Minireview

THE JOURNAL
OF BIOLOGICAL
CHEMISTRY
Vol. 269 No. 2 Issue of Janua 14, pp. 787-790,1994
0 1994 by The American sociity for Biwhemistry an?Molecular Biolo Inc.
Printed in%.S.A.

Cellular Responsesto Cisplatin

HZ0

++

Pt(NH3)2CI(HZO)+

PWH~ZCIZ

THE ROLES OF DNA-BINDING PROTEINS

AND DNA REPAIR*

'I-

b
H
+
Pt(NH&CI(OH)

Gilbert Chu
From the Department of Medicine, Stanford University
School of Medicine, Stanford, California 94305

&

Pf(NH&(H,0)2+2

bH+

PWHdz(OH)(HpO)+

REACTION
1

cyclobutanedicarboxylatogroup in carboplatin is much less labile

than thechloride in cis-DDP, so that the aquation reactions

proceed
The anticancer drug cisplatin provokes a complex response in the cell. A lethal dose of the drug kills cells more slowly (6). As a result, carboplatin is more stable and less
DNA adducts, causing6 2 arrest in reactive than cisplatin. The structures of cis-DDP, trans-DDP, and
primarily by forming
A sublethal carboplatin are shown in Fig. 1.
the cell cycle, and then triggering apoptosis.
dose induces drug resistance by several mechanisms,
Apoptoeis
including changes in drug uptake and efflux, glutathiHow does cis-DDP cause cell death? In the past, itwas believed
DNA repair. Cisplaone and metallothionein levels, and
tin-DNA adducts bind several cellular proteins, includ- that cytotoxicity was the result of inhibition of DNA synthesis (7,
8). However, DNA repair-deficient Chinese hamster ovary cells die
ing some that enhance survival of the cell by mediating
DNA repair and others that hastenits death by confer- a t concentrations of cis-DDP that failed to inhibit DNA synthesis
(9). and DNA repair proficient cells survive even high concentraring sensitivity to the drug.
tions of cis-DDP high enough to inhibit DNA synthesis and arrest
the cells in S phase. Thus cell death does not correlate with inhibition of DNA synthesis.
Instead, the analysis of cell death induced by cis-DDP (10, 11)
reveals DNA fragmentation into multimersof 180 base pairs, consistent with internucleosomal cleavage of chromatin by a n endonuclease, followed by loss of membrane integrity and cell shrinkage. This process is inhibited by cycloheximide, suggesting that cell
death requiresnew protein synthesis. Thesefindings are consistent
with the induction of programmed cell death or apoptosis (12).
Furthermore, studies with repair-deficient cells indicate that apoptosis is triggered by the presence of cis-DDP DNA adducts. The
signal transduction mechanism that links DNA damage to the cell
death pathway remainsunknown.

The biological activity of cisplatin was discovered serendipitously in 1965 during studiesof the effect of a n electric current on
Escherichia coli (1).Cell division was inhibited not by the electric
current butby the production of cis-diamminedichloroplatinum(I1)
(cis-DDP or cisplatin) from the platinum electrodes. Cisplatin was
found to have potent antitumor activity as well and now is widely
used for the treatmentof many malignancies, including testicular,
ovarian, head and neck, bladder, esophageal, and small cell lung
cancers. The related compound carboplatin has also been used increasingly in recent years.
When cisplatin isdissolved in aqueous solution, chlorideions are
displaced to allow the formation of aquated species, which are the
reactive forms of the compound (2), as shown in Reaction 1. The
Drug Resietance
concentration of chloride ions influences the reactivityof cisplatin.
Cell lines from many different tumors maydevelop resistance to
After intravenous administration it relatively
is
less reactive in the cisplatin. In most cases, the level of resistance is less than 50-fold,
extracellular space where the chloride concentration is -100 mM, although there are reports of up t o 1000-fold resistance (Table I).
but on crossing the plasma membrane, it is activated in the intraNevertheless, even a small increase in resistance of a tumor to
cellular space where the chloride concentration drops to -3 m. cisplatin can be clinically important, since a large dose escalation
Activated cisplatin isa potent electrophile that will react with any leads to severe multiorgantoxicities that mayinclude failure of the
nucleophile, including the sulfiydryl groups on proteins and nu- kidneys and bone marrow, intractable vomiting, peripheral neuropcleophilic groups on nucleic acids.
athy, deafness, seizures, and blindness (13).
The cytotoxicity of cisplatin is believed to be due to the
formation
Studies of the causes of cisplatin resistance have been summaof DNA adducts, which include DNA-protein cross-links, DNA rized in several excellent reviews (14-17). Postulated mechanisms
monoadducts, and interstrand and intrastrand DNA cross-links. include decreased drug accumulation, increased levelsof the intraQuantitativestudies
show that1,a-intrastrand
d(GpG), and cellular thiols, and increased DNA repair. Table I compiles results
d(ApG) cross-links account for 65 and 25%, respectively, of the from various cell lines derived from a single tumor type, human
cisplatin adducts formed in vitro (3, 4). X-ray diffraction of the ovarian cancer, for which cisplatin is a mainstay of treatment and
cross-linked dinucleotide cis-Pt(NH3)2(d(pGpG)) reveals that the
drug resistance is an important problem.
two guanines arecompletely destacked, and thedeoxyribose sugar
of the B'-deoxyguanosine is in a C3'-endo pucker. Thus, the intraIntracellular Accumulation
strand cisplatincross-link produces a severe local distortion in the
The
development
of cisplatin resistanceis sometimes associated
DNA double helix, leading to unwinding and kinking
(5).
The transisomer trans-DDP interacts withDNA in quite differ- with decreases in intracellular accumulation of the drug. When
ent ways from cis-DDP. It cannot form 1,2-intrastrand cross-links decreases do occur, they are usuallymodest, even when the level of
but does form 1,3-intrastrand cross-links and interstrand cross- resistance is quite high (Table I). So far, the search for a specific
links. Thus, trans-DDP may be less cytotoxic than the cis form cisplatin membrane transport system has been inconclusive. Uptake was not saturable up to the solubility limit of cisplatin (3.3
because of differences in the structuresof the DNA adducts.
Carboplatin has two amine groups in a cis configuration and mM), so that if a transport system exists it must be either a low
forms the same spectrum of DNA adducts as cisplatin. The 1,l- afhity site orvery abundant (14). Nevertheless, promising studies
of cisplatin-resistant cells with decreased drug accumulation have
identified two membrane proteins that may be involved in uptake
* This minireview will bereprinted in theMinireview Compendium, which and efflux, respectively: a 48-kDa protein with decreased expreswill be available in December, 1994. This work was supported in partby the
sion (18) and a 200-kDa glycoprotein with increased expression
National Institutesof Health, the RitaAllen Foundation, and fundsdonated
by Graham and JaneNissen.
(19). Both proteins are distinct from the membrane drug-efflux

787

Minireview: Cellular Responses to Cisplatin

788

more, although extractsfrom normal cells were capable of performing low levels of repair synthesis on DNA treated with cisplatin,
extracts from X P cells had no activity (34).
Cisplatin-resistant cell lines show increasedlevels of DNArepair
(Table I), as measured by the loss of platinum adducts (35, 36),
DNA repair synthesis (37), and reactivation of cisplatin-damaged
plasmid DNA (38, 39). When the removal of specific cisplatin adducts was measured, themost significant observation was the enFIG.
1. Structures of cis-DDP, trans-DDP,and carboplatin.
hanced repairof d(GpG) adducts in the resistantcells (35).Finally,
normal cells repair cisplatin interstrand cross-links preferentially
pump encoded by the mdr (multidrug resistance)gene, which is not in transcriptionally active genes, and resistant cells showed ininvolved in cisplatin resistance (20).
creases in this preferential repair (40).
Which DNA repair proteins might be responsible for the inIntracellular Thiols: Glutathioneand Metallothionein
creased repair of cisplatin lesions? Agel mobility shift assay using
Glutathione (GSH) is a tripeptide thiol, y-glutamylcysteinylglya W-damaged DNA probe identified a nuclear protein, XP group E
cine. Concentrations of 0.5-10 II~Mmake it the most abundant thiol binding factor(WE-BF), that
recognizes many DNAlesions includin the cell. Glutathione is synthesizedin a two-step pathway: Glu ing those induced by ultraviolet radiation and cisplatin (41-43).'
+ Cys + y-Glu-Cys y-Glu-Cys-Gly involving the ATP-dependent Binding activity was absent ina subset of patients from X P group
enzymes y-glutamylcysteine synthetase and glutathione syntheE (41,4345) but was
always present in other
X P groups or normal
tase, respectively (21). The first stepis rate-limiting andinhibited individuals. Tumor cell lines selected in cisplatinshowed increased
by glutathione itself and by buthionine sulfoximine (BSO).
DNA repair and expressed 5-fold increased levels of XPE-BF (39).
As a potent nucleophile, GSH reacts with alkylating agents and The increase inXPE-BF occurred early in thedevelopment of ciswith cisplatin. The reaction of GSH and cisplatin in a 2:l molar platin resistance. Further increases in
XPE-BF did not occur with
ratio forms a GSH-platinum complex that is then eliminated
from further selection, implying that other mechanisms must be actithe cell by a n ATP-dependent glutathione S-conjugate exportpump vated to achieve higher levels of resistance. Future studies will
(22). GSHmay protect cells by intercepting reactive platinum com- benefit from the recent isolation of a gene encoding the probable
plexes before they can react withDNA. GSH also protects cells by monkey equivalent of XPE-BF (74).
supporting DNArepair,possibly by stabilizing repairenzymes such
The DNA repair gene ERCCl contains a helix-turn-helix motif
as DNA polymerase (Y or by promoting the formation of deoxyribo- characteristic of DNA-binding proteins (46), and likeXPE-BF, may
nucleotides (23).
be involved in the recognition of cisplatin damage. ERCCl was
Increased glutathione levels have been found in some cisplatin- expressed at levels 2.6-fold higher in clinically resistant tumors
resistant cells but not in others(Table I). However, the capacity of than in sensitive tumors (47). In one case of particular interest, a
the cell to synthesize GSH in response to stress may
be more patient's tumor was initially sensitive to carboplatin but became
important than the steady state GSH level (24). BSO effectively resistant after the sixth treatment cycle, and ERCCl expression
inhibits GSH synthesis, but cisplatin resistance is not invariably increased more than 10-fold in the resistanttumor.
affected by BSO, suggesting that there are other cellular mechaWhat is the importance of DNA repair compared to the other
mechanisms discussed above? Drug resistance of a particular cell
nisms of resistance (25).
Mammalian metallothionein (MT) is a small protein of 61-62 may be due to several possible mechanisms, and a given cell may
amino acids that contains 20 cysteine residues (26). It has a pre- use more than one mechanism. For example, the A2780-CP70 subsumed role in the detoxification of heavy metal ions. MT gene line was found to have increasesin drug efflux and in glutathione
transcription is strongly induced by heavy metal ions, glucocorti- level, as well as in DNA repair activity (Table I). Nevertheless,
coids, interferon, and stress.Both cis-DDP and trans-DDPbind to many studies indicate that
DNA repair is almost alwaysincreased
MT, with 10 platinum atomdmolecule. The bindingrate constant is in resistant cells while drug uptake, efflux, glutathione level, or
much higher thanfor glutathione. Heavy metals may even induce metallothionein level may remain unchanged. There may be a n
the MT thiol content to
a level higher thanthat of glutathione (27). approximate ordering for the mechanisms that the cell uses to
When cis-DDP binds to MT, i t loses its amine ligands and
displaces protect itself from cisplatin. Since increased DNA repair occurs
early (39) andconsistently during cisplatinselection, it appearsto
heavy metal ions, as shown by Reaction 2.
be activated first. Then,
to achieve higher degrees of resistance, the
(Zn2+),-MT+ 10(NH,),Pt2+ (Pt2+),,-MT+ 20NH3+ 7Zn2+
cell may induce additional mechanisms affecting accumulation, efflux, glutathione,and metallothionein.Additional
mechanisms
REACTION 2
may also be involved, as discussed below.
Does MT play a role in cisplatin resistance? In
some studies (25,
Other Proteins That Bind to Cisplatin-damagedDNA
27, 28), cell lines selected with heavy metals showed increases in
MT and became cross-resistant to cisplatin. However, selection
Are there other proteins that bind to cisplatin damaged DNA?
with cisplatin often has no effect on MT (Table I). Transfection of Cisplatin-cross-linked DNA (CCD) probes have been used in gel
the
mouse C127 cells with a metallothionein gene construct yielded mobility shift assay to identify such proteins in cell extracts. One
inconsistent effects on cisplatin resistance (28, 29).
CCD binding factor boundcisplatin adducts but not
UV adducts, in
contrast toXPE-BF, which bound both typesof damage (39,411. A
DNA Repair
small mobility shift suggested involvement of a protein with a low
Human cells repair cis-DDP lesions much less efficiently than molecular mass. A second factor, cisplatin-DNA damage recognition
a molecular
trans-DDP lesions (30). In fact,cell-free extracts have undetectable protein, was alsospecific for cisplatin adducts and had
all complerepairsynthesis on substratescontaining asingle intrastrand mass of -100 kDa (48). Both factorswere present in XP
d(GpG) cross-link,the most abundant lesion produced by cisplatin mentation groups and were unchanged in cisplatin-resistantcells.
CCD probes were also usedto screen a human cDNA expression
(31).Poor repair of d(GpG) intrastrand cross-links may contribute
library, identifying a cDNA encoding an 81-kDa structure-specific
to the cytotoxicity of cisplatin.
Repair of cisplatin adducts occurs primarily by the nucleotide recognition protein (SSRP1) that bound to cis-DDP but not transexcision repair pathway that is defective in the inherited disease DDP adducts (48, 49). There were no changes in mRNA levels in
xeroderma pigmentosum (XP).This pathway mustinclude multiple resistant cell lines. Surprisingly, 75 amino acids in SSRPl were
proteins since there are seven genetic complementation groups in 47% identical to a portion of the highmobility group (HMG) protein
XP. Cells from X P group A were 4-fold more sensitive to cisplatin HMG1. This conserved region, known as the HMG box, imparts a
when survival was measured as a function of platinum bound to primary structure to HMGlconsisting of two tandem HMG boxes
cellular DNA (32). When plasmid DNA carrying a reporter gene in the N-terminal two-thirds of the protein. Subsequent studies
was damaged by cisplatin and transfected into cells, X P cells re* A. Payne and G. Chu, submitted for publication.
paired the plasmid less efficiently than normalcells (33). Further-

platin trans-DDP

cis-DDP

--f

"-f

~~

Minireview: Cellular Responses

Cisplatinto

789

TABLEI
Characteristics of cisplatin-resistant cell lines derived from human ovarian cancers
The cell lines were derived from previously untreated patients (A2780, 2008, COLO, and 41M) or from a patient aRer treatment with cisplatin, 5-fluorouracil,and
chlorambucil (PE04).The cell lines from untreated patients were developed in vitro by selective growth in media containing cisplatin, except for 2008"T, which was
selected in cadmium and zinc, and COLO-MT,which was selected in cadmium. Cisplatin resistance is expressed as the relative increase in LDso. Tables for other tumors
are found in Ref. 14.
Cell
lines

~~~~~~

-fold
A2780
-CP8
-CP
-CP70
X30
-E80
-C50-200
2008
-DDP
"T

0.5

COLO
-DDP

"T
0.18-0.31
41M-cisR
PE04

7-13
14
13-39"
34-280
68
500-1000
2.5-10
4
2-4
13
3
2-6
3

Uptake

Emux

Glutathione

-fold

-fold

-fold

2
3
13
15

Reversal of
resistance
by BSO

Metallothionein

DNA
repa,r

-fold

-fold

1
Partial
1
1
Increased

29,2"

20-50
0.5-0.7

1-1.5

1
1
1

1
3

None
None

1
23

1
2
3
1
2

None
Partial
None

Increasedb

1
2c

Ref.

29,64
65
23, 6736, 40, 64, 66,
29, 68
29
68
25, 27, 40, 69, 70
27
25
71
25
72
37, 73

* Relative resistance of the CP70 subline was only &fold when survival was measured under conditions of the DNA repair assay, which involved a 1-h exposure to
cisplatin. This is comparable to the 2-fold increase in DNA repair measured byDNA repair synthesis and removal of platinum from chromosomal DNA, and for
transcription-coupled repair.
Increased transcription-coupled repair.
E Increased DNA repair measured by unscheduled DNA synthesis.

showed that both HMGl and HMG2 also bind to CCD (50, 51). ase also bound to DNA damaged by cis-DDP (42),and deletions of
Therefore, it appears that the HMG box encodes a recognition the PHRl gene led to increased resistance tocis-DDP as well as to
element for cisplatin modified DNA. HMGl andHMGZ were iden- N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 4-nitrosotified long agoby their abundance and highelectrophoretic mobil- quinoline-N-oxide (4NQ013 Significantly, photolyase does not conity (52). The proteins are
highly conserved,present inboth nucleus tain an HMG box or homology to other domains of the HMG box
and cytoplasm, but have unknown roles in the cell. Their low mo- proteins.
lecularmassandcellular
location suggestthatHMGl
and/or
HMG2 may be identical to CCD binding factor.
A M&l for Protein Binding to cis-DDP Adducts
The mouse homolog of SSRPl was cloned independently by the
There
are severalpossible mechanisms for how binding proteins
ability of the encoded protein to bind to the recombination signal
sequences required for V(DM recombination in B-cells (53). It is might confer sensitivity to cis-DDP. For example, the proteins may
noteworthy that some models of V(DMrecombination involve DNA shield cis-DDPadducts from repair (59)or may signalfor apoptosis
intermediates with secondary structures. Thus, when SSRPl
binds (Fig. 2). Some proteins (eg.HMG box proteins) maybind with high
to signal sequences, it may also participate in the formation or affinity to cisplatin lesions but not to trans-DDP, UV, or other
lesions. By contrast, XPE-BF has a DNA binding activity flexible
resolution of such secondary structures.
enough to recognize many different lesions. The price for this flexYet another HMG box protein, human upstream binding factor
(541, binds to CCD.2 The protein alsobinds to the rRNA gene ibility is that the affinity of XPE-BF for cisplatin adducts is relapromoter to activate RNA polymerase I transcription. The HMG tively low, about 40-fold lower than for UV 1esions.l As a result,
box proteins now form an extensive family (55), and each member D E - B F can displace HMG box proteins from the cisplatin adduct
only with some difficulty and DNA repair proceeds slowly.
is a potential candidate for binding to cisplatin adducts.
Alternatively, the binding of HMG box or other proteins to the
Why do HMG box proteins bind to cisplatinDNA adducts? Many
a program
have other activities diverse
in
aspects of cellular metabolism. It is cisplatin adduct maylead to cell cycle arrest and trigger
for cell death. This mechanism might explain the paradoxical obdifficult to believe that HMG1, HMG2, SSRP1, and human upservation that MNNG and 4NQ0 induce the expression of yeast
stream binding factoralso have additional
functions in DNA repair.
"he HMG box proteins do have the common feature of binding to photolyase (60), whichthen confers sensitivity to the same drugs.3
DNA involved in structuraldeformations (55).For example, HMGl There may be pressure for cells, even in unicellular organisms, to
and HMG2 bind cruciform structures and unwind negatively su- evolve a pathway which couples DNA damage to apoptosis. In a
percoiled DNA (56). Thus, theaffinity of the HMG box proteins for milieu with incomplete mixing, genetically identical sibling cells
cisplatin adducts appears to
be a case of mistaken identity, Aesop's remain in close proximity competing for the same cache of nutrients. Thus, apoptosis of an injured cell would provide a survival
lamb following a wolf in sheep's clothing.
Do the affinities of HMG or other proteins for cisplatin adducts advantage to itssiblings, selectingfor genes encoding a death pathhave functional consequences? Recent studies in the yeast Saccha-way.
Of course, other mechanisms are also possible. The binding of
romyces cerevisiae have identified two different proteins, which
blocks to repactually confer sensitivity to cisplatin. The first protein, Ixrl (in- HMG box proteins to cisplatin adducts may enhance
trastrand cross-link recognition), contains two tandemly repeated lication or transcription ormay interfere withcontrol systems that
HMG boxes and was identified from a n expression library for its induce cell cycle arrest after DNA damage.
Lesions induced by trans-DDP and UV are not recognized by
ability to bind to DNA damaged by cis-DDP (57). When the Z X R l
gene was disrupted, the yeast mutant
grew normally but displayed HMG box proteins, while the DNA repair system is capable of
increased resistance to cis-DDI? The second protein, photolyase recognizing many lesions. For example, XPE-BF recognizes DNA
(Phrl) was already
well known as a DNA repair protein for its role damage induced by W, denaturation (41), nitrogen mustard, and
depurination.' Such lesions are rapidly repaired and the cell surintheenzymatic
photoreactivation of cyclobutanepyrimidine
dimers induced by ultraviolet radiation (58).Surprisingly, photoly- vives. Therefore, cellular resistance tocisplatin may be determined
D. Treiber and J. Essigmann, private communication.

M. Fox and G. Chu, submitted for publication.

Minireview: Cellular Responses to Cisplatin

790

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13137-13142

cis-DDP
damage
trans-DDP
or

UV damage

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