumula-
tion of, cisplatin, along with a marked increase in cellular glutathione levels (173),
and cisplatin has been shown to induce MRP2 gene expression (174). A range of
cisplatin-resistant cell lines have been shown to express increased levels of MRP2
(171), and some reports of MRP2 levels correlating with DNA platination exist in
melanoma and ovarian cancer lines (175, 176), although no definitive relationship
between MRP2 expression and resistance or accumulation defects has been reported
(177).
Analysis of clinical data gives mixed indications of MRP2 as a prognostic marker.
Immunohistochemical and RT-PCR analyses of ovarian (178–180) and lung (181,
182) cancer samples have shown no correlation with response to chemotherapy or
progression-free survival, although a study of Stage III ovarian carcinoma samples
demonstrated that an absence of MRP2 transcript was generally associated with im-
proved progression-free survival (183). One analysis of resected colon cancer found
that MRP2 expression was associated with resistance to cisplatin therapy, again
demonstrating the tissue-specific nature of transporter-mediated resistance (184).
It was recently reported that ABCC2 expression in the nuclear membrane, rather
than the plasma membrane, was associated with response to first-line chemotherapy
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ANRV333-PA48-18 ARI 4 December 2007 16:1
in ovarian carcinoma samples, and ABCC2 showed higher expression in cases with
relapse (185).
Of the remaining MRP transporters, Kool et al. initially reported that MRP3
(expressed mainly in the liver), MRP4 (low level expression), and MRP5 (widely
expressed in tissues including liver, kidney, heart, brain, and skeletal muscle) were not
correlated with drug resistance, although a number of cell lines expressed high levels
of MRP3 and MRP5 mRNA (186). Suzuki has reported increased MRP5 mRNA
levels in Pt-treated lung tumor samples, and that MRP5 expression correlated with
glutathione synthesis in said samples (7); little is known about the relationship between
MRP6 and MRP7 expression and Pt drug resistance.
Based on these observations, it seems that overexpression of MRP1 alone is not
sufficient to confer resistance to Pt drugs—a concomitant increase in glutathione
expression to complex Pt is required for efflux of the detoxified drug (187), and
the cross-resistance to metal ions observed in cisplatin-resistant cell lines is probably
conferred by the expression of GS-X pumps (described in the Supplemental Section
on Cross-Resistance to Metal Ions). Glutathione-conjugated Pt is already deactivated,
so efflux by the MRP (GS-X) transporters is not necessarily a part of the accumulation-
resistance phenotype; however, there is evidence in specific cases that their expression
is associated with clinical resistance to cisplatin, and more work needs to be done.
INTRACELLULAR TRAFFICKING
Examination of cellular accumulation of cisplatin is performed readily thanks to the
availability of elemental spectroscopies, such as atomic absorption spectroscopy (AAS)
and inductively-coupled plasma–optical emission spectroscopy (ICP-OES). Some
groups have also used radioactive 14C-labeled diamine groups (such as 14 C ethane-
1,2-diamine) or leaving groups (14C-cyclobutyldicarboxylic acid in carboplatin). Un-
fortunately, these spectroscopic handles do not readily lend themselves to imaging
of cisplatin. Few reports of electron microscopy (188) and synchrotron elemental
imaging (189–191; H.L. Daly, M. Zhang, R.A. Alderden, D.M. Pursche, “Monitor-
ing the biological reduction of cis and trans Pt(IV) complexes using X-ray absorption
near edge spectroscopy (XANES),” manuscript submitted) (SRIXE, XRF) that can
directly monitor Pt exist; however, live samples cannot be imaged, and in the case
of SRIXE, resolution is limiting (192). Chen et al. used SRIXE to show that ma-
lignant melanoma cells sequester Pt into large subcellular compartments (probably
melanosomes) that can then be extruded from the cell (193).
A number of Pt complexes have been synthesized with a fluorophore conjugated
to allow cellular trafficking studies. Although isolated subcellular components have
been assessed for Pt levels, little other information is available. The first of these,
reported by Reedijk and coworkers, incorporated carboyxyfluoresceindeacetate,
termed CFDA-Pt (Supplemental Figure 4) (194), to monitor cellular distribution
over time in U2-OS and U2-OS/Pt cisplatin-resistant osteosarcoma cell lines; im-
portantly, the U2-OS/Pt line demonstrates an accumulation defect (195). CFDA-Pt
demonstrated rapid uptake into cells, and there was no obvious difference in uptake
or localization between CFDA-Pt, or the nonplatinated fluorophore CFDA-Boc, for
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ANRV333-PA48-18 ARI 4 December 2007 16:1
6–8 h when CFDA-Pt levels are lower and punctate staining in the cytoplasm ap-
pears that colocalizes with a Golgi-specific stain (194). The U2-OS/Pt-resistant line
did not demonstrate differences in uptake, distribution, secretion, or localization of
drug compared with the parental cell line. Aside from the rapid uptake (indicated by
strong fluorescence intensity) (194), it is unlikely that CFDA-Pt behaves analogously
to cisplatin. The cytotoxicity of CFDA-Pt was not reported, the metabolism of the
fluorophore (breakdown or dissociation from the Pt center) is not known, and the
lack of differences in CFDA-Pt between resistant and sensitive cells may be due to the
large lipophilic fluorophore altering the pharmacokinetic properties of the complex
and allowing more rapid uptake.
Howell and coworkers addressed some of these issues, using another fluorescein-
conjugated Pt complex, FDDP (196). FDDP demonstrated cross-resistance with cis-
platin to 2008/C13∗5.25 ovarian cancer cells compared with parental lines (∼fivefold
resistance) in terms of both cytotoxicity and accumulation but lower absolute activ-
ity, and the free, nonplatinated fluorophore demonstrated altered localization (196).
FDDP was again noted to be concentrated in small vesicular structures in the cy-
toplasm, and FDDP colocalized in part with the copper efflux transporter ATP7B
(described above). FDDP was subsequently used to identify the vesicles as those
belonging to the Golgi, the secretory export pathway, and lysosomes (145).
Aside from the fluorescein-conjugated complexes above, Liang et al. utilized
the ULYSIS nucleic acid label (Molecular Probes) Alexa Fluor 546 (Supplemen-
tal Figure 4), developed by Houthoff and coworkers (197), which contains a Cy3
dye coordinated to the Pt center in lieu of a chloro ligand. Monofunctional com-
pounds, typified by [PtCl(dien)]+, have been shown to be inherently inactive (20),
and whether cellular processing of inactive complexes is physiologically relevant is
difficult to assess, although the decreased uptake in resistant cell lines argues for the
relevance of labeled Pt compounds. Liang observed lowered accumulation of Alexa
Fluor 546 in KB-CP.5 and KB-CP20 cells, the vesicular localization observed by
others, which merged with the signal from the fluid-phase endocytosis marker Flu-
orescein Dextran-10, and partial localization on the Golgi (as shown for CFDA-Pt)
(198). Liang et al. believe the vesicular localization is endocytic (as less was observed
in resistant cells); as such, the vesicles may represent a reservoir of drug available to
the cell over time. Other researchers observe decreasing vesicular staining over time,
consistent with a drug extrusion process.
A number of studies of novel Pt complexes tethered to bioactive intercalators
such as anthraquinones have utilized their native fluorescence to track uptake and
distribution in cells; however, these are beyond the scope of this review (199–202).
Yet the punctate staining of fluorescent drug observed in the above studies is anal-
ogous to that observed by Hambley and coworkers in A2780 cells with Pt-1C3
[cis-[PtCl2(NH3)(1-{[3-Aminopropyl]amino}-anthracene-9,10-dione}], which colo-
calizes with LysoTracker Green, a lysosome-specific stain ( J. Zhang and T.W.
Hambley, in preparation). Lysosomes are acidic vesicles with an intravesicular pH
around 4.8 that accumulate molecules (lysomotropic agents), especially weak bases,
via passive diffusion (predominately), autophagocytosis, active transport, and endo-
cytosis, and then trap them as charged species (owing to the acidic environment)
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ANRV333-PA48-18 ARI 4 December 2007 16:1
(204, 205). Lysosomes are then secreted to the plasma membrane and their contents
exocytosed. If the protonated species within the lysosome is protonated at low pH, as
aquated species of Pt drugs, and amines on fluorophores would be, the compounds are
effectively membrane impermeable and trapped by a process termed pH partitioning
(204).
There is certainly scope for greater use of fluorescent analogs of Pt drugs, especially
in comparing trafficking in sensitive and resistant cell lines to gain insight into altered
drug accumulation. Caution must be exercised to ensure that the effects observed are
related to the Pt center and not due to the fluorophore (i.e., that the tail is not wagging
the dog).
OCT1-3 Passive
diffusion
Fluid phase
endocytosis
MRP1-5
Pt
CTR1
ATP7A
?
Na+
Na+
Na+,K+-ATPase
K+
K+
a
b
c
Apoptosis
Repair
Pt
Pt
Pt
Pt
Pt
Pt
Pt Pt
ATP ADP
ATP 7B
Pt
Pt
Pt
Pt
Pt
Pt
Pt
Pt
Pt
Melanosomes
Vesicles
Nucleus
d
?
Pt
Pt
Pt
Pt
MT
GS–
GSH
H+
GS–
Pt
Pt
Pt Pt
Pt
Pt
Pt
Pt
Pt
Pt
Pt
Pt
Pt
ATP ADP
Pt
Pt
ATP ADP
Pt
[Cl–] = 4–50 mM
[CO32–] = ~10 mM
[PO43–] = ~80 mM
[Cl–] = ~100 mM
[CO32–] = 24 mM
[PO43–] = 9 mM
Pt
H
3
N
H
3
N
Cl
Cl
Pt
H
3
N
H
3
N
OH
2
Cl
Cl
–
H
2
O
H
+
+
H
+
Pt
Protein binding
(deactivation)
Pt
H
3
N
H
3
N
OH
Cl
Pt
Pt
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PLEIOTROPIC RESISTANCE TO PLATINUM DRUGS
Cells selected in our laboratory for cisplatin resistance, including single-step mutants,
express a complex pleiotropic phenotype consisting of cross-resistance to antimetabo-
lites and heavy metals, and reduced energy-dependent accumulation of cisplatin and
other compounds, including nutrients such as sugars and amino acids (11, 206, 207).
In examining this phenotype, it became clear that fluid-phase endocytosis was reduced
in these cells (208); proteins normally localized to the cell surface were mislocalized
to intracellular vesicular compartments because they fail to recycle back to the cell
surface (Supplemental Figure 5) (119); biophysical measurements of plasma mem-
brane function were altered (64); some genes, such as the folate binding protein gene,
were hypermethylated (209); actin and filamin networks were disorganized (210); and
endocytic recycling was abnormal (211). Fluorescent cisplatin complexes showed de-
layed uptake and altered distribution in the resistant cells, consistent with a role for
defective uptake and endocytosis in causing the reduced cisplatin accumulation (198).
Such a phenotype suggesting alterations in recycling of membranes and membrane
proteins would explain many of the observations reported in the literature, including
energy-dependence of uptake, a defect in several different putative cisplatin uptake
transporters, and reduced passive diffusion (i.e., decreased endocytosis) in cisplatin-
resistant cells.
In searching for a single abnormality that could account for such a pleiotropic phe-
notype, we examined the expression of several small GTPases thought to be involved
in membrane recycling and found decreases in several (209). Microarray experiments
suggested that K+channels were consistently altered in cisplatin-resistant cells, but
←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−
Figure 5
A schematic of the mechanisms affecting and controlling the cellular accumulation of platinum
chemotherapeutics (exemplified here by cisplatin). In the extracellular environment, cisplatin
can be aquated (shown) or react with biomolecules such as carbonate, resulting in a complex
speciation profile. These species may enter the cell or cross-react with extracellular proteins
such as serum albumin (shown), reducing the bioavailable pool of drug. Given the limited
knowledge of the ability of these species to be actively or passively translocated into cells, or
the intracellular speciation of exported drug, they are collectively represented here by yellow
squares (Pt). Neutral platinum drugs can enter the cell by passive diffusion across the lipid
bilayer, and a number of carrier-mediated import proteins have been identified, the main
players being organic cation transporters (OCT1–3, SLC22A1–3), CTR1, and an as-yet
unidentified sodium-dependent process, as well as a number of putative transporters such as
hMATE1 and the aquaporins. Inside the cell, (a) platinum drugs can be deactivated by binding
to the thiol-rich metallothionein (MT) or chelated by glutathione (GSH) and effluxed from
the cell via the GS-X pumps (MRP1–5). Platinum drugs can also be ensnared in subcellular
organelles such as (b) vesicles via ATP7B influx and/or acidic trapping followed by exocytosis
to expel the platinum from the cell, or (c) melanosomes in melanoma cells that are
subsequently exported from the cells. Drug that evades these detoxification and efflux
processes can enter the nucleus by an as-yet undetermined mechanism and bind to DNA,
eliciting apoptosis if the DNA lesion is not repaired. The global pool of platinum drug present
in the cell (and therefore the cytotoxic potential) is dependent on both the rate of influx and
efflux, and a shift in the balance of these in resistant cells (decreased influx, increased extrusion
processes) collectively leads to a reduction in cellular accumulation of cisplatin.
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ANRV333-PA48-18 ARI 4 December 2007 16:1
modulation of the function of these channels with antibodies had no effect on cis-
platin resistance (198). The amount of γ-catenin protein was strikingly reduced in
the resistant cells and relocalized away from the plasma membrane but re-expression
of gamma-catenin did not have a strong effect on cisplatin resistance (212). The com-
plex phenotype associated with cisplatin resistance has the hallmarks of a programmed
cellular response to environmental adversity, including reduced glucose uptake be-
cause of a reduction in Glut1 transporters on the cell surface, increased SirT1 levels
in mitochondria, and reduced oxygen utilization by mitochondria (X.J. Liang and
M.M. Gottesman, unpublished data), but the proximal trigger and/or regulator of
this response remains unknown.
CONCLUDING REMARKS
Cisplatin probably enters the cell via two pathways: (a) passive diffusion and (b) facili-
tated and active uptake by a number of transport proteins. The relative contributions
of the two uptake pathways to total cellular accumulation of drug are probably de-
pendent on the lipophilicity and speciation of the drug. Energy-dependent uptake is
supported by evidence that lowered accumulation of cisplatin is observed in energy-
depleted parental cells; if the accumulation defect were due to increased efflux, energy
depletion would result in increased drug accumulation. As a number of transporters
are probably capable of accommodating cisplatin uptake, molecular, genomic, and
proteomic studies of sensitive and resistant cell lines have not been able to identify a
single transporter whose decreased presence on the plasma membrane significantly
contributes to a reduction in accumulation. This is probably compounded by the fact
that, along with diminished expression, transporters are probably not recycled cor-
rectly in resistant cells, but mislocalized to intracellular compartments, making ready
detection of resistance candidates more difficult. Following this process of resistance
development, a drug can only enter resistant cells at a baseline rate owing to passive
diffusion and the modicum of transporters that persist on the membrane. Even this
mode of entry will be compromised by defects in fluid-phase endocytosis observed in
resistant cell lines.
Resolving cisplatin resistance with molecular biological or gene therapeutic ap-
proaches will rely on an understanding of drug design and the specific carriers present
at the cell surface to both control drug efficacy in cancer cells and ameliorate deactiva-
tion and drug clearance. The enthusiasm for creative Pt drug design and development
has cooled over the past 10 years, as a large number of analogs have been tested in
vitro that failed to offer any improvement over cisplatin, carboplatin, and their con-
geners. That the platinums will remain in the clinic for a long time to come appears
to be a certainty, particularly with the recent finding of carboplatin as an effective
therapy for colon cancer (213). If cellular resistance to cisplatin and its congeners can
be considered to be a generic response to Pt drugs, then features of resistance not as-
sociated with other drugs may point to potential non-Pt therapies that can circumvent
resistance. Given that accumulation of drug in resistant cells probably relies in large
part on passive diffusion, effective lipophilic drugs that do not compromise cytotoxic
potency should be considered, although increasing lipophilicity always raises the risk
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ANRV333-PA48-18 ARI 4 December 2007 16:1
of improving recognition by ATP-dependent efflux pumps. A greater understanding
of the homeostatic regulation of transporters at play in cisplatin accumulation could
also allow manipulation of their expression by coadministration of nontoxic substrates
that alter expression to favor drug accumulation.
Pt drugs probably enter cells by a number of influx transporters along with passive
diffusion, and they can be extruded after chelation by glutathione and in an unknown
form via the Cu efflux system (Figure 5). It is clear from a review of the literature
that disagreement exists about the relative importance of each of these transport
pathways to cisplatin accumulation, which is probably tissue-dependent. Additionally,
our understanding of the transportome and endocytic regulation is incomplete, and
almost certainly new candidates for Pt drug passage will appear. Although resistant
cells selected in vitro provide a good platform for probing accumulation resistance
defects, clinical resistance develops in tandem owing to combinations of Pt and natural
product drugs, and a comprehensive understanding of genome and proteome factors
that correlate with response to chemotherapy is required. Perhaps the most important
challenge for the future is to demonstrate in primary tumor samples that accumulation
defects owing to specific mechanisms correlate with clinical outcome.
DISCLOSURE STATEMENT
The authors are not aware of any biases that might be perceived as affecting the
objectivity of this review.
ACKNOWLEDGMENTS
This research was supported by the Intramural Research Program of the National
Institutes of Health, National Cancer Institute.
LITERATURE CITED
1. Szakacs G, Paterson JK, Ludwig JA, Booth-Genthe C, Gottesman MM. 2006.
Targeting multidrug resistance in cancer. Nat. Rev. Drug Discov. 5:219–34
2. Wernyj RP, Morin PJ. 2004. Molecular mechanisms of platinum resistance: still
searching for the Achilles’ heel. Drug Resist. Updates 7:227–32
3. Wang D, Lippard SJ. 2005. Cellular processing of platinum anticancer drugs.
Nat. Rev. Drug Discov. 4:307–20
4. Baird RD, Kaye SB. 2003. Drug resistance reversal—are we getting closer? Eur.
J. Cancer 39:2450–61
5. Kelland LR. 2000. Preclinical perspectives on platinum resistance. Drugs
59(Suppl. 4):1–8
6. Gately DP, Howell SB. 1993. Cellular accumulation of the anticancer agent
cisplatin: a review. Br. J. Cancer 67:1171–76
7. Suzuki T, Nishio K, Tanabe S. 2001. The MRP family and anticancer drug
metabolism. Curr. Drug Metab. 2:367–77
www.annualreviews.org •Pt Drug Accumulation and Resistance 521
Annu. Rev. Pharmacol. Toxicol. 2008.48:495-535. Downloaded from arjournals.annualreviews.org
by National Institute of Health Library on 01/17/08. For personal use only.

ANRV333-PA48-18 ARI 4 December 2007 16:1
8. Andrews PA, Howell SB. 1990. Cellular pharmacology of cisplatin: perspectives
on mechanisms of acquired resistance. Cancer Cells 2:35–43
9. Johnson SW, Shen D-W, Pastan I, Gottesman MM, Hamilton TC. 1996.
Cross-resistance, cisplatin accumulation, and platinum-DNA adduct formation
and removal in cisplatin-sensitive and -resistant human hepatoma cell lines. Exp.
Cell Res. 226:133–39
10. Koga H, Kotoh S, Nakashima M, Yokomizo A, Tanaka M, Naito S. 2000. Accu-
mulation of intracellular platinum is correlated with intrinsic cisplatin resistance
in human bladder cancer cell lines. Int. J. Oncol. 16:1003–7
11. Shen D-W, Goldenberg S, Pastan I, Gottesman MM. 2000. Decreased accu-
mulation of [14C]carboplatin in human cisplatin-resistant cells results from
reduced energy-dependent uptake. J. Cell Physiol. 183:108–16
12. Hector S, Bolanowska-Higdon W, Zdanowicz J, Hitt S, Pendyala L. 2001.
In vitro studies on the mechanisms of oxaliplatin resistance. Cancer Chemother.
Pharmacol. 48:398–406
13. Nicolson MC, Orr RM, O’Neill CF, Harrap KR. 1992. The role of platinum
uptake and glutathione levels in L1210 cells sensitive and resistant to cisplatin,
tetraplatin or carboplatin. Neoplasma 39:189–95
14. Holford J, Beale PJ, Boxall FE, Sharp SY, Kelland LR. 2000. Mechanisms of
drug resistance to the platinum complex ZD0473 in ovarian cancer cell lines.
Eur. J. Cancer 36:1984–90
15. de Jong WH, Steerenberg PA, Vos JG, Bulten EJ, Verbeek F, et al. 1983. Anti-
tumor activity, induction of cross-resistance, and nephrotoxicity of a new plat-
inum analogue, cis-1,1-diaminomethylcyclohexaneplatinum(II) sulfate, and of
cis-diamminedichloroplatinum(II) in an immunocytoma model in the LOU/M
rat. Cancer Res. 43:4927–34
16. Stewart DJ, Mikhael NZ, Nair RC, Kacew S, Montpetit V, et al. 1988. Platinum
concentrations in human autopsy tumor samples. Am. J. Clin. Oncol. 11:152–58
17. Andrews PA. 2000. Cisplatin accumulation. In Platinum-Based Drugs in Cancer
Therapy, ed. LR Kelland, NP Farrell, pp. 89–113. Totowa, NJ: Humana Press
18. Rosenberg B. 1999. The start. In Cisplatin: Chemistry and Biochemistry of a Leading
Anticancer Drug, ed. B Lippert, pp. 3–30. Weinheim, Ger.: Wiley-VCH
19. Alderden RA, Hall MD, Hambley TW. 2006. The discovery and development
of cisplatin. J. Chem. Educ. 83:728–34
20. Hambley TW. 1997. The influence of structure on the activity and toxicity of
Pt anticancer drugs. Coord. Chem. Rev. 166:181–223
21. Harris AL, Yang X, Hegmans A, Povirk L, Ryan JJ, et al. 2005. Synthesis,
characterization, and cytotoxicity of a novel highly charged trinuclear platinum
compound. Enhancement of cellular uptake with charge. Inorg. Chem. 44:9598–
600
22. Miller SE, House DA. 1990. The hydrolysis products of cis-dichlorodiammine-
platinum(II) 3. Hydrolysis kinetics at physiological pH. Inorg. Chim. Acta
173:53–60
23. Berners-Price SJ, Ronconi L, Sadler PJ. 2006. Insights into the mechanism
of action of platinum anticancer drugs from multinuclear NMR spectroscopy.
Prog. Nucl. Magn. Reson. Spectrosc. 49:65–98
522 Hall et al.
Annu. Rev. Pharmacol. Toxicol. 2008.48:495-535. Downloaded from arjournals.annualreviews.org
by National Institute of Health Library on 01/17/08. For personal use only.

ANRV333-PA48-18 ARI 4 December 2007 16:1
24. Jennerwein M, Andrews PA. 1995. Effect of intracellular chloride on the cellular
pharmacodynamics of cis-diamminedichloroplatinum(II). Drug Metab. Dispos.
23:178–84
25. Berners-Price SJ, Appleton TG. 2000. The chemistry of cisplatin in aqueous so-
lutions. In Platinum-Based Drugs in Cancer Therapy, ed. LR Kelland, NP Farrell,
pp. 3–35. Totawa, NJ: Humana Press
26. Martin RB. 1999. Platinum complexes: hydrolysis and binding to N(7) and N(1)
of purines. In Cisplatin: Chemistry and Biochemistry of a Leading Anticancer Drug,
ed. B Lippert, pp. 183–205. Weinheim, Ger.: Wiley-VCH
27. Platts JA, Hibbs DE, Hambley TW, Hall MD. 2001. Calculation of hydropho-
bicity of platinum drugs. J. Med. Chem. 44:472–74
28. Oldfield S, Hall MD, Platts JA. 2007. QSPR analysis of log Po/w values for a
large, diverse dataset of anti-cancer platinum complexes. J. Med. Chem. In press
29. Davies MS, Berners-Price SJ, Hambley TW. 1998. Rates of platination of AG
and GA containing double-stranded oligonucleotides: Insights into why cis-
platin binds to GG and AG but not GA sequences in DNA. J. Am. Chem. Soc.
120:11380–90
30. Davies MS, Berners-Price SJ, Hambley TW. 2000. Slowing of cisplatin aqua-
tion in the presence of DNA but not in the presence of phosphate: Improved
understanding of sequence selectivity and the roles of monoaquated and di-
aquated species in the binding of cisplatin to DNA. Inorg. Chem. 39:5603–13
31. Centerwall CR, Tacka KA, Kerwood DJ, Goodisman J, Toms BB, et al. 2006.
Modification and uptake of a cisplatin carbonato complex by Jurkat cells. Mol.
Pharmacol. 70:348–55
32. Palmer DA, Van Eldik R. 1983. The chemistry of metal carbonato and carbon
dioxide complexes. Chem. Rev. 83:651–731
33. Barnham KJ, Frey U, Murdoch PS, Ranford JD, Sadler PJ. 1994. [Pt(CBDCA-
O)(NH3)2(L-Methionine-S)]: Ring-opened adduct of the anticancer drug car-
boplatin (“paraplatin”). Detection of a similar complex in urine by NMR spec-
troscopy. J. Am. Chem. Soc. 116:11175–76
34. Di Pasqua AJ, Goodisman J, Kerwood DJ, Toms BB, Dubowy RL, Dabrowiak
JC. 2006. Activation of carboplatin by carbonate. Chem. Res. Toxicol. 19:139–49
35. Elferink F, van der Vijgh WJ, Klein I, Vermorken JB, Gall HE, Pinedo HM.
1987. Pharmacokinetics of carboplatin after i.v. administration. Cancer Treat.
Rep. 71:1231–37
36. Hall MD, Dolman RC, Hambley TW. 2004. Platinum(IV) anticancer com-
plexes. Met. Ions Biol. Syst. 42:297–322
37. Hall MD, Hambley TW. 2002. Platinum(IV) antitumour compounds: their
bioinorganic chemistry. Coord. Chem. Rev. 232:49–67
38. Mashima R, Nakanishi-Ueda T, Yamamoto Y. 2003. Simultaneous determi-
nation of methionine sulfoxide and methionine in blood plasma using gas
chromatography-mass spectrometry. Anal. Biochem. 313:28–33
39. Timerbaev AR, Hartinger CG, Aleksenko SS, Keppler BK. 2006. Interactions
of antitumor metallodrugs with serum proteins: advances in characterization
using modern analytical methodology. Chem. Rev. 106:2224–48
www.annualreviews.org •Pt Drug Accumulation and Resistance 523
Annu. Rev. Pharmacol. Toxicol. 2008.48:495-535. Downloaded from arjournals.annualreviews.org
by National Institute of Health Library on 01/17/08. For personal use only.

ANRV333-PA48-18 ARI 4 December 2007 16:1
40. Melvik JE, Dornish JM, Pettersen EO. 1992. The binding of cis-
dichlorodiammineplatinum(II) to extracellular and intracellular compounds in
relation to drug uptake and cytotoxicity in vitro. Br. J. Cancer 66:260–65
41. Takahashi K, Seki T, Nishikawa K, Minamide S, Iwabuchi M, et al. 1985. An-
titumor activity and toxicity of serum protein-bound platinum formed from
cisplatin. Jpn. J. Cancer Res. 76:68–74
42. Schuldes H, Bade S, Knobloch J, Jonas D. 1997. Loss of in vitro cytotoxicity
of cisplatin after storage as stock solution in cell culture medium at various
temperatures. Cancer 79:1723–28
43. Dolman RC, Deacon GB, Hambley TW. 2002. Studies of the binding of a series
of platinum(IV) complexes to plasma proteins. J. Inorg. Biochem. 88:260–67
44. Guchelaar HJ, de Vries EG, Meijer C, Esselink MT, Vellenga E, et al. 1994.
Effect of ultrafilterable platinum concentration on cisplatin and carboplatin cy-
totoxicity in human tumor and bone marrow cells in vitro. Pharm. Res. 11:1265–
69
45. Hegedus L, van der Vijgh WJ, Klein I, Kerpel-Fronius S, Pinedo HM. 1987.
Chemical reactivity of cisplatin bound to human plasma proteins. Cancer
Chemother. Pharmacol. 20:211–12
46. Jennerwein M, Andrews PA. 1994. Drug accumulation and DNA platination in
cells exposed to aquated cisplatin species. Cancer Lett. 81:215–20
47. Pereira-Maia E, Garnier-Suillerot A. 2003. Impaired hydrolysis of cisplatin
derivatives to aquated species prevents energy-dependent uptake in GLC4 cells
resistant to cisplatin. J. Biol. Inorg. Chem. 8:626–34
48. Shirazi HH, Molepo JM, Stewart DJ, Ng CE, Raaphorst GP, Goel R. 1996.
Cytotoxicity, accumulation, and efflux of cisplatin and its metabolites in human
ovarian carcinoma cells. Toxicol. Appl. Pharmacol. 140:211–18
49. Yachnin JR, WallinI, Lewensohn R, Sirzen F, Ehrsson H. 1998. The kinetics and
cytotoxicity of cisplatin and its monohydrated complex. Cancer Lett. 132:175–80
50. Laurencot CM, Kennedy KA. 1995. Influence of pH on the cytotoxicity of
cisplatin in EMT6 mouse mammary tumor cells. Oncol. Res. 7:371–79
51. Andrews PA, Mann SC, Velury S, Howell SB. 1988. Cisplatin uptake mediated
cisplatin-resistance in human ovarian carcinoma cells. In Platinum and Other
Metal Coordination Compounds in Cancer Chemotherapy (Developments in Oncology),
ed. M Nicolini, pp. 248–54. Boston: Martinus Nijhoff
52. Andrews PA, Velury S, Mann SC, Howell SB. 1988. cis-Diamminedichloro-
platinum(II) accumulation in sensitive and resistant human ovarian carcinoma
cells. Cancer Res. 48:68–73
53. Eichholtz-Wirth H, Hietel B. 1986. The relationship between cisplatin sensi-
tivity and drug uptake into mammalian cells in vitro. Br. J. Cancer 54:239–43
54. Gale GR, Morris CR, Atkins LM, Smith AB. 1973. Binding of an antitumor
platinum compound to cells as influenced by physical factors and pharmacolog-
ically active agents. Cancer Res. 33:813–18
55. Hecquet B, Leroy A, Lefebvre JL, Peyrat JP, Adenis L. 1986. Uptake of platinum
compounds in human tumors. In vitro study. Bull. Cancer 73:535–41
56. Hromas RA, North JA, Burns CP. 1987. Decreased cisplatin uptake by resistant
L1210 leukemia cells. Cancer Lett. 36:197–201
524 Hall et al.
Annu. Rev. Pharmacol. Toxicol. 2008.48:495-535. Downloaded from arjournals.annualreviews.org
by National Institute of Health Library on 01/17/08. For personal use only.

ANRV333-PA48-18 ARI 4 December 2007 16:1
57. Ogawa M, Gale GR, Keirn SS. 1975. Effects of cis-diamminedichloroplatinum
(NSC 119875) on murine and human hemopoietic precursor cells. Cancer Res.
35:1398–401
58. Binks SP, Dobrota M. 1990. Kinetics and mechanism of uptake of platinum-
based pharmaceuticals by the rat small intestine. Biochem. Pharmacol. 40:1329–36
59. Waud WR. 1987. Differential uptake of cis-diamminedichloroplatinum(II) by
sensitive and resistant murine L1210 leukemia cells. Cancer Res 47:6549–
55
60. Marverti G, Andrews PA. 1996. Stimulation of cis-diamminedichloro-
platinum(II) accumulation by modulation of passive permeability with genis-
tein: an altered response in accumulation-defective resistant cells. Clin. Cancer
Res. 2:991–99
61. Ghezzi A, Aceto M, Cassino C, Gabano E, Osella D. 2004. Uptake of antitumor
platinum(II)-complexes by cancer cells, assayed by inductively coupled plasma
mass spectrometry (ICP-MS). J. Inorg. Biochem. 98:73–78
62. Wang K, Lu J, Li R. 1996. The events that occur when cisplatin encounters
cells. Coord. Chem. Rev. 151:53–88
63. Liang X, Huang Y. 2002. Physical state changes of membrane lipids in human
lung adenocarcinoma A549 cells and their resistance to cisplatin. Int. J. Biochem.
Cell Biol. 34:1248–55
64. Liang XJ, Yin JJ, Zhou JW, Wang PC, Taylor B, et al. 2004. Changes in biophys-
ical parameters of plasma membranes influence cisplatin resistance of sensitive
and resistant epidermal carcinoma cells. Exp. Cell Res. 293:283–91
65. Mann SC, Andrews PA, Howell SB. 1988. Comparison of lipid content, surface
membrane fluidity, and temperature dependence of cis-diamminedichloro-
platinum(II) accumulation in sensitive and resistant human ovarian carcinoma
cells. Anticancer Res. 8:1211–15
66. Mellish KJ, Kelland LR. 1994. Mechanisms of acquired resistance to the
orally active platinum-based anticancer drug bis-acetato-ammine-dichloro-
cyclohexylamine platinum (i.v.) (JM216) in two human ovarian carcinoma cell
lines. Cancer Res. 54:6194–200
67. Hall MD, Amjadi S, Zhang M, Beale PJ, Hambley TW. 2004. The mechanism
of action of platinum(IV) complexes in ovarian cancer cell lines. J. Inorg. Biochem.
98:1614–24
68. Kikkawa F, Kojima M, Oguchi H, Maeda O, Ishikawa H, et al. 1993. Potenti-
ating effect of amphotericin B on five platinum anticancer drugs in human cis-
diamminedichloroplatinum(II) sensitive and resistant ovarian carcinoma cells.
Anticancer Res. 13:891–96
69. Jekunen AP, Shalinsky DR, Hom DK, Albright KD, Heath D, Howell SB.
1993. Modulation of cisplatin cytotoxicity by permeabilization of the plasma
membrane by digitonin in vitro. Biochem. Pharmacol. 45:2079–85
70. Lindner PG, Heath D, Howell SB, Naredi PL, Hafstrom LR. 1997. Digitonin
enhances the efficacy of carboplatin in liver tumour after intra-arterial admin-
istration. Cancer Chemother. Pharmacol. 40:444–48
71. Byfield JE, Calabro-Jones PM. 1981. Carrier-dependent and carrier-
independent transport of anticancer alkylating agents. Nature 294:281–83
www.annualreviews.org •Pt Drug Accumulation and Resistance 525
Annu. Rev. Pharmacol. Toxicol. 2008.48:495-535. Downloaded from arjournals.annualreviews.org
by National Institute of Health Library on 01/17/08. For personal use only.

ANRV333-PA48-18 ARI 4 December 2007 16:1
72. Dornish JM, Pettersen EO, Oftebro R, Melvik JE. 1984. Reduction of cis-
dichlorodiammineplatinum-induced cell inactivation by benzaldehyde. Eur. J.
Cancer Clin. Oncol. 20:1287–93
73. Dornish JM, Melvik JE, Pettersen EO. 1986. Reduced cellular uptake of cis-
dichlorodiammine-platinum by benzaldehyde. Anticancer Res. 6:583–88
74. Dornish JM, Pettersen EO. 1985. Protection from cis-dichlorodiammine-
platinum-induced cell inactivation by aldehydes involves cell membrane amino
groups. Cancer Lett. 29:235–43
75. Dornish JM, Pettersen EO. 1990. Requirement of a reactive aldehyde moiety for
aldehyde-mediated protection against cis-dichlorodiammineplatinum-induced
cell inactivation. Biochem. Pharmacol. 39:309–18
76. Andrews PA, Mann SC, Huynh HH, Albright KD. 1991. Role of the Na+,
K(+)-adenosine triphosphatase in the accumulation of cis-diamminedichloro-
platinum(II) in human ovarian carcinoma cells. Cancer Res. 51:3677–81
77. Kasahara K, Fujimara M, Bando T, Shibata K, Shirasaki H, Matsuda T. 1996.
Modulation of sensitivity to cis-diamminedichloroplatinum(II) by thromboxane
A2 receptor antagonists in nonsmall-cell lung cancer cell lines. Br. J. Cancer
74:1553–58
78. Katz AI. 1986. Distribution and function of classes of ATPases along the
nephron. Kidney Int. 29:21–31
79. Cheng PW, Liu SH, Hsu CJ, Lin-Shiau SY. 2005. Correlation of increased
activities of Na+,K
+-ATPase and Ca2+-ATPase with the reversal of cisplatin
ototoxicity induced by D-methionine in guinea pigs. Hear Res. 205:102–9
80. Smith E, Brock AP. 1989. The effect of reduced osmolarity on platinum drug
toxicity. Br. J. Cancer 59:873–75
81. Cheng PW, Liu SH, Young YH, Lin-Shiau SY. 2006. D-Methionine attenu-
ated cisplatin-induced vestibulotoxicity through altering ATPase activities and
oxidative stress in guinea pigs. Toxicol. Appl. Pharmacol. 215:228–36
82. Kroning R, Lichtenstein AK, Nagami GT. 2000. Sulfur-containing amino acids
decrease cisplatin cytotoxicity and uptake in renal tubule epithelial cell lines.
Cancer Chemother. Pharmacol. 45:43–49
83. Deegan PM, Pratt IS, Ryan MP. 1994. The nephrotoxicity, cytotoxicity and
renal handling of a cisplatin-methionine complex in male Wistar rats. Toxicology
89:1–14
84. Sharp SY, Rogers PM, Kelland LR. 1995. Transport of cisplatin and bis-acetato-
ammine-dichlorocyclohexylamine platinum(IV) ( JM216) in human ovarian car-
cinoma cell lines: identification of a plasma membrane protein associated with
cisplatin resistance. Clin. Cancer Res. 1:981–89
85. Ohmori T, Morikage T, Sugimoto Y, Fujiwara Y, Kasahara K, et al. 1993.
The mechanism of the difference in cellular uptake of platinum derivatives in
nonsmall cell lung cancer cell line (PC-14) and its cisplatin-resistant subline
(PC-14/CDDP). Jpn. J. Cancer Res. 84:83–92
86. Ma J, Maliepaard M, Kolker HJ, Verweij J, Schellens JH. 1998. Abrogated
energy-dependent uptake of cisplatin in a cisplatin-resistant subline of the hu-
man ovarian cancer cell line IGROV-1. Cancer Chemother. Pharmacol. 41:186–92
526 Hall et al.
Annu. Rev. Pharmacol. Toxicol. 2008.48:495-535. Downloaded from arjournals.annualreviews.org
by National Institute of Health Library on 01/17/08. For personal use only.

ANRV333-PA48-18 ARI 4 December 2007 16:1
87. Kishimoto S, Kawazoe Y, Ikeno M, Saitoh M, Nakano Y, et al. 2006. Role
of Na+,K
+-ATPase α1 subunit in the intracellular accumulation of cisplatin.
Cancer Chemother. Pharmacol. 57:84–90
88. Bando T, Fujimura M, Kasahara K, Matsuda T. 1998. Significance of
Na+,K
(+)-ATPase on intracellular accumulation of cis-diamminedichloro-
platinum(II) in human nonsmall-cell but not in small-cell lung cancer cell lines.
Anticancer Res. 18:1085–89
89. Uozumi J, Litterst CL. 1985. The effect of cisplatin on renal ATPase activity
in vivo and in vitro. Cancer Chemother. Pharmacol. 15:93–96
90. Viadiu H, Gonen T, Walz T. 2007. Projection map of aquaporin-9 at 7 A
resolution. J. Mol. Biol. 367:80–88
91. Gonen T, Walz T. 2006. The structure of aquaporins. Q. Rev. Biophys. 39:361–
96
92. Leung J, Pang A, Yuen WH, Kwong YL, Tse EW. 2007. Relationship of ex-
pression of aquaglyceroporin 9 with arsenic uptake and sensitivity in leukemia
cells. Blood 109:740–46
93. Helleman J, Jansen MP, Span PN, van Staveren IL, Massuger LF, et al.
2006. Molecular profiling of platinum resistant ovarian cancer. Int. J. Cancer
118:1963–71
94. Natile G, Coluccia M. 2004. Antitumor active trans-platinum compounds. Met.
Ions Biol. Syst. 42:209–50
95. Cheng TC, Manorek G, Samimi G, Lin X, Berry CC, Howell SB. 2006. Iden-
tification of genes whose expression is associated with cisplatin resistance in
human ovarian carcinoma cells. Cancer Chemother. Pharmacol. 58:384–95
96. Hough CD, Sherman-Baust CA, Pizer ES, Montz FJ, Im DD, et al. 2000. Large-
scale serial analysis of gene expression reveals genes differentially expressed in
ovarian cancer. Cancer Res. 60:6281–87
97. Le Moguen K, Lincet H, Deslandes E, Hubert-Roux M, Lange C, et al. 2006.
Comparative proteomic analysis of cisplatin sensitive IGROV1 ovarian carci-
noma cell line and its resistant counterpart IGROV1-R10. Proteomics 6:5183–92
98. Macleod K, Mullen P, Sewell J, Rabiasz G, Lawrie S, et al. 2005. Altered ErbB
receptor signaling and gene expression in cisplatin-resistant ovarian cancer.
Cancer Res. 65:6789–800
99. Roberts D, Schick J, Conway S, Biade S, Laub PB, et al. 2005. Identification of
genes associated with platinum drug sensitivity and resistance in human ovarian
cancer cells. Br. J. Cancer 92:1149–58
100. Sakamoto M, Kondo A, Kawasaki K, Goto T, Sakamoto H, et al. 2001. Analysis
of gene expression profiles associated with cisplatin resistance in human ovarian
cancer cell lines and tissues using cDNA microarray. Hum. Cell 14:305–15
101. Stewart JJ, White JT, Yan X, Collins S, Drescher CW, et al. 2006. Proteins
associated with Cisplatin resistance in ovarian cancer cells identified by quanti-
tative proteomic technology and integrated with mRNA expression levels. Mol.
Cell Proteomics 5:433–43
102. Zhang P, Zhang Z, Zhou X, Qiu W, Chen F, Chen W. 2006. Identification of
genes associated with cisplatin resistance in human oral squamous cell carcinoma
cell line. BMC Cancer 6:224
www.annualreviews.org •Pt Drug Accumulation and Resistance 527
Annu. Rev. Pharmacol. Toxicol. 2008.48:495-535. Downloaded from arjournals.annualreviews.org
by National Institute of Health Library on 01/17/08. For personal use only.

ANRV333-PA48-18 ARI 4 December 2007 16:1
103. Zhu K, Fukasawa I, Fujinoki M, Furuno M, Inaba F, et al. 2005. Profiling
of proteins associated with cisplatin resistance in ovarian cancer cells. Int. J.
Gynecol. Cancer 15:747–54
104. Luk E, Jensen LT, Culotta VC. 2003. The many highways for intracellular
trafficking of metals. J. Biol. Inorg. Chem. 8:803–9
105. Pena MM, Puig S, Thiele DJ. 2000. Characterization of the Saccharomyces cere-
visiae high affinity copper transporter Ctr3. J. Biol. Chem. 275:33244–51
106. Aller SG, Unger VM. 2006. Projection structure of the human copper trans-
porter CTR1 at 6-A resolution reveals a compact trimer with a novel channel-
like architecture. Proc. Natl. Acad. Sci. USA 103:3627–32
107. Safaei R. 2006. Role of copper transporters in the uptake and efflux of platinum
containing drugs. Cancer Lett. 234:34–39
108. Safaei R, Howell SB. 2005. Copper transporters regulate the cellular pharma-
cology and sensitivity to Pt drugs. Crit. Rev. Oncol. Hematol. 53:13–23
109. Ishida S, Lee J, Thiele DJ, Herskowitz I. 2002. Uptake of the anticancer drug
cisplatin mediated by the copper transporter Ctr1 in yeast and mammals. Proc.
Natl. Acad. Sci. USA 99:14298–302
110. Lin X, Okuda T, Holzer A, Howell SB. 2002. The copper transporter
CTR1 regulates cisplatin uptake in Saccharomyces cerevisiae. Mol. Pharmacol.
62:1154–59
111. Song IS, Savaraj N, Siddik ZH, Liu P, Wei Y, et al. 2004. Role of human
copper transporter Ctr1 in the transport of platinum-based antitumor agents in
cisplatin-sensitive and cisplatin-resistant cells. Mol. Cancer Ther. 3:1543–49
112. Zisowsky J, Koegel S, Leyers S, Devarakonda K, Kassack MU, et al. 2007.
Relevance of drug uptake and efflux for cisplatin sensitivity of tumor cells.
Biochem. Pharmacol. 73:298–307
113. Holzer AK, Manorek GH, Howell SB. 2006. Contribution of the major copper
influx transporter CTR1 to the cellular accumulation of cisplatin, carboplatin,
and oxaliplatin. Mol. Pharmacol. 70:1390–94
114. Samimi G, Howell SB. 2006. Modulation of the cellular pharmacology of
JM118, the major metabolite of satraplatin, by copper influx and efflux trans-
porters. Cancer Chemother. Pharmacol. 57:781–88
115. Guo Y, Smith K, Lee J, Thiele DJ, Petris MJ. 2004. Identification of
methionine-rich clusters that regulate copper-stimulated endocytosis of the hu-
man Ctr1 copper transporter. J. Biol. Chem. 279:17428–33
116. Jiang J, Nadas IA, Kim MA, Franz KJ. 2005. A Mets motif peptide found in
copper transport proteins selectively binds Cu(I) with methionine-only coordi-
nation. Inorg. Chem. 44:9787–94
117. Guo Y, Smith K, Petris MJ. 2004. Cisplatin stabilizes a multimeric com-
plex of the human Ctr1 copper transporter: requirement for the extracellular
methionine-rich clusters. J. Biol. Chem. 279:46393–99
118. Holzer AK, Samimi G, Katano K, Naerdemann W, Lin X, et al. 2004. The
copper influx transporter human copper transport protein 1 regulates the up-
take of cisplatin in human ovarian carcinoma cells. Mol. Pharmacol. 66:817–
23
528 Hall et al.
Annu. Rev. Pharmacol. Toxicol. 2008.48:495-535. Downloaded from arjournals.annualreviews.org
by National Institute of Health Library on 01/17/08. For personal use only.

ANRV333-PA48-18 ARI 4 December 2007 16:1
119. Liang XJ, Shen DW, Garfield S, Gottesman MM. 2003. Mislocalization of
membrane proteins associated with multidrug resistance in cisplatin-resistant
cancer cell lines. Cancer Res. 63:5909–16
120. Hediger MA, Romero MF, Peng JB, Rolfs A, Takanaga H, Bruford EA. 2004.
The ABCs of solute carriers: physiological, pathological and therapeutic im-
plications of human membrane transport proteins. Introduction. P߬ugers Arch.
447:465–68
121. Koepsell H, Endou H. 2004. The SLC22 drug transporter family. Eur. J. Physiol.
447:666–76
122. Nelson JA, Santos G, Herbert BH. 1984. Mechanisms for the renal secretion
of cisplatin. Cancer Treat. Rep. 68:849–53
123. Briz O, Serrano MA, Rebollo N, Hagenbuch B, Meier PJ, et al. 2002. Carriers
involved in targeting the cytostatic bile acid-cisplatin derivatives cis-diammine-
chloro-cholylglycinate-platinum(II) and cis-diammine-bisursodeoxycholate-
platinum(II) toward liver cells. Mol. Pharmacol. 61:853–60
124. Ciarimboli G, Ludwig T, Lang D, Pavenstadt H, Koepsell H, et al. 2005.
Cisplatin nephrotoxicity is critically mediated via the human organic cation
transporter 2. Am. J. Pathol. 167:1477–84
125. Criado JJ, Dominguez MF, Medarde M, Fernandez ER, Macias RI, Marin JJ.
2000. Structural characterization, kinetic studies, and in vitro biological activ-
ity of new cis-diamminebis-cholylglycinate(O,O) Pt(II) and cis-diamminebis-
ursodeoxycholate(O,O) Pt(II) complexes. Bioconjug. Chem. 11:167–74
126. Criado JJ, Macias RI, Medarde M, Monte MJ, Serrano MA, Marin JJ.
1997. Synthesis and characterization of the new cytostatic complex cis-
diammineplatinum(II)-chlorocholylglycinate. Bioconjug. Chem. 8:453–58
127. Kolb RJ, Ghazi AM, Barfuss DW. 2003. Inhibition of basolateral transport and
cellular accumulation of cDDP and N-acetyl-L-cysteine-cDDP by TEA and
PAH in the renal proximal tubule. Cancer Chemother. Pharmacol. 51:132–38
128. Pan BF, Sweet DH, Pritchard JB, Chen R, Nelson JA. 1999. A transfected cell
model for the renal toxin transporter, rOCT2. Toxicol. Sci. 47:181–86
129. Yonezawa A, Masuda S, Nishihara K, Yano I, Katsura T, Inui K. 2005. Asso-
ciation between tubular toxicity of cisplatin and expression of organic cation
transporter rOCT2 (Slc22a2) in the rat. Biochem. Pharmacol. 70:1823–31
130. Yonezawa A, Masuda S, Yokoo S, Katsura T, Inui KI. 2006. Cisplatin and ox-
aliplatin, but not carboplatin and nedaplatin, are substrates for human organic
cation transporters (SLC22A1–3 and MATE family). J. Pharmacol. Exp. Ther.
319:879–86
131. Zhang S, Lovejoy KS, Shima JE, Lagpacan LL, Shu Y, et al. 2006. Organic
cation transporters are determinants of oxaliplatin cytotoxicity. Cancer Res.
66:8847–57
132. Vermorken JB. 2001. The integration of paclitaxel and new platinum com-
pounds in the treatment of advanced ovarian cancer. Int. J. Gynecol. Cancer
11(Suppl. 1):21–30
133. Gottesman MM, Pastan I. 1988. The multidrug transporter, a double-edged
sword. J. Biol. Chem. 263:12163–66
www.annualreviews.org •Pt Drug Accumulation and Resistance 529
Annu. Rev. Pharmacol. Toxicol. 2008.48:495-535. Downloaded from arjournals.annualreviews.org
by National Institute of Health Library on 01/17/08. For personal use only.

ANRV333-PA48-18 ARI 4 December 2007 16:1
134. Chau Q, Stewart DJ. 1999. Cisplatin efflux, binding and intracellular pH in
the HTB56 human lung adenocarcinoma cell line and the E-8/0.7 cisplatin-
resistant variant. Cancer Chemother. Pharmacol. 44:193–202
135. Fujii R, Mutoh M, Niwa K, Yamada K, Aikou T, et al. 1994. Active efflux system
for cisplatin in cisplatin-resistant human KB cells. Jpn. J. Cancer Res. 85:426–33
136. Fujii R, Mutoh M, Sumizawa T, Chen ZS, Yoshimura A, Akiyama S. 1994.
Adenosine triphosphate-dependent transport of leukotriene C4 by membrane
vesicles prepared from cisplatin-resistant human epidermoid carcinoma tumor
cells. J. Natl. Cancer Inst. 86:1781–84
137. Mann SC, Andrews PA, Howell SB. 1990. Short-term cis-diamminedichloro-
platinum(II) accumulation in sensitive and resistant human ovarian carcinoma
cells. Cancer Chemother. Pharmacol. 25:236–40
138. Martelli L, Di Mario F, Ragazzi E, Apostoli P, Leone R, et al. 2006. Different ac-
cumulation of cisplatin, oxaliplatin and JM216 in sensitive and cisplatin-resistant
human cervical tumour cells. Biochem. Pharmacol. 72:693–700
139. Kuo MT, Chen HH, Song IS, Savaraj N, Ishikawa T. 2007. The roles of copper
transporters in cisplatin resistance. Cancer Metastasis Rev. 26:71–83
140. Rae TD, Schmidt PJ, Pufahl RA, Culotta VC, O’Halloran TV. 1999. Unde-
tectable intracellular free copper: the requirement of a copper chaperone for
superoxide dismutase. Science 284:805–8
141. Komatsu M, Sumizawa T, Mutoh M, Chen ZS, Terada K, et al. 2000. Copper-
transporting P-type adenosine triphosphatase (ATP7B) is associated with cis-
platin resistance. Cancer Res. 60:1312–16
142. Nakayama K, Miyazaki K, Kanzaki A, Fukumoto M, Takebayashi Y. 2001.
Expression and cisplatin sensitivity of copper-transporting P-type adenosine
triphosphatase (ATP7B) in human solid carcinoma cell lines. Oncol. Rep. 8:1285–
87
143. Katano K, Kondo A, Safaei R, Holzer A, Samimi G, et al. 2002. Acquisition of
resistance to cisplatin is accompanied by changes in the cellular pharmacology
of copper. Cancer Res. 62:6559–65
144. Samimi G, Varki NM, Wilcyznski S, Safaei R, Alberts DS, Howell SB. 2003.
Increase in expression of the Copper Transporter ATP7A during platinum drug-
based treatment is associated with poor survival in ovarian cancer patients. Clin.
Cancer Res. 9:5853–59
145. Safaei R, Katano K, Larson BJ, Samimi G, Holzer AK, et al. 2005. Intracellular
localization and trafficking of fluorescein-labeled cisplatin in human ovarian
carcinoma cells. Clin. Cancer Res. 11:756–67
146. Owatari S, Akune S, Komatsu M, Ikeda R, Firth SD, et al. 2007. Copper-
transporting P-type ATPase, ATP7A, confers multidrug resistance and its ex-
pression is related to resistance to SN-38 in clinical colon cancer. Cancer Res.
67:4860–68
147. Mufti AR, Burstein E, Csomos RA, Graf PC, Wilkinson JC, et al. 2006. XIAP
is a copper binding protein deregulated in Wilson’s disease and other copper
toxicosis disorders. Mol. Cell 21:775–85
148. Mufti AR, Burstein E, Duckett CS. 2007. XIAP: Cell death regulation meets
copper homeostasis. Arch. Biochem. Biophys. 463:168–74
530 Hall et al.
Annu. Rev. Pharmacol. Toxicol. 2008.48:495-535. Downloaded from arjournals.annualreviews.org
by National Institute of Health Library on 01/17/08. For personal use only.

ANRV333-PA48-18 ARI 4 December 2007 16:1
149. Safaei R, Larson BJ, Cheng TC, Gibson MA, Otani S, et al. 2005. Abnor-
mal lysosomal trafficking and enhanced exosomal export of cisplatin in drug-
resistant human ovarian carcinoma cells. Mol. Cancer Ther. 4:1595–604
150. Katoh R, Takebayashi Y, Takenoshita S. 2005. Expression of copper-
transporting P-type adenosine triphosphatase (ATP7B) as a chemoresistance
marker in human solid carcinomas. Ann. Thorac. Cardiovasc. Surg. 11:143–45
151. Nakayama K, Kanzaki A, Terada K, Mutoh M, Ogawa K, et al. 2004. Prognostic
value of the Cu-transporting ATPase in ovarian carcinoma patients receiving
cisplatin-based chemotherapy. Clin. Cancer Res. 10:2804–11
152. Kabolizadeh P, Ryan J, Farrell N. 2007. Differences in the cellular response and
signaling pathways of cisplatin and BBR3464 ([{trans-PtCl(NH(3))(2)}(2)mu-
(trans-Pt(NH(3))(2)(H(2)N(CH(2))(6)-NH(2))(2))](4+)) influenced by copper
homeostasis. Biochem. Pharmacol. 73:1270–79
153. Safaei R, Katano K, Samimi G, Naedemann W, Stevenson JL, et al. 2004.
Cross-resistance to cisplatin in cells with acquired resistance to copper. Cancer
Chemother. Pharmacol. 53:239–46
154. Pearson RG. 1963. Hard and soft acids and bases. J. Am. Chem. Soc. 85:3533–39
155. Pearson RG. 1968. Hard and soft acids and bases. HSAB, Part I. Fundamental
principles. J. Chem. Ed. 45:581–87
156. Pearson RG. 1968. Hard and soft acids and bases. HSAB, Part II. Underlying
theories. J. Chem. Ed. 45:643–48
157. Ishikawa T, Ali-Osman F. 1993. Glutathione-associated cis-diamminedichloro-
platinum(II) metabolism and ATP-dependent efflux from leukemia cells. Molec-
ular characterization of glutathione-platinum complex and its biological signif-
icance. J. Biol. Chem. 268:20116–25
158. Godwin AK, Meister A, O’Dwyer PJ, Huang CS, Hamilton TC, Anderson
ME. 1992. High resistance to cisplatin in human ovarian cancer cell lines is
associated with marked increase of glutathione synthesis. Proc. Natl. Acad. Sci.
USA 89:3070–74
159. Kawai K, Kamatani N, Georges E, Ling V. 1990. Identification of a mem-
brane glycoprotein overexpressed in murine lymphoma sublines resistant to
cis-diamminedichloroplatinum(II). J. Biol. Chem. 265:13137–42
160. Ishikawa T, Wright CD, Ishizuka H. 1994. GS-X pump is functionally over-
expressed in cis-diamminedichloroplatinum (II)-resistant human leukemia HL-
60 cells and down-regulated by cell differentiation. J. Biol. Chem. 269:29085–
93
161. Cole SP, Bhardwaj G, Gerlach JH, Mackie JE, Grant CE, et al. 1992. Overex-
pression of a transporter gene in a multidrug-resistant human lung cancer cell
line. Science 258:1650–54
162. Zaman GJR, Borst P. 1996. MRP, mode of action and role in MDR. In Multidrug
Resistance in Cancer Cells, ed. S Gupta, T Tsuruo, pp. 95–107. Chichester, UK:
Wiley
163. Grant CE, Valdimarsson G, Hipfner DR, Almquist KC, Cole SP, Deeley RG.
1994. Overexpression of multidrug resistance-associated protein (MRP) in-
creases resistance to natural product drugs. Cancer Res. 54:357–61
www.annualreviews.org •Pt Drug Accumulation and Resistance 531
Annu. Rev. Pharmacol. Toxicol. 2008.48:495-535. Downloaded from arjournals.annualreviews.org
by National Institute of Health Library on 01/17/08. For personal use only.

ANRV333-PA48-18 ARI 4 December 2007 16:1
164. Cole SP, Sparks KE, Fraser K, Loe DW, Grant CE, et al. 1994. Pharmacological
characterization of multidrug resistant MRP-transfected human tumor cells.
Cancer Res. 54:5902–10
165. Hamaguchi K, Godwin AK, Yakushiji M, O’Dwyer PJ, Ozols RF, Hamilton
TC. 1993. Cross-resistance to diverse drugs is associated with primary cisplatin
resistance in ovarian cancer cell lines. Cancer Res. 53:5225–32
166. Lewandowicz GM, Britt P, Elgie AW, Williamson CJ, Coley HM, et al. 2002.
Cellular glutathione content, in vitro chemoresponse, and the effect of BSO
modulation in samples derived from patients with advanced ovarian cancer.
Gynecol. Oncol. 85:298–304
167. Ikuta K, Takemura K, Sasaki K, Kihara M, Nishimura M, et al. 2005. Expres-
sion of multidrug resistance proteins and accumulation of cisplatin in human
nonsmall cell lung cancer cells. Biol. Pharm. Bull. 28:707–12
168. de Cremoux P, Jourdan-Da-Silva N, Couturier J, Tran-Perennou C, Schleier-
macher G, et al. 2007. Role of chemotherapy resistance genes in outcome of
neuroblastoma. Pediatr. Blood Cancer 48:311–17
169. Nicholson DL, Purser SM, Maier RH. 1998. Differential cytotoxicity of trace
metals in cisplatin-sensitive and -resistant human ovarian cancer cells. Biometals
11:259–63
170. Jedlitschky G, Leier I, Buchholz U, Hummel-Eisenbeiss J, Burchell B, Keppler
D. 1997. ATP-dependent transport of bilirubin glucuronides by the multidrug
resistance protein MRP1 and its hepatocyte canalicular isoform MRP2. Biochem.
J. 327(Part 1):305–10
171. Borst P, Kool M, Evers R. 1997. Do cMOAT (MRP2), other MRP homologues,
and LRP play a role in MDR? Semin. Cancer Biol. 8:205–13
172. Taniguchi K, Wada M, Kohno K, Nakamura T, Kawabe T, et al. 1996. A
human canalicular multispecific organic anion transporter (cMOAT) gene is
overexpressed in cisplatin-resistant human cancer cell lines with decreased drug
accumulation. Cancer Res. 56:4124–29
173. Koike K, Kawabe T, Tanaka T, Toh S, Uchiumi T, et al. 1997. A canalicular
multispecific organic anion transporter (cMOAT) antisense cDNA enhances
drug sensitivity in human hepatic cancer cells. Cancer Res. 57:5475–79
174. Kauffmann H-M, Keppler D, Kartenbeck J, Schrenk D. 1997. Induction of
cMrp/cMoat gene expression by cisplatin, 2-acetylaminofluorene, or cyclohex-
imide in rat hepatocytes. Hepatology 26:980–85
175. Liedert B, Materna V, Schadendorf D, Thomale J, Lage H. 2003. Overex-
pression of cMOAT (MRP2/ABCC2) is associated with decreased formation of
platinum-DNA adducts and decreased G2-arrest in melanoma cells resistant to
cisplatin. J. Invest. Dermatol. 121:172–76
176. Materna V, Liedert B, Thomale J, Lage H. 2005. Protection of platinum-DNA
adduct formation and reversal of cisplatin resistance by anti-MRP2 hammerhead
ribozymes in human cancer cells. Int. J. Cancer 115:393–402
177. Helleman J, Burger H, Hamelers IH, Boersma AW, de Kroon AI, et al. 2006.
Impaired cisplatin influx in an A2780 mutant cell line: evidence for a putative,
cis-configuration-specific, platinum influx transporter. Cancer Biol. Ther. 5:943–
49
532 Hall et al.
Annu. Rev. Pharmacol. Toxicol. 2008.48:495-535. Downloaded from arjournals.annualreviews.org
by National Institute of Health Library on 01/17/08. For personal use only.

ANRV333-PA48-18 ARI 4 December 2007 16:1
178. Arts HJ, Katsaros D, de Vries EG, Massobrio M, Genta F, et al. 1999. Drug
resistance-associated markers P-glycoprotein, multidrug resistance-associated
protein 1, multidrug resistance-associated protein 2, and lung resistance pro-
tein as prognostic factors in ovarian carcinoma. Clin. Cancer Res. 5:2798–
805
179. Ohishi Y, Oda Y, Uchiumi T, Kobayashi H, Hirakawa T, et al. 2002. ATP-
binding cassette superfamily transporter gene expression in human primary
ovarian carcinoma. Clin. Cancer Res. 8:3767–75
180. Nakayama K, Kanzaki A, Ogawa K, Miyazaki K, Neamati N, Takebayashi Y.
2002. Copper-transporting P-type adenosine triphosphatase (ATP7B) as a cis-
platin based chemoresistance marker in ovarian carcinoma: comparative anal-
ysis with expression of MDR1, MRP1, MRP2, LRP and BCRP. Int. J. Cancer
101:488–95
181. Oguri T, Fujiwara Y, Miyazaki M, Takahashi T, Kurata T, et al. 1999. In-
duction of γ-glutamylcysteine synthetase gene expression by platinum drugs
in peripheral mononuclear cells of lung cancer patients. Ann. Oncol. 10:455–
60
182. Oguri T, Fujiwara Y, Ochiai M, Fujitaka K, Miyazaki M, et al. 1998. Expression
of lung-resistance protein gene is not associated with platinum drug exposure
in lung cancer. Anticancer Res. 18:4159–62
183. Materna V, Pleger J, Hoffmann U, Lage H. 2004. RNA expression of MDR1/P-
glycoprotein, DNA-topoisomerase I, and MRP2 in ovarian carcinoma patients:
correlation with chemotherapeutic response. Gynecol. Oncol. 94:152–60
184. Hinoshita E, Uchiumi T, Taguchi K, Kinukawa N, Tsuneyoshi M, et al. 2000.
Increased expression of an ATP-binding cassette superfamily transporter, mul-
tidrug resistance protein 2, in human colorectal carcinomas. Clin. Cancer Res.
6:2401–7
185. Surowiak P, Materna V, Kaplenko I, Spaczynski M, Dolinska-Krajewska B, et al.
2006. ABCC2 (MRP2, cMOAT) can be localized in the nuclear membrane
of ovarian carcinomas and correlates with resistance to cisplatin and clinical
outcome. Clin. Cancer Res. 12:7149–58
186. Kool M, de Haas M, Scheffer GL, Scheper RJ, van Eijk MJ, et al. 1997. Analysis
of expression of cMOAT (MRP2), MRP3, MRP4, and MRP5, homologues of
the multidrug resistance-associated protein gene (MRP1), in human cancer cell
lines. Cancer Res. 57:3537–47
187. de Vries EG, Muller M, Meijer C, Jansen PL, Mulder NH. 1995. Role of
the glutathione S-conjugate pump in cisplatin resistance. J. Natl. Cancer Inst.
87:537–38
188. Beretta GL, Righetti SC, Lombardi L, Zunino F, Perego P. 2002. Electron
microscopy analysis of early localization of cisplatin in ovarian carcinoma cells.
Ultrastruct. Pathol. 26:331–34
189. Hall MD, Alderden RA, Zhang M, Beale PJ, Cai Z, et al. 2006. The fate of
platinum(II) and platinum(IV) anticancer agents in cancer cells and tumours. J.
Struct. Biol. 155:38–44
190. Deleted in proof
www.annualreviews.org •Pt Drug Accumulation and Resistance 533
Annu. Rev. Pharmacol. Toxicol. 2008.48:495-535. Downloaded from arjournals.annualreviews.org
by National Institute of Health Library on 01/17/08. For personal use only.

ANRV333-PA48-18 ARI 4 December 2007 16:1
191. Shimura M, Saito A, Matsuyama S, Sakuma T, Terui Y, et al. 2005. Element
array by scanning X-ray fluorescence microscopy after cis-diamminedichloro-
platinum(II) treatment. Cancer Res. 65:4998–5002
192. Hall MD, Dillon CT, Zhang M, Beale P, Cai Z, et al. 2003. The cellular
distribution and oxidation state of platinum(II) and platinum(IV) antitumour
complexes in cancer cells. J. Biol. Inorg. Chem. 8:726–32
193. Chen KG, Valencia JC, Lai B, Zhang G, Paterson JK, et al. 2006. Melanosomal
sequestration of cytotoxic drugs contributes to the intractability of malignant
melanomas. Proc. Natl. Acad. Sci. USA 103:9903–7
194. Molenaar C, Teuben JM, Heetebrij RJ, Tanke HJ, Reedijk J. 2000. New insights
in the cellular processing of platinum antitumor compounds, using fluorophore-
labeled platinum complexes and digital fluorescence microscopy. J. Biol. Inorg.
Chem. 5:655–65
195. Perego P, Caserini C, Gatti L, Carenini N, Romanelli S, et al. 1999. A novel
trinuclear platinum complex overcomes cisplatin resistance in an osteosarcoma
cell system. Mol. Pharmacol. 55:528–34
196. Katano K, Safaei R, Samimi G, Holzer A, Tomioka M, et al. 2004. Confocal
microscopic analysis of the interaction between cisplatin and the copper trans-
porter ATP7B in human ovarian carcinoma cells. Clin. Cancer Res. 10:4578–88
197. Heetebrij RJ, Talman EG, van Velzen MA, van Gijlswijk RP, Snoeijers SS,
et al. 2003. Platinum(II)-based coordination compounds as nucleic acid labeling
reagents: synthesis, reactivity, and applications in hybridization assays. Chem-
biochem 4:573–83
198. Liang XJ, Shen DW, Chen KG, Wincovitch SM, Garfield SH, Gottesman MM.
2005. Trafficking and localization of platinum complexes in cisplatin-resistant
cell lines monitored by fluorescence-labeled platinum. J. Cell Physiol. 202:635–
41
199. Alderden RA, Mellor HR, Modok S, Hambley TW, Callaghan R. 2006. Cy-
totoxic efficacy of an anthraquinone linked platinum anticancer drug. Biochem.
Pharmacol. 71:1136–45
200. Kalayda GV, Jansen BA, Molenaar C, Wielaard P, Tanke HJ, Reed-
ijk J. 2004. Dinuclear platinum complexes with N, N-bis(aminoalkyl)-1,4-
diaminoanthraquinones as linking ligands. Part II. Cellular processing in A2780
cisplatin-resistant human ovarian carcinoma cells: new insights into the mech-
anism of resistance. J. Biol. Inorg. Chem. 9:414–22
201. Kalayda GV, Jansen BA, Wielaard P, Tanke HJ, Reedijk J. 2005. Dinu-
clear platinum anticancer complexes with fluorescent N,N-bis(aminoalkyl)-
1,4-diaminoanthraquinones: cellular processing in two cisplatin-resistant cell
lines reflects the differences in their resistance profiles. J. Biol. Inorg. Chem.
10:305–15
202. Kalayda GV, Zhang G, Abraham T, Tanke HJ, Reedijk J. 2005. Application of
fluorescence microscopy for investigation of cellular distribution of dinuclear
platinum anticancer drugs. J. Med. Chem 48:5191–202
203. Deleted in proof
204. Kaufmann AM, Krise JP. 2006. Lysosomal sequestration of amine-containing
drugs: Analysis and therapeutic implications. J. Pharm. Sci. 96:729–46
534 Hall et al.
Annu. Rev. Pharmacol. Toxicol. 2008.48:495-535. Downloaded from arjournals.annualreviews.org
by National Institute of Health Library on 01/17/08. For personal use only.

ANRV333-PA48-18 ARI 4 December 2007 16:1
205. Larsen AK, Escargueil AE, Skladanowski A. 2000. Resistance mechanisms as-
sociated with altered intracellular distribution of anticancer agents. Pharmacol.
Ther. 85:217–29
206. Shen DW, Pastan I, Gottesman MM. 1998. Cross-resistance to methotrexate
and metals in human cisplatin-resistant cell lines results from a pleiotropic defect
in accumulation of these compounds associated with reduced plasma membrane
binding proteins. Cancer Res. 58:268–75
207. Shen DW, Akiyama S, Schoenlein P, Pastan I, Gottesman MM. 1995. Char-
acterisation of high-level cisplatin-resistant cell lines established from a human
hepatoma cell line and human KB adenocarcinoma cells: cross-resistance and
protein changes. Br. J. Cancer 71:676–83
208. Chauhan SS, Liang XJ, Su AW, Pai-Panandiker A, Shen D-W, et al. 2003.
Reduced endocytosis and altered lysosome function in cisplatin-resistant cell
lines. Br. J. Cancer 88:1327–34
209. Shen DW, Su A, Liang XJ, Pai-Panandiker A, Gottesman MM. 2004. Reduced
expression of small GTPases and hypermethylation of the folate binding protein
gene in cisplatin-resistant cells. Br. J. Cancer 91:270–76
210. Shen DW, Liang XJ, Gawinowicz MA, Gottesman MM. 2004. Identification
of cytoskeletal [14C]carboplatin-binding proteins reveals reduced expression
and disorganization of actin and filamin in cisplatin-resistant cell lines. Mol.
Pharmacol. 66:789–93
211. Liang XJ, Mukherjee S, Shen DW, Maxfield FR, Gottesman MM. 2006. Endo-
cytic recycling compartments altered in cisplatin-resistant cancer cells. Cancer
Res. 66:2346–53
212. Liang XJ, Shen DW, Gottesman MM. 2004. Down-regulation and altered local-
ization of γ-catenin in cisplatin-resistant adenocarcinoma cells. Mol. Pharmacol.
65:1217–24
213. Muggia FM, Fojo T. 2004. Platinums: extending their therapeutic spectrum. J.
Chemother. 16(Suppl. 4):77–82
www.annualreviews.org •Pt Drug Accumulation and Resistance 535
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Annual Review of
Pharmacology and
Toxicology
Volume 48, 2008
Contents
The Tangle of Nuclear Receptors that Controls Xenobiotic
Metabolism and Transport: Crosstalk and Consequences
Jean-Marc Pascussi, Sabine Gerbal-Chaloin, C´edric Duret,
Martine Daujat-Chavanieu, Marie-Jos´e Vilarem, and Patrick Maurel pppppppppppppppp1
Mechanisms of Placebo and Placebo-Related Effects Across Diseases
and Treatments
Fabrizio Benedetti ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp 33
Pharmacotherapy for the Treatment of Choroidal Neovascularization
Due to Age-Related Macular Degeneration
Gary D. Novack ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp61
Nicotinic Acid: Pharmacological Effects and Mechanisms of Action
Andreas Gille, Erik T. Bodor, Kashan Ahmed, and Stefan Offermanns pppppppppppppppp79
Activation of G Protein–Coupled Receptors: Beyond Two-State
Models and Tertiary Conformational Changes
Paul S.-H. Park, David T. Lodowski, and Krzysztof Palczewski pppppppppppppppppppppp107
Apoptin: Therapeutic Potential of an Early Sensor of Carcinogenic
Transformation
Claude Backendorf, Astrid E. Visser, A.G. de Boer, Rhyenne Zimmerman,
Mijke Visser, Patrick Voskamp, Ying-Hui Zhang, and Mathieu Noteborn ppppppppppp143
Chemokines and Their Receptors: Drug Targets in Immunity
and Inflammation
Antonella Viola and Andrew D. Luster ppppppppppppppppppppppppppppppppppppppppppppppppp171
Apoptosis Signal-Regulating Kinase 1 in Stress and Immune Response
Kohsuke Takeda, Takuya Noguchi, Isao Naguro, and Hidenori Ichijo ppppppppppppppppp199
Pharmacogenetics of Anti-HIV Drugs
A. Telenti and U.M. Zanger ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp227
Epigenetics and Complex Disease: From Etiology to New Therapeutics
Carolyn Ptak and Arturas Petronis ppppppppppppppppppppppppppppppppppppppppppppppppppppp257
Vesicular Neurotransmitter Transporters as Targets for Endogenous
and Exogenous Toxic Substances
Farrukh A. Chaudhry, Robert H. Edwards, and Frode Fonnum pppppppppppppppppppppp277
v
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AR333-FM ARI 4 December 2007 16:4
Mechanism-Based Concepts of Size and Maturity in Pharmacokinetics
B.J. Anderson and N.H.G. Holford ppppppppppppppppppppppppppppppppppppppppppppppppppppp303
Role of CYP1B1 in Glaucoma
Vasilis Vasiliou and Frank J. Gonzalez ppppppppppppppppppppppppppppppppppppppppppppppppp333
Caveolae as Organizers of Pharmacologically Relevant Signal
Transduction Molecules
Hemal H. Patel, Fiona Murray, and Paul A. Insel pppppppppppppppppppppppppppppppppppp359
Proteases for Processing Proneuropeptides into Peptide
Neurotransmitters and Hormones
Vivian Hook, Lydiane Funkelstein, Douglas Lu, Steven Bark,
Jill Wegrzyn, and Shin-Rong Hwang pppppppppppppppppppppppppppppppppppppppppppppppppp393
Targeting Chemokine Receptors in HIV: A Status Report
Shawn E. Kuhmann and Oliver Hartley ppppppppppppppppppppppppppppppppppppppppppppppp425
Biomarkers of Acute Kidney Injury
Vishal S. Vaidya, Michael A. Ferguson, and Joseph V. Bonventre pppppppppppppppppppp463
The Role of Cellular Accumulation in Determining Sensitivity to
Platinum-Based Chemotherapy
Matthew D. Hall, Mitsunori Okabe, Ding-Wu Shen, Xing-Jie Liang,
and Michael M. Gottesman pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp495
Regulation of GPCRs by Endocytic Membrane Trafficking and Its
Potential Implications
Aylin C. Hanyaloglu and Mark von Zastrow pppppppppppppppppppppppppppppppppppppppppp537
PKC Isozymes in Chronic Cardiac Disease: Possible Therapeutic
Targets?
Eric Churchill, Grant Budas, Alice Vallentin, Tomoyoshi Koyanagi,
and Daria Mochly-Rosen pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp569
G Protein–Coupled Receptor Sorting to Endosomes and Lysosomes
Adriano Marchese, May M. Paing, Brenda R.S. Temple, and JoAnn Trejo ppppppppp601
Strategic Approach to Fit-for-Purpose Biomarkers in Drug Development
John A. Wagner ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp631
Metabolomics: A Global Biochemical Approach to Drug Response and Disease
Rima Kaddurah-Daouk, Bruce S. Kristal, and Richard M. Weinshilboum pppppppppp653
Indexes
Contributing Authors, Volumes 44–48 pppppppppppppppppppppppppppppppppppppppppppppppppp685
Chapter Titles, Volumes 44–48 pppppppppppppppppppppppppppppppppppppppppppppppppppppppppp688
Errata
An online log of corrections to Annual Review of Pharmacology and Toxicology
articles may be found at http://pharmtox.annualreviews.org/errata.shtml
vi Contents
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